Neuroblast lineage-specific origin of the neurons of the Drosophila larval olfactory system

Abstract

The complete neuronal repertoire of the central brain of Drosophila originates from only approximately 100 pairs of neural stem cells, or neuroblasts. Each neuroblast produces a highly stereotyped lineage of neurons which innervate specific compartments of the brain. Neuroblasts undergo two rounds of mitotic activity: embryonic divisions produce lineages of primary neurons that build the larval nervous system; after a brief quiescence, the neuroblasts go through a second round of divisions in larval stage to produce secondary neurons which are integrated into the adult nervous system. Here we investigate the lineages that are associated with the larval antennal lobe, one of the most widely studied neuronal systems in fly. We find that the same five neuroblasts responsible for the adult antennal lobe also produce the antennal lobe of the larval brain. However, there are notable differences in the composition of larval (primary) lineages and their adult (secondary) counterparts. Significantly, in the adult, two lineages (lNB/BAlc and adNB/BAmv3) produce uniglomerular projection neurons connecting the antennal lobe with the mushroom body and lateral horn; another lineage, vNB/BAla1, generates multiglomerular neurons reaching the lateral horn directly. lNB/BAlc, as well as a fourth lineage, vlNB/BAla2, generate a diversity of local interneurons. We describe a fifth, previously unknown lineage, BAlp4, which connects the posterior part of the antennal lobe and the neighboring tritocerebrum (gustatory center) with a higher brain center located adjacent to the mushroom body. In the larva, only one of these lineages, adNB/BAmv3, generates all uniglomerular projection neurons. Also as in the adult, lNB/BAlc and vlNB/BAla2 produce local interneurons which, in terms of diversity in architecture and transmitter expression, resemble their adult counterparts. In addition, lineages lNB/BAlc and vNB/BAla1, as well as the newly described BAlp4, form numerous types of projection neurons which along the same major axon pathways (antennal tracts) used by the antennal projection neurons, but which form connections that include regions outside the “classical” olfactory circuit triad antennal lobe-mushroom body-lateral horn. Our work will benefit functional studies of the larval olfactory circuit, and shed light on the relationship between larval and adult neurons.

Introduction

The olfactory system of Drosophila has become a useful genetic model for understanding the mechanisms of neural circuit formation. Notably, the adult olfactory system has been studied intensively and its component central and peripheral neurons have been characterized in terms of their anatomy, function, and development (reviwed by Vosshall and Stocker, 2007). The peripheral sensory component consists of approximately 1,300 olfactory sensory neurons (OSNs) located on each antenna. These sensory neurons establish precise neuronal circuitry in the antennal lobes, which represent the primary olfactory processing center of the brain. Each antennal lobe (AL) consists of approximately 50 subunits called glomeruli and each glomerulus receives synaptic input from olfactory sensory neurons that express a given odorant receptor (Axel, 1995; Vosshall, 2000). In the antennal lobe, olfactory sensory neurons make connections to two types of interneurons, projection neurons and local interneurons. There are approximately 200 projection neurons (PNs), many of which typically target their dendrites in a highly stereotyped manner to specific glomeruli and project their axons to higher brain centers in the mushroom body and lateral horn (Jefferis et al., 2001). The approximately 100 local interneurons (LNs, also called local neurons) restrict all of their neural arborizations to the antennal lobe where they make connections with projection neurons, olfactory sensory neurons as well as other local interneurons (Huang et al., 2010; Yaksi and Wilson, 2010). Recent analyses have revealed that both projection neurons and local interneurons can be further divided into several different neural subtypes which differ in glomerular innervation patterns, neurotransmitter type, and neurophysiological properties (Carlsson et al., 2010; Chou et al., 2010; Das et al., 2008; Lai et al., 2008).

A great deal of insight has been obtained regarding the development of adult olfactory circuitry of Drosophila primarily due to the availability of neuron type-specific driver lines combined with clonal MARCM labeling and mutational methods (reviewed in Brochtrup and Hummel, 2010; Rodrigues and Hummel, 2008). This type of clonal analysis has revealed that projection neurons and local interneurons derive in a lineage-specific manner from a small set of identified deutocerebral stem cells called neuroblasts (Das et al., 2011; Das et al., 2008; Jefferis et al., 2001; Lai et al., 2008). These neuroblasts, like all other neuroblasts in the Drosophila brain, each generate a specific lineage of neural progeny during two phases of proliferation. During embryonic development, neuroblasts generate the primary neurons of the larval CNS and during larval development, they generate the secondary (adult-specific) neurons that differentiate during metamorphosis to form the mature circuitry of the adult CNS. Published work shows that the adult-specific projection neurons and local interneurons arise from at least four neuroblasts, denoted as anterodorsal (adNB), lateral (lNB), ventral (vNB), and ventrolateral (vlNB) (Das et al., 2011; Das et al., 2008; Jefferis et al., 2001; Lai et al., 2008). Each NB generates an invariant lineage containing one or more interneuron types, and can be targeted by specific GAL4 driver lines. Lineages formed by adNB and lNB express the genetic markers GH146-GAL4 and Acj6-GAL4 (Fig. 1A, B). The adNB gives rise to uniglomerular projection neurons as well as a few multiglomerular projection neurons (Yu et al., 2010). The lNB, in contrast, generates a more complex set of progeny comprising uniglomerular and diverse atypical projection neurons as well as various types of local interneurons (Das et al., 2008; Lai et al., 2008). The vNB, labeled by MZ699-GAL4 and per-GAL4 (Fig. 1C) generates multiglomerular and oligoglomerular projection neurons (Lai et al., 2008). The vlNB, expressing OK371-GAL4 (Fig. 1D), gives rise to unilateral and bilateral local interneurons (Das et al., 2011). Together with the adult-specific olfactory sensory neurons, which derive from numerous sensory organ precursors in the antennal disc, the ensemble of these projection neurons and local interneurons form the adult specific olfactory circuitry of the mature antennal lobe.

Figure 1. Lineage specific labeling of adult olfactory interneurons by GAL4 lines.

Panels A–D show z-projection of frontal confocal sections showing cell bodies and proximal (antennal lobe) branches of labeled neurons; A′-D′ depict posterior levels, showing distal (posterior) branches of labeled neurons. Diagrams A″-E″ show schematic representation of lineages and their projections.

(A and A′) GAL4-GH146 labels 3 projection neuron clusters around the adult antennal lobe: antero-dorsal projection neurons belonging to the adNB/BAmv3 lineage, lateral projection neurons belonging to the lNB/BAlc lineage, and ventral projection neurons corresponding to the vNB/BAla1 lineage. They send out axonal tracts to the higher olfactory centers, the calyx of the mushroom body (MB) and the lateral horn of the protocerebrum through two antenno-cerebral tracts (ACTs): lNB/BAlc neurons send their axonal projections through the inner antenno-cerebral tract (iACT) and vNB/BAla1 neurons through the medial antenno-cerebral tract (mACT).

(B and B′) Acj6-GAL4 labels two sets of projection neurons: adNB/BAmv3 neurons, and lateral atypical projection neurons of the lNB/BAlc lineage (B) sending axonal projections through the iACT and oACT (outer antenno-cerebral tract white arrow in B′) respectively.

(C and C′) GAL4-MZ699 labels the ventral projection neurons of the vNB/BAla1 lineage, sending axons to the lateral horn through the mACT.

(D and D′) GAL4-OK371 labels local interneurons belonging to the ventrolateral local interneuron (vlLN/BAla2) lineage; several of these local interneurons send axons to the contralateral antennal lobe through the antennal lobe commissure (white arrow in D′). Inset in D shows MARCM clone in BAla2 visualized by OK371. Neuron on contralateral side (asterisk) is not part of the clone.

(E and E′) GAL4-VGN9281 labels several projection neurons of the BAlp4 lineage, located ventro-lateral to the antennal lobe. They send their dendrites mostly to the ventro-medial glomeruli of the antennal lobe and also to the SOG (white arrow in E). The axon tract of these projection neurons follow the iACT (white arrow in E′) at least initially, before terminating in the protocerebral neuropil.

Note: Cell bodies of neurons belonging to specific lineages are circled by lines rendered in colors representing distinct lineages: BAmv3: cyan, BAlc: magenta, BAla1: blue, BAla2: red and BAlp4: yellow.

Scale bar represents 10 μm.

A much simpler version of the adult olfactory system is formed in the larval brain of Drosophila. The larval olfactory system lacks the cellular complexity of the adult and yet fulfills all the major functional requirements of olfactory processing circuitry. Only 21 olfactory sensory neurons located in the dorsal organ of the antenno-maxillary complex project their axons to the 21 glomeruli of the larval antennal lobe (Heimbeck et al., 1999; Ramaekers et al., 2005; Tissot et al., 1997). These olfactory sensory neurons are thought to make connections with a set of approximately 21 uniglomerular and biglomerular projection neurons as well as with approximately 21 putative multiglomerular local interneurons (Ramaekers et al., 2005). The projection neurons of the larval olfactory system project from the larval antennal lobe to the mushroom body calyx and lateral horn and form stereotyped connectivity with Kenyon cells (Masuda-Nakagawa et al., 2009; Masuda-Nakagawa et al., 2005). When compared with the olfactory system of the adult fly, the larval olfactory system appears remarkably streamlined and manifests a 1:1:1 relationship among olfactory sensory neuron number, glomerular number, and projection neuron number (Ramaekers et al., 2005).

Little is known about the lineage composition and development of the larval olfactory system, which prompted the current study. A total of 18 uniglomerular projection neurons and 1 polyglomerular projection neuron of the larval projection neurons derive from the adNB during embryogenesis (Marin et al., 2005; Yu et al., 2010). The larval local interneurons are reported to be grouped in three cell body clusters; however, their neuroblasts of origin are not known (Ramaekers et al., 2005). In this paper, we study the lineage-specific development of the primary projection neurons and local interneurons that contribute to the larval antennal lobe. We use the lineage specific drivers marking the adult antennal lobe lineages, and clonal MARCM and cis-flip-out based labeling of single neurons to investigate the diversity of primary projection neurons and local interneurons produced in the embryo. We find that the adNB, similar to its adult counterpart, comprises uniglomerular, biglomerular, and multiglomerular primary projection neurons. The lNB lineage produces various multiglomerular and oligoglomerular primary local interneurons, many of which also project processes to the neighboring suboesophageal neuropile, as well as atypical primary projection neurons. The vNB lineage contains a few oligoglomerular and multiglomerular primary projection neurons, but, unlike in the adult, mostly produces neurons with only short branches into the antennal lobe, and more extensive ones in the basal brain compartments posteriorly adjacent to the antennal lobe. The vlNB comprises primary local interneurons of diverse multiglomerular and oligoglomerular types. The new fifth NB lineage produces novel primary projection neurons, which send processes to antennal lobe, suboesophageal ganglion (SOG) and the vertical lobe of the mushroom body. In addition to these four lineages known for the adult, we identified a novel fifth lineage, BAlp4, which generates primary and secondary olfactory projection neurons. Both in the larva and adult, these neurons innervate part of the posterior antennal lobe, and the adjacent SOG, and project to higher brain centers other than calyx and lateral horn. Taken together, our findings reveal a complexity of different primary projection neuron and local interneuron types that is comparable to that of the antennal lobe of the adult.

Material and methods

Fly stocks

All flies were raised at 25°C on standard cornmeal-agar medium unless otherwise mentioned. Following are the GAL4 lines used in this study with their source:

GAL4 Lines	Source
C155-GAL4	Bloomington Drosophila Stock Center, Indiana, USA.
GAL4-GH146	R. F. Stocker, University of Fribourg, Switzerland.
Acj6-GAL4	Tzumin Lee, University of Massachusetts, Worcester, USA.
GAL4-LN2	Kei Ito and NP consortium, The University of Tokyo, Japan.
GAL4-MZ699	Tzumin Lee, University of Massachusetts, Worcester, USA.
GAL4-OK371	Hermann Aberle, Max Plank Institute, Tubingen, Germany.
0.3ChaT-GAL4	(See methods)
VGN9281-GAL4	(see Brierley et al., 2009)
Per-GAL4	(see Larsen et al., 2009)

MARCM and flip-out experiments

In order to generate MARCM clones from different GAL4 lines, the following crosses were set up:

GAL4 line	Females	Males
C155-GAL4	GAL4-C155,UASmCD8::GFP,hsflp; FRT40A,tubGAL80/CyO	FRT40A
GAL4-GH146	y,w,hsflp; GAL4-GH146, UAS-mCD8::GFP/CyO; FRT82B,tub- GAL80	UAS-mCD8::GFP,UAS-LacZ/CyO; FRT82B
Acj6-GAL4	Acj6-GAL4, UAS- mCD8::GFP/FM7a; FRTG13,UASmCD8::GFP/CyO	y,w,hsflp,UAS-mCD8::GFP; FRTG13,tub-GAL80/CyO
GAL4-OK371	y,w,hsflp,UAS-mCD8::GFP; FRTG13,tub-GAL80/CyO	UAS-mCD8::GFP,OK371,FRTG13

Embryos from the above crosses were collected at 2 hrs intervals and reared at 25°C. Neuroblast or single cell clones were induced at various stages of early embryonic development between 1–12 hrs after egg laying (AEL) by heat shocks for 30 min to 1 hour in a water bath maintained at 37°C.

In order to generate single cell flip-out (cis-flip) clones of the primary neurons of the larval brain from different GAL4 lines (GAL4-LN2, GAL4-MZ699, 0.3ChaT-GAL4, GAL4-VGN9281), males from the GAL4 lines were crossed to the virgin females of the yhsflp; UAS>CD2y+>mCD8::GFP/CyO; TM2/TM6 line. Embryos were collected every 4 hours and kept in 25°C for larval hatching. For labeling single primary neurons, heat shock was given to the second instar larvae (between 48–60 hrs after egg laying) for 30 min to 1 hour in a 37°C water bath. Cultures were returned to 25°C incubators and animals were allowed to develop to the third instar larval stage when their brains were dissected and immunolabeled.

Immunohistochemistry and Imaging

Dissection of third instar larval brains was carried out in 1X PBS and fixed in 4% PFA for 30 minutes. The fixative was then removed and the brain samples were washed and simultaneously blocked with 0.1% PBTX (0.1% BSA and 0.3% TritonX-100 containing 1X PBS) 4 times 15 minutes each at room temperature. Primary antibodies were diluted in 0.1% PBTX and added to the tissue samples and incubated in 4°C for two overnights. Primary antibodies used were: rabbit anti-GFP (1:10,000; Molecular Probes, Invitrogen, Delhi, India), chicken anti-GFP (1:500; AbCam, Cambridge, UK), mouse anti-Bruchpilot (mAbnc82, 1:20; DSHB, Iowa, USA), mouse anti Dlg (1:100, DSHB, Iowa, USA), mouse anti-neurotactin (Nrt, BP106, 1:10; DSHB, Iowa, USA), rabbit anti-GABA (1:500; cat#A2052, Sigma, St Louis, MO, USA), mouse anti-ChaT (4B1, 1:1000; DSHB, Iowa, USA), rabbit anti-C-term DVGLUT (1:500; a gift from Hermann Aberle and Aaron DiAntonio, Washington University in St. Louis, MO, USA) Rat anti N-Cadherin (1:50, DSHB). After primary antibody incubation, brains were washed in 0.3% PTX (0.3% TritonX-100 containing PBS) for 4 times of 15 minutes each. Secondary antibodies were prepared in 0.3% PTX and incubation was done overnight in 4°C. Secondary antibodies used were: Alexa-488, Alexa-568, and Alexa-647 coupled antibodies generated in goat (Molecular Probes) – all were used at 1:400 dilutions. Following 4 rounds (15 min each) of wash with 0.3% PTX, larval brains were mounted in 70% glycerol. Brain samples were imaged on Olympus Fluoview FV1000 Laser Scanning Confocal microscope at 1 μm intervals with a size of 512 × 512 pixels or 1024 × 1024 pixels; data was processed using Olympus Fluoview (version 2.0), ImageJ, and Adobe Photoshop CS3 softwares. 3-D reconstructions were made by using Amira 5.2.0 software (Germany).

Generation of the 0.3Cha-GAL4 line

The cholinergic GAL4 driver line (0.3Cha-GAL4) were created by amplifying a 300bp fragment of the regulatory region of ChaT gene via Polymerase Chain Reaction (PCR). The 300bp (−520bp to −817bp) fragment was amplified using the Gateway primers attB1- ggc aaa atg gca aaa gca cgc aat g and attB2- Cgc atg cgc ctg cgc ctc cgc cg. PCR products were introduced into the pDONRTM221-vector (Invitrogen) via Gateway cloning (Invitrogen) to create the adequate pEntry-vector. In a subsequent Gateway reaction, the regulatory region was cloned upstream of the GAL4 open reading frame of the pCaSpeR-Destination3 vector. Successful introduction was proven by sequencing. DNA was purified using the Qiagen Midi Kit and used to establish transgenic lines via germ line transformation by BestGene Inc., (Chino Hills, CA, USA). These lines were screened for expression in the larval antennal lobe. We found the fragment 0.3Cha-GAL4 to be expressed in a population of cholinergic local interneurons.

Results

Lineages associated with the larval antennal lobe

We utilized the GAL4-markers known to be expressed in specific clusters of neurons of the adult antennal lobe (Fig 1) to identify the corresponding secondary lineages in the late larval brain. Furthermore, following a screen for suitable markers among existing GAL4-line collections, a total of 8 additional GAL4 lines (along with the pan-neuronal driver C155-GAL4) were selected and used to first identify the antennal lobe associated lineages in the larva, and subsequently to generate single cell clones of the primary neurons in these lineages. A summary scheme of these GAL4 lines and their lineage-specific labeling properties is shown in figure 2A; expression patterns of these GAL4 lines in the larval interneurons (local interneurons and projection neurons) surrounding the larval antennal lobe are shown in Figure S1.

Figure 2. Antennal lobe-associated lineages in the late larval brain.

(A) Schematic diagram showing lineage-specific expression of the 8 GAL4 lines used in this study. Dark magenta colored circle (at the middle) represents the larval antennal lobe neuropil and cell body clusters (smaller circles around the lobe) denote primary projection neurons and local interneurons belonging to different lineages. All GAL4 lines are listed on the right hand side with specific color codes to indicate the lineages where they are expressed. Some GAL4 lines are restricted to very few lineages, for example OK371 is seen to express only in the BAla2 lineage, GH146 is restricted to BAmv3 and BAlc, LN2 expression is seen in BAlc and BAla2 lineages; whereas some lines show much broader expression, for example, 0.3Cha line is expressed in primary local interneurons and projection neurons belonging to all 5 lineages.

(B–F) Characteristic axonal projection of the five antennal lobe-associated lineages. B: Overview. Z-projection of anterior part of confocal stack of a third instar larval brain hemisphere, expressing membrane tagged GFP in antennal lobe projection neurons driven by GAL4-GH146 (green) in the background of anti-neurotactin immunolabeling to show the developing secondary neurons and their axonal tracts. Midline (ml) to the left. Rectangular frame indicates the area of the brain, centered on the antennal lobe, which is shown in panels C–F. These panels show enlargements of z-projections of the same hemisphere at different antero-posterior levels. The levels are indicated by hatched lines in the digital model presented in panel H. Outline of antennal lobe is shaded (green). The five antennal lobe-associated lineages are indicated in colors that are used throughout the following panels of this figure. C: anterior surface of antennal lobe, BAmv3 tract enters the antennal lobe at its anterior-dorsal surface (ad); BAlc tract enters laterally (al); BAla1 and BAla2 run close to each other and enter ventro-laterally (av). D, E: Level 5–10 micron into the antennal lobe. The tracts of BAmv3 and BAlc have approached each other and turn dorsally to form the anterior root of the antennal tract (ar in E). The tracts of BAla1 and BAla2 also turn upward (D); BAla2 (local interneurons) does not leave the antennal lobe, whereas BAla1 forms the posterior root of the ACT (pr in E). The fifth lineage, BAlp4, enters the antennal lobe near its posterior-ventral surface (pv in E). It projects straight dorsally and joins BAla1 in the posterior root of the ACT. F: level shortly posterior to the antennal lobe. Tracts of BAmv3, BAlc and BAlp4 have come close and form the inner ACT (iACT); axons of BAla1 veer off laterally and form the mACT.

(G–I) Digital 3-dimentional models of the five antennal lobe-associated lineages and projection pattern of their SATs with respect to the antennal lobe (gray). Round spheres indicate location of cell body clusters of the lineages; pipes show projection of axon tracts of the corresponding lineages. The same color code is used as in panels C–F. Aside from antennal lobe, the lobes, peduncle, and calyx of the mushroom body are shown for reference. G: anterior view; medial to the left (ml: midline), dorsal up; H: dorsal view; medial to the left, anterior up; I: lateral view; anterior to the left, dorsal up. Hatched lines in H indicate the anterior-posterior levels represented by photographs in panels C–F.

As is the case for all other lineages of the brain, the secondary (adult-specific) interneurons of the antennal lobe lineages are generated during larval development and form axons which fasciculate with each other to form secondary axon tracts (SATs) that project towards the neuropile forming a highly stereotyped axon tract system. Based on the invariant and characteristic nature of the SAT for each neuroblast lineage in the larval brain, a comprehensive lineage-based map of all the secondary neurons in the larval brain has been generated (Pereanu and Hartenstein, 2006). Moreover, a comprehensive three dimensional digital atlas has been made based on lineage location and projection as well as a neuroanatomical nomenclature for identification of all of these lineages has been established (Cardona et al., 2010).

Five secondary lineages are associated with the larval antennal lobe. Four of these can be identified with the previously described (Das et al., 2011; Lai et al., 2008) adult lineages. The adNB lineage (GH146, Acj6) corresponds to BAmv3 (Fig. 1A,B); the lNB lineage (GH146, Acj6) to BAlc (Fig. 1A,B); the vNB lineage (MZ699, per) to BAla1 (Fig. 1C); and vlNB (OK371) to BAla2 (Fig. 1D). In the rest of the paper the larval nomenclature system has been followed for defining these lineages (i.e., BAmv3, BAlc, BAla1, BAla2 and Balp4) as per Pereanu and Hartenstein, 2006. The BAmv3 and BAlc tracts enter the antennal lobe antero-dorsally and laterally, respectively (ad and al in Fig. 2). These tracts travel across the dorsal surface of the antennal lobe, and project straight medially, before turning posterior and upward to exit the antennal lobe as the anterior root (ar in Fig. 2C, E and G) of the antenno-cerebral tract (ACT). Within the antenno-cerebral tract, BAmv3 and BAlc neurons form the most medial or inner group of axons (inner antenno-cerebral tract, iACT) which project to the calyx and lateral horn (Fig. 2C, G). The neurons of BAla1 and BAla2 form clusters located laterally and dorsally of the antennal lobe. Their axons form two closely apposed, parallel tracts that pass over the anterior surface of the antennal lobe, before entering at a ventral position (av in Fig. 2C, E, G, I). They project posteriorly, near the ventral surface of the antennal lobe, and then turn upward. One tract (BAla2) terminates within the antennal lobe; the other, BAla1, continues dorsally as the posterior root of the antenno-cerebral tract (pr in Fig. 2C, E, G, I). Within the ACT, BAla1 axons form a separate, lateral bundle; this bundle later branches off and follows a lateral trajectory towards the lateral horn (medial antenno-cerebral tract, mACT; Fig. 2C, G, H). One should note that the lateral horn, as a anatomically distinct compartment similar to that in the adult brain, does not exist in the larva. Here, the entire dorsal protocerebrum appears as a thin layer of neuropile termed “CPLd”; the large lateral horn and superior lateral protocerebrum of the adult emerge during metamorphosis from the CPLd (Pereanu et al., 2010). However, the future position of the lateral horn within the CPLd is clearly demarcated by the projection of GH146-labeled neurons, as shown by previous authors and in this study.

A fifth secondary lineage associated with the larval antennal lobe, BAlp4, is located further posterior and ventral of the other lineages (Pereanu and Hartenstein, 2006; Fig. 3A, A′, E). The BAlp4 tract follows a straight, dorso-medial trajectory and joins BAla1 as it enters the posterior root of the ACT (Fig. 2D, E, H). The BAlp4 tract can be followed throughout pupal development into the adult (Fig. 3A–D). Characteristically, from late larval stages onward, one can recognize a ventrally directed branch towards the SOG (arrowheads in Fig. 3C′, C″, E′). Thus, as shown in greater detail below, BAlp4 projection neurons form connections between the antennal lobe and SOG, as well as antennal lobe and protocerebrum. A driver line, VGN9281-GAL4, is expressed in multiple groups of antennal lobe-associated neurons that include (in part) the BAlp4 lineage (Fig 1E and 3F).

Figure 3. Development of the fifth antennal lobe-associated lineage (BAlp4) through larval and pupal stages.

(A–D) Identification of the BAlp4 lineage and its axon tracts through development (L3, 18 hrs APF, 48 hrs APF, and adult brain). Anti-Neurotactin (Anti-Nrt, BP106) is used to label all secondary lineages and their SATs in the larval brain (red in A–D and gray in A′-D′) and neuropils are labeled by anti-DN-Cadherin (blue). The boxed area in A–D shows the antennal lobe region of the brain in each panel, shown at higher magnification in A′-D′. The BAlp4 lineage is identifiable as a cell cluster located ventral and lateral of the antennal lobe, sending its axon tract straight dorsally along the posterior surface of the antennal lobe. This tract can be followed throughout development into the adult. The BAlp4 tract forms a characteristic ventrally directed branch (red arrowhead) towards the SOG, visibly most clearly at pupal stages (C′, C″). (E and E′) Neuroblast clone of the BAlp4 lineage in L3 showing cell body location and axon tract. Note tuft of filopodia (red arrowhead) which foreshadow the ventral branch depicted in C-C″. (F) The GAL4-VGN9281 driver line labels a subset of neurons of the BAlp4 lineage (yellow arrowheads) in the adult brain, sending primary neurites (white arrowheads) into the ventro-medial glomeruli of the antennal lobe (red arrowhead). Ventral branch (red arrow) exits the antennal lobe towards the SOG.

Identification and lineage relationship of primary interneurons in the olfactory system

To label the primary olfactory interneuron types that are involved in the larval antennal lobe, two types of labeling methods were employed (see Methods). First, single-cell MARCM (Lee and Luo, 1999) clones were induced at different stages during embryogenesis and recovered in the late third larval instar stage. The time window of clone induction ranged from 1h AEL (after egg laying) to 12h AEL in order to label primary neurons born at different times during embryonic neurogenesis. Second, a cis-flip-out technique (Wong et al., 2002) was used to generate single cell clones in post-mitotic neurons. Heat shocks were given during the second larval instar and clones were recovered in the late third instar larval brain.

The single cell clones of the primary neurons that make up the larval olfactory system can be unambiguously distinguished from secondary neurons by the fact that the primary neurons are fully differentiated in the larva and have formed mature dendrites, axons, and terminal arbors which arborize in the larval brain neuropile (Fig. 4A, C). In contrast, single cell clones of secondary neurons in the larval brain are immature (Fig. 4B, D) and only differentiate during the pupal period when proximal and terminal arbors are formed. Primary neurons also typically have larger somata than secondary neurons in the larval brain (compare the cell body sizes of primary projection neurons, yellow arrowhead, and secondary projection neurons, blue arrowhead in Fig. 4A).

Figure 4. Spatial relationship between primary and secondary neurons of the same lineage.

(A and B) Neuroblast clones of the BAmv3 lineage induced at early (A) and late (B) embryo and recovered in L3. In A, both primary projection neurons (yellow arrowhead) and secondary projection neurons (blue arrowhead) are present within the clone (induced at early embryo); whereas in B, the neuroblast clone contains only secondary projection neurons. The larval antennal lobe is innervated only by the primaries (A) and not by the secondaries. The primary axon tract (PAT), containing axons of differentiated primary BAmv3 neurons, projects via the iACT and forms branches in the calyx (CX) and lateral horn (LH) neuropils (outlined by yellow dotted lines in A, A′); the secondary axon tract (SAT) follows the same route through the iACT, but does not form terminal arbors in these neuropils (yellow dotted line in B).

(C and D) Single cell clones of primary and secondary projection neurons in 3^rd instar larval brain. The primary projection neuron sends terminal neurite branches to a single antennal lobe glomerulus (red arrow) and the axon terminates in the MB calyx and lateral horn neuropils (C, C′). The single secondary projection neuron has no branches in the antennal lobe; instead a few immature branches are observed in the prospective adult antennal lobe region (demarcated by cyan circular line, dorsal to the larval antennal lobe demarcated by yellow dotted line, in D, D′). No terminal branches of the SAT reach within the calyx or lateral horn (yellow dotted lines in D, D′).

(E) BAmv3 neuroblast clone induced in early embryo, labeled by GAL4-GH146, showing close association between primary and secondary projection neuron cell bodies and also between PAT and SAT (yellow arrowhead) running parallel to each other. Only the primary neurons form mature axon terminals in the neuropils of the MB calyx and the lateral horn (yellow dotted lines).

(F–H) BAmv3 projection neurons labeled by GAL4-GH146 (green) in the background of neurotactin immunolabeling (red) at late larval stage. GAL4-GH146 expresses strongly in primary neurons (yellow arrowhead) and less strongly in secondary neurons (cyan arrowhead). By contrast, only secondary neurons are Neurotactin (Nrt) immunoreactive. The PAT and SAT run parallel to each other (H-H″) en route to the higher center neuropils; the SAT is Nrt+ve (cyan arrowheads) and the PAT is Nrt-ve (yellow arrowheads).

Abbreviations: AL= antennal lobe; CX=calyx; LH= lateral horn; PAT=primary axon tract; SAT=secondary axon tract.

Scale bar represents 10 μm.

To relate the diverse single cell clones of primary olfactory interneurons to one of the five identified olfactory lineages, we took advantage of the close association of primary and secondary axon tracts in larval neuroblast lineages (Spindler and Hartenstein, 2010). This intimate association of primary and secondary neuron tracts in a given lineage is shown for the BAmv3 lineage in Fig. 4E, which depicts a MARCM labeled neuroblast clone induced during embryogenesis that comprises both the primary and secondary neurons of this lineage. The close association between the primary neuron axons and the SAT of the secondary neurons is further documented in Fig. 4F–H where anti-Neurotactin (red) labels only the cell bodies and axon tracts of secondary neurons (Fig. 4G′, H′), while GH146 expression (green) is strongest in primary neurons and their axons (Fig. 4G, H; yellow arrowheads). It is evident that primary and secondary axon tracts remain closely associated and follow the same route towards the higher centers.

Given that the axons of the primary neurons are closely associated with the same lineage-specific SAT that comprises the axons of their lineally related secondary neurons, the lineage of origin of any labeled primary neuron can be determined by its point of entry into the antennal lobe and its specific axonal trajectory. With these methods, single cell clones of primary olfactory interneurons were characterized anatomically and related to specific neuroblast lineages. These findings are presented below for each of the five olfactory interneuron lineages.

Primary olfactory projection neurons of the BAmv3 lineage

The BAmv3 lineage contains the majority of primary antennal projection neurons. This lineage is labeled by the GAL4 line GH146 starting from first instar larval stage. Fig. 5 illustrates the expression pattern of GH146 in the BAmv3 lineage of first (Fig. 5A,B) and third instar larvae (Fig. 5C). Single cell clones of primary neurons belonging to the BAmv3 lineage were recovered, all of which project their axons through the larval iACT, and make terminal arbors in the calyx of the mushroom body and lateral horn of the larval brain (as previously shown in Masuda-Nakagawa et al., 2009; Masuda-Nakagawa et al., 2005) (Fig. 5D–F). Morphologically, these projection neurons correspond to three different types. As observed by Masuda-Nakagawa et al. (2009), who used the GH146 driver for their clonal analysis, the most frequently recovered projection neuron type has a uniglomerular antennal lobe innervation pattern (Fig. 5D). The second type has a biglomerular antennal lobe innervation pattern (Fig. 5E). The third, most infrequently recovered type makes multiglomerular or large-field dendritic innervation of the antennal lobe (Fig. 5F). All three types typically make axon terminal arbors in 1–2 mushroom body calyx glomeruli.

Figure 5. BAmv3 produces projection neurons of the larval antennal lobe.

(A–C) Larval projection neurons of the BAmv3 lineage labeled by GAL4-GH146. The neuropile is labeled by anti-Brp (red). A, B: First instar larva; z-projection of anterior brain hemisphere, including antennal lobe (AL in A), and of posterior hemisphere with calyx (CX) and lateral horn (LH). Medial to the left, dorsal up. C: anterior part of third instar larval brain. Note the increase in cell number in BAmv3 lineage in third instar larval stage due to addition of secondary projection neurons which form a dense cluster of small cell bodies (arrowhead in C). (D–F) Third instar larva, z-projection of one brain hemisphere, medial to the left, dorsal up. Three types of single cell MARCM clones of the BAmv3 projection neurons: uniglomerular (D), biglomerular (E), and large-field or poly-glomerular projection neuron (F) (blue arrows in the antennal lobe). All single cell clones send their axon through the iACT (yellow arrow) and only one or two glomeruli in the MB calyx are innervated (cyan arrows in calyx) by a single projection neuron irrespective of their innervation pattern in the antennal lobe glomeruli.

Abbreviations: AL= antennal lobe; CX= calyx of mushroom body; CPLd= centro-posterior-lateral compartment, dorsal domain

Scale bar represents 10 μm (for all panels).

Primary neurons of BAla1 include antennal projection neurons and interneurons branching outside the antennal lobe

Among the primary neurons of the BAla1 (vNB) lineage we also find projection neurons connecting the antennal lobe to the superior protocerebrum. Similar to their adult counterparts, primary projection neurons of the BAla1 lineage form oligo or multiglomerular (dendritic) branches in the antennal lobe, and project via the mACT, which passes underneath the peduncle. Interestingly, at least one of these primary BAla1 projection neurons crosses the peduncle medially and dorsally, rather than ventrally/laterally (Fig. 6A–C). The axon of the neuron shown terminates in two distinct glomerulus-like structures in the lateral horn (termed dorsal domain of centro-posterior-lateral compartment, CPLd, in the larva) (Fig. 6D). The BAla1 neuron shown in Fig. 6E–G forms branches that reach beyond the boundaries of the antennal lobe into the adjacent neuropile compartments (baso-posterior medial compartment, BPM) and SOG; Fig. 6E, E′, F). Furthermore, the axon of this neuron, after initially extending along the iACT (Fig. 6F), projects straight dorsally to terminate around the proximal peduncle and the lobes of the mushroom body, rather than the lateral horn (Fig. 6E, G).

Figure 6. Primary neurons of the BAla1 lineage.

All panels show z-projections of third instar larval brain hemispheres; medial to the left, dorsal up. Neuropile is labeled by anti-Brp (red). (A–A′) GAL4–MZ699 labeled single cell clone of a projection neuron innervating 2–3 glomeruli of the posterior antennal lobe; the axonal projection ascends through the mACT (white arrow) to terminate in the CPLd (larval forerunner of the lateral horn; yellow dotted line) at its boundary with the MB calyx. (B–D) Same clone as in A; B shows z-projection of anterior part of brain hemisphere, including cell body and proximal arbors in antennal lobe; C contains a segment of the axon crossing over the peduncle (p); D shows terminal arborization in the form of two glomeruli located at the boundary between CX and CPLd. (E–E′) A single projection neuron labeled by GAL4-LN2 line sends oligo-glomerular projections to the larval antennal lobe (yellow dotted line), as well as to the SOG (yellow arrow). The axon tract initially follows the iACT, but then deviates from this tract to form terminal branches (yellow arrowhead) around the proximal peduncle and the dorsal lobe of the MB (dl, blue dotted line). (F–G) Same clone as in E; F shows z-projection of anterior brain hemisphere with cell body and projections to antennal lobe and SOG; G shows terminal axonal boutons in the neuropile surrounding the peduncle and dorsal lobe (yellow arrowhead). (H–K) Period-GAL4 labels the extra-antennal lobe BAla1 projection neurons. H: First instar larval brain, lateral view, anterior to the left, dorsal up. I–K: first instar larval brain; medial to the left, dorsal up. Anterior level (I), central level (J), posterior level (K). Labeled BAla1 neurons do not form branches in the antennal lobe (AL); axons follow the mACT and from branches in the BPM compartment posterior to the antennal lobe and in the vicinity of the peduncle (yellow arrowheads in H and K).

Abbreviations: AL= antennal lobe; SOG= suboesophageal ganglion; CX= calyx of mushroom body; dl= dorsal lobe of mushroom body; ml= medial lobe of mushroom body; p= peduncle of mushroom body; CPL= centro-posterior-lateral compartment ; CPLd= centro-posterior-lateral compartment, dorsal domain; BPM= baso-posterior medial compartment; LAL= lateral accessory lobe.

Scale bars represent 10 μm (for all panels).

Aside from the antenno-protocerebral projection neurons, the larval BAla1 lineage included projection neurons that did not innervate the antennal lobe. These cells, called extra-antennal BAla1 projection neurons in the following, are highlighted by the Per-GAL4 driver line (Fig. 6H–K). Axons of these neurons enter the neuropile like the antenno-protocerebral projection neurons, via the posterior root of the ACT (white arrows in Fig. 6H, I). They then give off profuse branches towards posterior into the BPM compartment that is located posterior of the antennal lobe (yellow arrows in Fig. 6H, J). At least some extra-antennal projection neurons send fibers into the mACT; however, these fibers do not appear to reach the superior protocerebrum, but terminate in the CPI compartment around the peduncle (yellow arrowheads in Fig. 6H, K).

Primary olfactory interneurons of the BAla2 lineage are local interneurons

Similar to what has been reported for the adult brain (Das et al., 2011; Das et al., 2008; Lai et al., 2008), local interneurons of the larval antennal lobe are formed by two lineages, BAla2 and BAlc. The driver line OK371-GAL4, restricted to glutamatergic neurons (Fig. S2A), is expressed exclusively in a subset of BAla2 neurons and was used to induce single cell clones. In addition, the Cha-GAL4 driver, labeling cholinergic neurons (Fig. S2B), and the LN2-GAL4, induced BAla2 single cell clones. BAla2 local interneurons are located antero-dorsally of the antennal lobe. The main axon of a BAla2 neuron skirts the anterior surface of the antennal lobe and enters at the antero-ventral entry point (white arrowheads in Fig. 7A–E). In many neurons, branches are given off the main axon as it passes over the anterior antennal lobe. BAla2 local interneurons can be assigned to six different types based on morphology and neurotransmitter characteristics. The first three types of BAla2 local interneurons are glutamatergic and can be visualized by using OK371-GAL4:

Figure 7. BAla2 lineage produces primary local interneurons of diverse architectures.

All panels show z-projections of third instar larval brain hemispheres; medial to the left, dorsal up. Neuropile is labeled by anti-Brp (red). The antennal lobe is outlined by yellow dotted line; SOG by blue dotted line. Small white arrowheads point at entry point of BAla2 neuronal fibers into antennal lobe. (A–C) local interneurons labeled by GAL4-OK371. Neuron with multiglomerular projections restricted mainly to the central glomeruli (A); multiglomerular projections restricted to peripheral glomeruli (B); oligoglomerular local interneurons with projections to a few glomeruli located centrally in the antennal lobe (C). In the first two cases, neurites extend to the SOG as well (white arrows). (D) local interneuron labeled by 0.3Cha-GAL4, with arbors thinly branching throughout the antennal lobe, as well as the SOG (white arrow). Antennal lobe neurites are denser in the medial side of the lobe (magenta arrowhead).

(E) GAL4-LN2 labeled single multiglomerular local interneuron with arbors occupying the ventro-lateral side of the larval antennal lobe (cyan arrowhead) which also sends some projections to the SOG (white arrow).

(F) Another type of single local interneuron clone labeled by GAL4-LN2 with oligo-glomerular antennal lobe projections mostly restricted to a few ventro-medial glomeruli (blue arrowhead) and prominent branches to the SOG (white arrow).

Abbreviations: AL= antennal lobe; LAL= lateral accessory lobe; SOG= suboesophageal ganglion.

Scale bar represents 10 μm (for all panels).

1. Local interneurons with prominent multiglomerular arborization in the center of the antennal lobe. These cells also form sparse branches in the suboesophageal neuropile medially adjacent to the antennal lobe (Fig. 7A).

2. Local interneurons innervating the peripheral glomeruli of the antennal lobe and forming small arbors in the SOG (Fig. 7B).

3. Local interneurons with branches restricted to a few glomeruli in the posterior antennal lobe and lacking projections to the SOG (Fig. 7C).

A fourth type of BAla2 neuron is cholinergic, has a panglomerular innervation pattern in the antennal lobe and projects neurites to the SOG (Fig. 7D).

Two additional types of BAla2 neurons were visualized by using the LN2-GAL4 driver. Both of these LN2 neurons are oligoglomerular in their projection to the antennal lobe, and have sparse branches in the SOG. In one type, antennal lobe innervation was concentrated in the ventrolateral glomeruli; the other type focused on ventromedial glomeruli (Fig. 7E, F).

It is possible that the expression of LN2-GAL4 and Cha-GAL4 overlaps, in which case these two types of neurons could both be cholinergic.

Primary olfactory interneurons of the BAlc lineage are projection neurons and local neurons

The primary neurons of the BAlc lineage show the highest degree of diversity, similar to what is seen with BAlc secondary neurons in the adult. As in the adult, larval BAlc neurons include both local neurons and projection neurons (Das et al., 2008; Lai et al., 2008). BAlc local interneurons are similar in projection and shape to the BAla2 neurons described above; however, they can be unambiguously distinguished from those by their characteristic lateral-posterior entry point into the antennal lobe (white arrowheads in Fig. 8A–D; see also Fig. 2). BAlc local interneurons are visualized using the GAL4 drivers LN2 and 0.3Cha. LN2-GAL4 which, in the BAlc lineage, overlaps with GABA immunoreactivity (Fig. S2C), labels two types of neurons:

Figure 8. Diverse primary interneuron types formed by the BAlc lineage.

All panels show z-projections of third instar larval brain hemispheres; medial to the left, dorsal up. Neuropile is labeled by anti-Brp (red). The antennal lobe is outlined by yellow dotted line; SOG by blue dotted line. White arrowheads mark entry points of BAlc fibres. Panels of upper row (A–C) show three different types of local interneurons belonging to the BAlc lineage. (A) A pan-antennal lobe local interneuron labeled by GAL4-LN2, with all neurites restricted to the antennal lobe. (B) Another pan-antennal lobe local interneuron labeled by the same driver with few projections to the SOG (white arrow). (C) An oligo-glomerular local interneuron labeled by 0.3Cha-GAL4 with strong projections to the SOG (white arrow). Panels of lower three rows (D–F, G–I, J–L) show three types of atypical projection neurons. Left panel of each row represents z-projection of entire brain hemisphere; panels in center show partial z-projection of anterior brain, including antennal lobe and SOG; panels at the right represent posterior brain. (D–F) Atypical projection neuron labeled by Acj6-GAL4 sending a small number of branches to the larval antennal lobe; significant amount of neurites are detected in the ipsilateral and contralateral SOG (white arrows). The long axon projects via oACT to the CPLd (forerunner of lateral horn). (G–I) Another type of projection neuron has sparse branches in the posterior antennal lobe; it projects its axon along the iACT, and forms terminal branches medial and dorsal of the peduncle in a domain called the central posterior intermediate compartment (CPI; forerunner of inferior protocerebrum of adult brain). (J–L) A third type of BAlc projection neuron with bilateral antennal lobe innervation. It innervates the ipsilateral antennal lobe with sparse neurites, the axon tract ascends through the iACT to form widespread branches in the higher centers, crosses the midline to send out branches to the contralateral higher centers, and finally descends through the iACT towards the contralateral antennal lobe, where it sends out pan-glomerular branches, some even terminating in the SOG neuropil. This neuron corresponds to the contralaterally projecting serotonin-immunoreactive deutocerebral (CSD) interneuron.

Abbreviations: AL= antennal lobe; BPL= baso-posterior lateral compartment; BPM= baso-posterior medial compartment; CPI= centro-posterior intermediate compartment; CPLd= centro-posterior-lateral compartment, dorsal domain; CX= calyx of mushroom body; dl= dorsal lobe of mushroom body; LAL= lateral accessory lobe; oACT= outer antenno-cerebral tract; iACT= inner antenno-cerebral tract; ml= medial lobe of mushroom body; p= peduncle of mushroom body; SOG= suboesophageal ganglion.

Scale bars represent 10 μm, correspond to specific rows.

1. Local interneurons with panglomerular innervation of the antennal lobe (Fig. 8A).

2. Local interneurons with panglomerular antennal lobe innervation and additional arbors in the SOG (Fig. 8B).

The same two types of local interneurons were obtained when using the 0.3Cha-GAL4 driver (data not shown) which are anti-ChaT immunoreactive (Fig S2B). In addition, using this driver, we recovered local interneurons with an oligoglomerular innervation pattern in the antennal lobe and pronounced arbors in the SOG (Fig. 8C).

We could recognize three different types of BAlc projection neurons connecting the antennal lobe with the superior protocerebrum. Two types were labeled when using Acj6-GAL4 to visualize single cell clones; a third type could be visualized using the RN2 driver line. These three types are:

1. Neurons that form sparse projections in the posterior antennal lobe, near the point of entry of the cell body fibre with more profuse arbors reaching into the SOG medial and ventral of the antennal lobe. These projections are of one of two types: ipsilateral or ipsi- and contralateral, as shown in Fig. 8D–F. Their ascending axons leave the antennal lobe lateral and ventral of the main ACT and project via the outer antenno-cerebral tract (oACT) to the CPLd compartment, the larval forerunner of the lateral horn (Fig. 8D–F).

2. The second type of BAlc projection neuron (Fig. 8G–I) also has sparse branches in the posterior antennal lobe, projects its axon along the iACT, and leaves this tract at a fairly anterior level, forming terminal branches medial and dorsal of the peduncle in a domain called the central posterior intermediate compartment (CPI; Younossi-Hartenstein et al., 2003).

3. The third type of BAlc projection neuron (Fig. 8J–L) has widespread, bilateral arborization, and corresponds to the (only) serotonergic interneuron in the olfactory system, named contralaterally projecting serotonin-immunoreactive deutocerebral (CSD) interneurons (Roy et al., 2007). This neuron innervates a restricted, small domain of the dorso-posterior ipsilateral antennal lobe (Fig. 8J, K) and sends its axon via the iACT to the higher brain centers; unlike other projection neurons, it crosses the midline and turns back ventrally to reach the contralateral antennal lobe via the iACT (Fig. 8J, L). Profuse branches are given off on both sides in the neuropile domain medial and dorsal of the peduncle (CPI and dorso-posterior (DP) compartments); a dense projection also reaches the baso-posterior medial compartment (BPM). Few, if any branches contact the “canonical” targets of the antennal lobe projection neurons, i.e., the calyx and lateral horn (CPLd in the larva). Pan-glomerular axonal endings are formed in the contralateral antennal lobe (Fig. 8J, K).

Note that the main type of adult BAlc projection neurons, the uniglomerular projection neuron projection to the calyx and lateral horn, is not represented at all among larval BAlc neurons.

Primary neurons of the BAlp4 lineage

None of the driver lines used in this study had a strong, exclusive expression in BAlp4. However, LN2 and VGN 9281 were expressed in a pattern that included few BAlp4 neurons (Fig. S1H). An early embryonic clone using C155-GAL4 (pan neuronal driver) labeled the full lineage in the late third larval instar brain, including primary and secondary neurons (Fig. 9A–C). Primary BAlp4 neurons are projection neurons with long axons in the iACT. Their cell bodies are located ventro-laterally of the antennal lobe and enter at the postero-ventral entry point (see Fig. 2). Single BAlp4 primaries labeled by 0.3Cha-GAL4 (Fig. 9D–F) shows that their proximal branches are given off towards the posterior glomeruli of the antennal lobe; however, more prominent branches are directed ventrally towards the SOG (Fig. 9A, C and D–F). Two primaries labeled by VGN9281 show very sparse neurites within antennal lobe or SOG (Fig. 9G–I). Axons entering the iACT reach the superior protocerebrum and form sparse arborizations in the CPI and CPL compartments (Fig. 9C, F, I); none of these branches enters the calyx. VGN 9281 revealed small groups of BAlp4 neurons in the late larva (Fig. 9G–I) and adult (Fig. 9J–O), confirming proximal arborization in the antennal lobe plus SOG, and distal projections in the CPI/CPL (central posterior intermediate and central posterior lateral compartments respectively, corresponding to the inferior protocerebrum of the adult brain). Distal branches had thick varicosities (Fig. 9M), indicating a preponderance of output synapses.

Figure 9. Projection neurons of the BAlp4 lineage.

All panels except J–O show z-projections of third instar larval brain hemispheres; medial to the left, dorsal up. J–O represent adult brain hemisphere. Neuropile is labeled by anti-Brp (red). The antennal lobe (AL) is outlined by yellow dotted line; SOG by cyan dotted line. All four rows of panels (A–C, D–F, G–I, J–L) are organized such that left panel of each row represents z-projection of entire brain hemisphere; panels in center show partial z-projection of anterior brain, including antennal lobe and SOG; panels at the right represent posterior brain. (A–C) A full BAlp4 NB clone induced at early embryonic stage and recovered at the late third instar larval stage showing cell bodies (both primaries and secondaries) located ventro-lateral of the antennal lobe, sparse proximal projections in the antennal lobe, and profuse branching in the SOG; axons project through the iACT to the CPI and CPL compartments of the protocerebrum.

(D–F) A single BAlp4 projection neuron clone labeled by 0.3Cha-GAL4 line sending neurites into larval antennal lobe and SOG; the axon termini are largely observed in the CPI compartment.

(G–I) Two primary projection neurons of BAlp4 lineage are labeled by GAL4-VGN9281 line. Antennal lobe and SOG received very sparse neurite projections whereas the axons follow the iACT to project to CPI and CPL compartments.

(J–O) Adult brain showing BAlp4 lineage labeled partly by GAL4-VGN9281. The cell bodies are located latero-ventral to the antennal lobe, sending oligo-glomerular dendritic innervation to the posterior antennal lobe and to the SOG; axons terminate in the inferior protocerebrum of the brain, terminal varicosities are shown in M–O.

Abbreviations: AL= antennal lobe; SOG= suboesophageal ganglion; CX= calyx of mushroom body; dl= dorsal lobe of mushroom body; ml= medial lobe of mushroom body; p= peduncle of mushroom body; CPI= central posterior intermediate compartment; CPL= centro-posterior-lateral compartment ; CPLd= centro-posterior-lateral compartment, dorsal domain; BPL= baso-posterior lateral compartment; LAL= lateral accessory lobe.

Scale bars = 10 μm, correspond to specific rows.

Discussion

The olfactory system of Drosophila has been intensively studied in terms of formation and function as a model for understanding olfactory information processing. Most investigations have focused on the adult olfactory system since it manifests a remarkably similar general organization as the olfactory system in vertebrates yet is simpler in its numerical complexity. This reduced complexity of the peripheral odorant receptors, olfactory sensory neurons, and central interneuronal elements has contributed to the experimental success of this model in unraveling the genetic basis of circuit formation (Brochtrup and Hummel, 2010; Rodrigues and Hummel, 2008). Compared to the adult, the olfactory system of the larva is even simpler in numerical complexity, while maintaining a comparable functional organization. Only 21 olfactory sensory neurons project into the larval antennal lobe (Heimbeck et al., 1999; Ramaekers et al., 2005; Tissot et al., 1997) and interconnect with less than 50 interneuronal elements, comprising both projection neurons and local interneurons (Ramaekers et al., 2005). Given its reduced, streamlined nature, the larval olfactory system of Drosophila may become an equally important model for understanding circuit formation, since it can be analyzed in terms of all of its constituting peripheral and central neural cells. However, currently very little is known about the developmental origin of the larval olfactory system. In this report, we analyze the developmental origin and morphological diversity of the primary projection neurons and local interneurons that contribute to the circuitry of the larval antennal lobe. We find an unexpectedly large diversity of projection neuron and local interneuron cell types, map these different cell types to their lineage of origin, and, in doing so, identify a novel fifth olfactory interneuron lineage. Below we discuss the significance of these findings.

Lineage-specific origin of projection neuron and local interneuron types in the larval and adult olfactory system

Previous work has shown that neuroblast lineages form the fundamental genetic, developmental, and structural units of the brain (see Hartenstein et al., 2008; Ito and Awasaki, 2008). Neuroblast lineages are clusters of neurons, usually comprising 100–150 cells, which remain associated throughout development. The axons of the primary and secondary neurons in a given lineage generally form a coherent fascicle with a common trajectory and, hence, represent a unit of projection. Moreover, the neurite arborizations of a lineage are often spatially restricted to individual neuropile compartments/subcompartments.

This lineage-specific organization of the brain is exemplified by recent studies on the antennal lobe circuitry of the adult olfactory system (Das et al., 2011; Das et al., 2008; Lai et al., 2008; Yu et al., 2010). Four neuroblast lineages have been shown to give rise to the secondary projection neurons and local interneurons of the adult antennal lobe. Two of these, the adNB-derived BAmv3 lineage and the vNB-derived BAla1 lineages are composed exclusively of projection neurons, and for each of these lineages, their cell bodies cluster together and their axons project together in the same axon tract (iACT for BAmv3 and mACT for BAla1) to common targets in higher brain centers (Fig. 10). The vlNB-derived BAla2 lineage is composed exclusively of local interneurons; their cell bodies remain clustered together and their neurites arborize ipsilateral as well as the contralateral antennal lobe (Fig. 10). The lNB-derived BAlc lineage is more heterogeneous and comprises both local interneurons and projection neurons of different types. Aside from uniglomerular projection neurons with “canonical” projection to the calyx and lateral horn (through iACT), lNB neurons included a large group that project via iACT to the inferior protocerebrum, as well as multiglomerular neurons with “atypical” projections to various protocerebral compartments outside the calyx/lateral horn (Lai et al., 2008).

Figure 10. A schematic depicting comparison of anatomical features of the primary and secondary interneurons born from each of the five olfactory interneuron lineages.

(A) BAmv3 lineage produces olfactory projection neurons, primaries and secondaries are nearly identical. They are mostly uniglomerular in nature (with minor variations in both cases); axons follow the iACT to reach the MB calyx and lateral horn of the protocerebrum.

(B) The BAlc interneurons in both larval and adult stages are similar. (i) Local interneurons (orange) of the larva often invade the neighbouring SOG neuropil, but in the adult they are restricted to the antennal lobe. (ii) Projection neurons (light green) are uniglomerular in the adult, axons follow the iACT to reach the MB calyx and lateral horn; in larva they are oligo-glomerular and follow the iACT where they terminate in the CPI compartment surrounding the peduncle of the MB. (iii) Atypical projection neurons (dark green) of both the adult and larval brain follow the oACT to the reach the CPLd/LH and CPI/IP compartments.

(C) Larval and adult BAla1 neurons show considerable differences. In the adult, they are uniglomerular and multiglomerular projection neurons that project through the mACT. In the larval brain the primaries are oligoglomerular or multiglomerular. Their axons project through either the mACT or iACT. Only a fraction of larval BAla1 primaries connect AL and CPLd/LH; most neurons have atypical connections with the SOG, CPI/IP, and BPM/VMC compartments.

(D) Both primaries and secondaries of the BAla2 lineage are either oligoglomerular or multiglomerular local interneurons, though the primaries often connect with the larval SOG and secondaries often cross-over to connect with the contra-lateral adult antennal lobe.

(E) BAlp4 neurons are projection neurons in both larval and adult brains and have comparable features. They send oligoglomerular or sparse branches to the antennal lobe as well as prominent branches to the SOG; axons follow the iACT and form terminal connections in the protocerebrum.

Our findings indicate that the lineage-specific organization of the larval antennal lobe circuitry is similar to that of the corresponding adult system. However, when looking at details of their neurite tree, several larval primary lineages contain types of projection neurons that are not seen in the corresponding secondary lineages (Fig. 10). Only in case of BAmv3, all primary neurons form a relatively homogenous group which resembles its adult counterpart. Both primary and secondary BAmv3 projection neurons have uniglomerular or oligoglomerular dendritic branches within the antennal lobe (Ramaekers et al., 2005; Thum et al., 2011; Yu et al., 2010; this study) and project via iACT to the calyx and lateral horn. As mentioned above, similar projection neurons are formed by the secondary BAlc lineage, but are not part of the primary BAlc lineage. Primary BAlc projection neurons either fall into the group that uses the outer ACT (also described in adult), or constitute novel types not encountered among the secondary BAlc neurons. Thus, one type of primary BAlc projection neurons exits the antennal lobe via the iACT, but then terminates in the neuropile surrounding the peduncle (CPI) before reaching the calyx. Another type, probably corresponding to the previously identified antennal serotonergic neuron (Roy et al., 2007), crosses the midline in the supraesophageal commissure and forms bilateral projections in the antennal lobe and protocerebral neuropiles.

Our data reveal several types of projection neurons for the primary BAla1 lineage, which contrasts with the homogeneity of secondary BAla1 neurons. Before going into detail it should be noted that this stated difference notwithstanding, primary and secondary neurons share a common location (ventral of the antennal lobe), enter the antennal lobe at the same position (ventro-lateral entry point), and form a common proximal tract, the posterior root of the ACT, which is clearly set apart from the anterior root constituted by axons of BAmv3 and BAlc projection neurons. Furthermore, as a result of the posterior entry of the neurites, dendritic branches of primary and secondary BAla1 neurons alike enter the antennal lobe from posterior side. However, after entering the posterior root and giving off dendrites into the antennal lobe, all secondary neurons continue to project medially (mACT) towards/underneath the peduncle, and terminate in the lateral horn. By contrast, only a few primary BAla1 projection neurons show this kind of projection pattern. Many primary BAla1 neurons do not have dendrites in the antennal lobe, but instead project further posteriorly to the BPM compartment. Another class that does not exist among the secondary BAla1 neurons, forms branches in both the antennal lobe and adjacent SOG, and projects axons via the inner ACT towards the lobes of the mushroom body (see Fig. 6 and Fig10).

BAla2 primary neurons resemble their secondary neuron counterparts when considering the absence of long axons that project to “higher brain centers”, i.e., neuropile compartments of the protocerebrum. Most branches of the neurite tree of BAla2 neurons are restricted to the antennal lobe. However, unlike their secondary siblings, a large fraction of primary BAla2 neurons forms branches outside the antennal lobe in the medially and ventrally adjacent SOG. The same holds true for many primary BAlc neurons, which project to parts of the SOG close to the antennal lobe (Thum et al., 2011 and this study), unlike secondary BAlc local interneurons, which are confined to the antennal lobe.

Local interneurons and projection neurons of the larval olfactory system link antennal lobe and suboesophageal neuropile

Our analysis of the lineages which generate projection neurons or local interneurons that innervate the antennal lobe has identified a fifth olfactory neuroblast lineage, BAlp4. This lineage appears to be composed exclusively of projection neurons, which have a similar morphology in the larval and the adult olfactory system. Both primary and secondary BAlp4 projection neurons project their axons via the iACT to protocerebral compartments outside the calyx and lateral horn. These compartments include the deeper layers of the superior protocerebrum as well as the inferior protocerebrum. Likewise, primary BAlp4 projection neurons of the larva form terminal branches in the CPI and CPL compartments, which are the forerunners of the inferior protocerebrum (Pereanu et al., 2010). Furthermore, BAlp4 projection neurons form profuse arborizations in the neuropile of the SOG. Since the SOG is the primary site of gustatory information processing, this morphological feature suggests that the BAlp4 projection neurons might integrate both olfactory and gustatory information and convey this to higher brain centers. However, more detailed anatomical and functional investigations of this projection neuron type are necessary before this notion can be confirmed.

As mentioned above, the combination of glomerular antennal lobe innervation with innervation of the SOG is also seen in many of the primary olfactory local interneurons, as well as in some of the primary BAla1 projection neurons. Numerous primary local interneurons which form arborizations in the antennal lobe and SOG are seen in the BAlc and BAla2 lineages, confirming previous reports of local interneurons with suboesophageal arbors in the larval antennal system (Python and Stocker, 2002). In their recent study of BAlc interneurons, Thum et al (2011) have used markers for presynaptic (dendritic) sites to distinguish whether the synapses formed by neurons branching in both antennal lobe and SOG were input or output synapses. Their data indicate that both types of synapses are intermingled: the same interneuron forms both output and input synapses in the antennal lobe and SOG. In this regard, the neurons resemble the type of non spiking local interneurons studied in detail in the thoracic ganglia of locusts (Siegler and Burrows, 1979). In the adult olfactory system, neither secondary projection neurons nor secondary local interneurons make arborizations in the SOG (except for the adult projection neurons of the fifth olfactory lineage BAlp4, which has not yet been studied in the adult). Indeed the formation of both antennal lobe and SOG arborizations by many primary local interneurons and projection neurons is probably the most conspicuous difference in the morphology of primary versus secondary olfactory neurons.

Single cell analysis reveals a diverse set of projection neurons and local interneurons in the larval olfactory system

The single-cell MARCM clones of olfactory projection neurons and local interneurons reported in this study are striking in their diversity. Our data indicate that there are at least 8 projection neuron types present (Fig. 10). Owing to the use of multiple GAL4 lines a larger variety of projection neuron types are identified compared to earlier studies (Masuda-Nakagawa et al., 2009; Masuda-Nakagawa et al., 2005; Ramaekers et al., 2005). A few of these types, namely those formed by neurons of BAmv3, a subset of BAla1, and a subset of BAlc, have been described for the adult (Lai et al., 2008; Yu et al., 2010). They represent the uniglomerular, bi/oligoglomerular, and multiglomerular projection neurons that target the calyx and lateral horn. The remainder of primary projection neuron types project to protocerebral targets other than the calyx and lateral horn, as well as to the SOG. Our data also indicate that at least 9 local interneuron types with different morphological and putative neurotransmitter phenotypes are represented in the larval antennal lobe. We find oligoglomerular, multiglomerular, and panglomerular local interneurons, which can be GABA-ergic, cholinergic or glutamatergic (Fig. S2). The existence of excitatory larval local interneurons prompts us to think that the lateral interglomerular excitation of projection neurons proposed in adult (Shang et al., 2007) might be relevant to the larva as well. Thus, although the larval circuitry may be reduced in terms of cell number, it shows a surprising diversity of projection neuron and local interneuron cell types that is comparable to that of the complex adult olfactory system.

This morphological and neurotransmitter diversity is all the more remarkable since previous studies of total cell numbers suggest that there may only be 21 projection neurons and 21 local interneurons present in the larval olfactory system (Ramaekers et al., 2005). This in turn suggests that each cell type identified in our study may only be represented by a small number of cells. Indeed, in the case of projection neurons, a given cell type/uniglomerular subtype may be uniquely represented by a single cell. If this is also the case for local interneurons, then the larval olfactory system would represent an ideal model system for the investigation of complex sensory processing circuitry on the basis of individually identifiable (and potentially genetically accessible) cells. The power of this type of identified cell approach for understanding complex circuit function has been amply demonstrated in other invertebrate models such as the stomatogastric nervous system of the dacapod crustaceans (e.g. Marder and Bucher, 2007). Thus, further investigations using additional molecular markers to investigate if each local interneuron in the larval olfactory system does indeed represent a single identifiable neuron type will be important to carry out.

Supplementary Material

Sup. Fig.S1. Figure S1. Larval brain expression pattern of GAL4 lines used in this study.

(A) GAL4-GH146 is expressed in two clusters of cells around the larval antennal lobe, a dorso-lateral cluster containing both primary and secondary projection neurons (belonging to BAmv3 lineage, cyan oval) sending PATs and SATs through the iACT (yellow arrowhead); and a lateral cluster containing immature secondary projection neurons sending a SAT through the iACT (magenta oval) and 1–2 primary atypical projection neurons (cyan arrow) sending axons through the oACT (cyan arrowhead). The antennal lobe gets dendritic branches from the primary projection neurons of the BAmv3 lineage and the atypical projection neurons of BAlc lineage which also project to the SOG (white arrowhead).

(B) Acj6-GAL4 expression is detected in two clusters around the larval antennal lobe; the dorso-lateral cluster contains both primary and secondary projection neurons of the BAmv3/adPN lineage (cyan oval) and the ventro-lateral cluster contains primary and secondary atypical projection neurons which belong to the lNB/BAlc lineage (cyan arrow, magenta oval). (B′) The adPNs project their PATs and SATs to higher brain centers (MB calyx and lateral horn) through the iACT (yellow arrow); the atypical projection neurons of the BAlc lineage project their axons through the oACT (cyan arrow). Some amount of innervation is observed in the SOG as well (white arrowhead), probably from the primary atypical projection neurons of the BAlc lineage.

(C) GAL4-MZ699 labels a number of neurons that project to the larval antennal lobe, which are located in a lateral cluster (purple oval). These cells also send axons to higher brain centers through the mACT (yellow arrowheads). These neurons belong to the vNB/BAla1 lineage.

(D) Expression of 0.3Cha-GAL4 is observed in several clusters of larval antennal interneurons. A dorsal cluster of 3–4 cells (cyan oval), two lateral clusters containing ~7–8 cells (red oval), and a ventro-lateral cluster of ~8–10 neurons (magenta oval) are visible.

(E) GAL4-LN2 is expressed in two clusters of local interneurons located lateral (red oval) and ventro-lateral (magenta oval) to the larval antennal lobe, each containing ~4–6 cell bodies. The neurons send profuse branches throughout the larval antennal lobe and also form prominent branches in the SOG (white arrowheads).

(F) GAL4-OK371 labels a single cluster of local interneurons at the lateral periphery of the larval antennal lobe (red oval), containing both primary as well as secondary local interneurons.

(G) Per-GAL4 shows expression in some primary and secondary projection neurons of BAla2 lineage in L3. They send their axon through the iACT (yellow arrowheads in the inset).

(H) GAL4-VGN9281 labels few projection neurons belonging to the BAlp4 lineage. These projection neurons are located ventral to antennal lobe and their axons follow the iACT (yellow arrowheads) towards the higher brain centers. The larval antennal lobe receives sparse projections of dendrites which also connects to SOG (white arrowhead).

In each panel, the larval antennal lobe is demarcated with a yellow dotted line, clusters of antennal lobe interneurons are indicated with oval shaped lines of different colors indicating their lineages of origin: cyan- BAmv3, magenta- BAlc, blue- Bala1, and red- BAla2 and yellow- BAlp4.

Scale bars= 10 μm.

Sup. Fig.S2. Figure S2. Neurotransmtter identities of the local interneurons labeled by different GAL4 lines.

(A) OK371-labeled BAla2 local interneurons are glutamatergic; anti-DVGLUT staining in OK371-GAL4 shows the cell bodies of the BAla2 local interneurons are DVGLUT +ve (white arrows in A1–A3).

(B) 0.3Cha-labeled BAlc local interneurons are cholinergic; anti-ChaT staining of 0.3Cha line in B1-B3 shows the ventro-lateral local interneurons marked by this line to be cholinergic.

(C) LN2 labels two clusters of local interneurons in BAlc and BAla2; among which BAlc-local interneurons (ventro-lateral cluster) are GABA+ve (white arrows in C1–C3) and BAla2-local interneurons (lateral cluster) are GABA-ve (cyan arrowheads in B4–B6).