The ventral nerve cord (VNC) of Drosophila melanogaster is home to circuits coding for vital behaviors, such as walking, jumping, and flight (Harris et al., 2015; Gowda et al., 2018). It is composed of about 16,000 neurons, all of which arise from a set of segmentally repeated 30 paired and one unpaired neural stem cells (Neuroblasts [NBs]) (Bossing et al., 1996; Schmid et al., 1999; Lacin and Truman, 2016; Shepherd et al., 2016). NBs generate unique progeny via undergoing two rounds of proliferation: a brief embryonic and an extended postembryonic phase (Truman and Bate, 1988; Truman et al., 2004; Truman et al., 2010). Embryonic neurogenesis generates the neurons of the larval CNS and many of these cells are then remodeled to function in the adult CNS (Truman, 1990; Hartenstein and Wodarz, 2013). 90–95% of the adult neurons, however, are adult-specific and arise during the post-embryonic phase of neurogenesis.

The VNC contains only NBs that show a Type I pattern of proliferation (Truman et al., 2010). Each NB divides repeatedly via asymmetric cell division to renew itself and to generate a secondary precursor cell, called a ganglion mother cell (GMC) (Figure 1A). Each GMC, in turn, divides terminally to form two neurons, each of which acquires a unique identity due to the presence or absence of active Notch signaling (Spana and Doe, 1996; Skeath and Doe, 1998; Truman et al., 2010). Within a NB progeny, the Notch-ON neurons are called the ‘A’ hemilineage; their Notch-OFF siblings are called the ‘B’ hemilineage (Figure 1B).

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By progressing through the temporal transcriptional cascade, Hunchback →Kruppel → Pdm, NBs generate diverse ‘A’ and ‘B’ neurons during the early embryonic phase (Figure 1B), (Kambadur et al., 1998; Isshiki et al., 2001). Subsequently, NBs express Castor and Grainyhead in late embryonic stages and many NBs maintain this Castor/Grainyhead expression into the postembryonic stages (Almeida and Bray, 2005; Maurange et al., 2008; Lacin and Truman, 2016). Correlated with this shared gene expression, neurons of a hemilineage (‘A’ or’ B’) that are born during the late embryonic and early postembyonic stages often adopt similar fates (Truman et al., 2010; Lacin and Truman, 2016). Recent studies characterized the morphologies of postembryonic hemilineages in their immature states in the larva and mature states in the adult (Truman et al., 2004; Truman et al., 2010; Harris et al., 2015; Shepherd et al., 2016). These studies revealed that in the larva, the immature neurons of each hemilineage cluster together and extend their initial processes as a bundle to the same region and that after metamorphosis, in the adult, they continue to be clustered and share common anatomical and functional features (Figure 1C).

In addition to similar morphology, neurons within a postembryonic hemilineage share patterns of transcription factor expression. In the larval VNC, each hemilineage cluster can be identified with a specific combination of transcription factor expression (Lacin et al., 2014). Interestingly, vertebrate homologs of many of these hemilineage-specific transcription factors are expressed in the spinal cord and are required for fate determination (Thor and Thomas, 1997; Jessell, 2000). For example, in flies, the combinatorial expression of Lim3, Islet, and Nkx6 is observed uniquely in hemilineage 15B, which is composed of leg motor neurons (Lacin et al., 2014). In mice, homologs of these three factors are essential for the identity of spinal motor neurons (Tsuchida et al., 1994; Lacin et al., 2014). Indeed, interneurons in the vertebrate spinal cord are also organized into discrete cardinal classes that share developmental origins, transcription factor and neurotransmitter expression, and functional roles (Briscoe et al., 2000; Jessell, 2000; Zhang et al., 2008; Arber, 2012; Lu et al., 2015). The fly VNC appears to be organized in an analogous manner, with the developmental origins of neuronal clusters providing the basis for their transcription factor expression and functional properties. The relationship between specific stem cells and neurotransmitter expression in their progeny, though, has yet to be resolved.

Studies on grasshoppers (Siegler et al., 2001) and Manduca sexta (Witten and Truman, 1991) showed that clusters of GABAergic interneurons were based on their lineage of origin. Likewise, cholinergic and glutamatergic neurons are also typically found as clusters in the VNC and the brain consistent with a shared lineage (Salvaterra and Kitamoto, 2001; Liu and Wilson, 2013) In the fly, as a prelude to studies that seek to dissect the developmental basis of behavior, we wanted to map neurotransmitter usage comprehensively across the VNC to determine how neurotransmitter selection relates to lineage identity. A similar comprehensive neurotransmitter map was generated for the C. elegans nervous system and proved to be beneficial in identifying regulatory mechanisms that control neurotransmitter identity and circuit assembly (Serrano-Saiz et al., 2013; Pereira et al., 2015; Gendrel et al., 2016; Serrano-Saiz et al., 2017). Here, by using molecular and genetic tools, we mapped neurotransmitter usage in all hemilineages in the adult fly VNC. Our results revealed that, as found in the neurons of the vertebrate cardinal classes, all neurons within a fly hemilineage use the same neurotransmitter. In agreement with our earlier findings (Harris et al., 2015), this study further shows that hemilineages are not just developmental units but also functional units that drive animal behavior.

The vast majority of fly neurons use one of the three fast acting neurotransmitters: GABA, glutamate, or acetylcholine. First, we wanted to know the relative usage of these neurotransmitters in the VNC and asked if neurons use more than one neurotransmitter, a possibility suggested for the fly brain based on transcriptomic studies (Croset et al., 2018). We visualized mRNAs of neurotransmitter-specific genes glutamate decarboxylase 1 (gad1), vesicular glutamate transporter (VGlut), and choline acetyltransferase (ChAT) in the same animal via RNA in situ hybridization (Long et al., 2017). gad1 and ChAT encode enzymes that are required for the production of GABA and acetylcholine, respectively, while VGlut encodes a glutamate transporter (Slemmon et al., 1991; Kulkarni et al., 1994; Daniels et al., 2004). We observed mutually exclusive expression patterns among these mRNAs across the entire VNC (Figure 1D), suggesting that most if not all neurons in the VNC use only one of these neurotransmitters. One caution, though, is that some of the GABAergic and glutamatergic neurons show low levels of ChAT transcripts. We address this issue below.

Considering the obvious clustering of neurons expressing a given transmitter type in the VNC (Figure 1D), we asked if these clusters correspond to the postembryonic hemilineages and if all the neurons within a hemilineage use the same neurotransmitter. Thus, we decided to characterize all postembryonic hemilineages in terms of their choice of neurotransmitters.

Visualizing adult lineages

We employed several different approaches to visualize specific hemilineages in the adult VNC, each of which can be identified based on cell body position and axonal projection (Harris, 2013; Harris et al., 2015; Shepherd et al., 2016). First, we used a set of transcription factors that are lineage-specific molecular markers that identify immature neurons of specific hemilineages in the larval VNC (Lacin et al., 2014). Expression of many of these factors are maintained through metamorphosis and identify the corresponding mature neurons in the adult (Figure 1E; Figure 2). We also generated split-GAL4 halves reporting the expression of some of these transcription factors via converting available MIMIC lines with the TROJAN method or via CRISPR tagging (Venken et al., 2011; Diao et al., 2015). We used these split-GAL4 lines to reveal overlapping expression patterns. Simply, we generated reporter lines expressing either the DNA-binding domain (DBD) or activation domain (AD) of the GAL4 transcription factor with the idea that both the AD and DBD need to be present in the same cell to form a functional GAL4 reporter.

Figure 2

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We used these genetic lines in combination with one another or with neurotransmitter-specific split-GAL4 halves (Diao et al., 2015) to mark specific hemilineages and reveal their neurotransmitter identity (Figure 1F–J). Of note, intersectional combinations of these split -GAL4 halves proved to be extremely useful for developmental studies because they mark specific hemilineages throughout the development and adult life unlike many of the enhancer driven GAL4 lines from the Janelia (Jenett et al., 2012) or Vienna (Kvon et al., 2014) collection, which show dynamic expression through metamorphosis (Figure 1—figure supplement 1). In addition to lineage specific transcription factor expression, we employed NB intersected reporter immortalization to visualize specific neuronal lineages in the adult (Figure 1K: Awasaki et al., 2014; Lacin and Truman, 2016). This technique employs a cascade of recombinases and allows us to visualize the entire progeny of a NB when used with NB-specific GAL4 lines (Table 1). Simply, it maintains (i.e. immortalizes) transient reporter expression in the NB and forces the progeny of the NB to express the reporter in an irreversible and lineage-specific manner. This technique can unambiguously identify hemilineages in which the sibling hemilineage was eliminated by apoptosis (e.g. 2A, 10B, and 17A) without any additional markers since they are the only surviving lineages (Figure 2; Truman et al., 2010). For the lineages in which both hemilineages survive, we used molecular markers, the location of cell bodies, and axonal projections to distinguish between sibling hemilineages (Lacin et al., 2014; Harris et al., 2015; Shepherd et al., 2016).

Table 1

Line	Neuroblast	Lineages
16A05AD;28H10DBD	NB1-2	1A-1B
70D06AD;28H10DBD	NB2-1	2A
10C12	NB2-3	15B
65G02	NB2-4	18B
19B03	NB2-5, NB2-4	17A, 18B
21E09AD;16H11DBD	NB3-2	7B
ems-GAL4	NB2-2, NB3-3, NB3-5	10B, 8A-8B, 9A-9B
59E09	NB3-5	9A-9B
VT48571	NB4-1	14A
81C12A;42F01DBD	NB4-2	13A-13B
16H11AD-19B03DBD	NB4-3	21A
VT205490	NB4-4	24A
45D04	NB5-2	6A-6B
54B10	NB5-3	5B
19B03AD;45D04DBD	NB5-4, NB5-7	20-22A
81F01 or 70D06AD;42F01DBD	NB6-1	12A-12B
R76D11	NB6-2	19A-19B
R51B04	NB7-1	3A-3B
35B12AD;28H10DBD	NB7-2	11A-11B
19B03AD;18F07DBD	NB7-4	23B
70D06	MNB	0B

1. For detailed information about these lines, see Lacin and Truman, 2016.

Utilizing the tools mentioned above we systematically visualized each hemilineage in the VNC and then mapped the fast-acting neurotransmitter used by the neurons of each hemilineage (Figure 2). We found that all neurons within a hemilineage use the same neurotransmitter (see below). Of the 34 major hemilineages per thoracic ganglia, eight hemilineages are Glutamatergic, 12 hemilineages are GABAergic, and 14 hemilineages are cholinergic. For easier access for the readers, the data for each hemilineage will be presented below in groups based on the neurotransmitter type although the neurotransmitter identity of each hemilineage was identified independently in an unbiased fashion.

Glutamatergic lineages

Glutamate can serve as an inhibitory or excitatory neurotransmitter in the fly CNS depending on the neuronal type (Daniels et al., 2004; Liu and Wilson, 2013). To identify neurons and neuronal lineages that use glutamate, we used genetic reporters- VGlut-GAL4, VGlut^DBD, VGlut-LexA (Diao et al., 2015) and VGlut^AD (this study), and a vGLUT specific antibody (Daniels et al., 2004). Figure 3 shows an example of how we used multiple approaches to identify the neurotransmitter identity of neurons in the 14A and 21A hemilineages. Msh marks lineages 1A, 9A, 14A and 21A in both the larval and adult nerve cords (Lacin and Truman, 2016; this study). We used msh^AD in combination with VGlut^DBD to see if any of the Msh⁺ lineages were glutamatergic as GAL4 mediated expression will only occur in cells that co-express both transgenes. This split-GAL4 combination identified the 14A (67 ± 2 neurons; n = 5 hemisegments) and 21A (63 ± 1 neurons; n = 3 hemisegments) hemilineages as likely glutamatergic (Figure 3A,B). The VGlut-LexA line similarly marked the 14A and 21A neurons, which were revealed by Msh antibody staining (Figure 3C and not shown). We confirmed these findings with an antibody staining against VGlut and showed that all 14A and 21A neurons do indeed express VGlut protein (Figure 3D; Figure 4—figure supplement 1D,G). Interestingly, a msh^AD-ChAT ^DBD combination also marked 14A neurons (Figure 1H), suggesting 14A neurons might also be cholinergic. To resolve such discrepancies, we stained the nerve cords against ChAT and GABA and failed to detect any cholinergic or GABAergic neurons among the 14A or 21A neuronal populations (Figure 3E,F; Figure 4—figure supplement 1G). Thus, our results clearly show that all neurons within 14A and 21A hemilineages acquire a glutamatergic fate. The interesting mismatch between ChAT reporters and the immunocytochemical staining will be addressed below.

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Motor neurons in Drosophila use glutamate as the choice of neurotransmitter for muscle excitation (Johansen et al., 1989). 15B and 24A hemilineages are made of entirely motor neurons, which innervate the leg musculature (Truman et al., 2004; Baek and Mann, 2009; Truman et al., 2010). As expected, antibodies detected vGLUT protein in the motor neurons of lineage 15B and 24A, which were visualized via NB intersected reporter immortalization (Figure 4A,B; Table 1), but neither GABA nor ChAT protein was detected in these lineages (Figure 1—figure supplement 1E,H). Transcription factor expression profile for adult 24B neurons was studied in detail, and combinatorial expression of six transcription factors was shown to be responsible for the identity of the 24B motor neurons (Enriquez et al., 2015). 15B motor neurons express Lim3, Nkx6, and Islet (Isl) during larval development (Lacin et al., 2014). Based on antibody staining and genetic reporters (lim3^DBD and nkx6^DBD), we found that most of the mature 15B motor neurons in the adult co-express Lim3, Isl and Nkx6 (Figure 1F; Figure 4—figure supplement 1E). Interestingly, the level of Nkx6 expression was not uniform across 15B neurons: some neurons expressed it at high levels while some expressed it at low level (Figure 4—figure supplement 1E). The amount of Nkx6 might be regulating differentiation of 15B neurons.

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In addition to lineages 14A, 15B, 21A and 24A, we identified four more glutamatergic hemilineages: 2A, 8A, 9B, and 16B (Figure 4G). NB2-2 generates only 2A neurons since their 2B sibs die soon after birth (Truman et al., 2010). We used NB2-2 intersected reporter immortalization (Table 1) or R50G08-GAL4 expression to mark 2A neurons, which reside medially and in a close proximity to 14A neurons. Labeling with both antibodies and genetic tools showed that all 2A neurons (34 ± 3, n = 3 hemisegments) express vGLUT, but neither ChAT nor GABA (Figure 4C; Figure 4—figure supplement 1A).

Neurons of the 8A cluster are located laterally and innervate the leg neuropil (Harris et al., 2015). We visualized the 8A neurons by ey^DBD-tub^AD or OK107-GAL4, a reporter line inserted into ey gene (Adachi et al., 2003). We observed 55 ± 2 (n = 5 hemisegments) 8A neurons per cluster and all appeared to express VGlut (Figure 4D; Figure 4—figure supplement 1B). Moreover, the intersection of ey^AD-VGlut^DBD drivers also marked the 8A neurons further confirming their glutamatergic identity (Figure 1G). In agreement with these results, we did not detect any ChAT or GABA expression in neurons of the 8A hemilineage (Figure 4—figure supplement 1B).

It was previously thought that only the ‘A’ sibs in lineage nine survived and that this 9A cluster included a large group of ipsilateral cells and a much smaller group of cells that projected to the contralateral side of the segment (Truman et al., 2010). We found, though, that the contralateral cells are actually surviving neurons of the 9B hemilineage based on wild type lineage clones and MARCM clones of Notch signaling mutants (Figure 4—figure supplement 1C; not shown). 9B hemilineage includes 18 ± 3 neurons (n = 4 hemisegments) that express Lim3 and Acj6 in the adult VNC (Figure 4E and not shown). Both VGlut antibody and VGlut-LexA mark 9B neurons (Figure 4—figure supplement 1C).

Neurons of the16B hemilineage express Hb9 and Lim3 in the adult VNC and are located laterally to the other cluster of Hb9⁺ Lim3⁺ neurons that constitute the 10B cluster (Figure 4—figure supplement 1F; Shepherd et al., 2016). Both VGlut antibody and VGlut-LexA driver mark the 16B neurons (44 ± 4, n = 3 hemisegments), and ChAT and GABA antibodies fail to mark them (Figure 4F; Figure 4—figure supplement 1A).

In conclusion, we identified eight hemilineages that use glutamate as their neurotransmitter (Figure 4G). Two clusters are composed of leg motor neurons, and the other six are composed of interneurons. Five of these interneuronal hemilineages (8A, 9B, 14A, 16B, and 21A) innervate the leg neuropil while the 2A neurons innervate flight-related neuropils.

GABAergic lineages

GABA is the major fast inhibitory neurotransmitter in the Drosophila CNS (Wilson and Laurent, 2005). To identify GABAergic neurons, we used genetic reporters (gad1^AD, gad1-LexA (Diao et al., 2015); gad1^DBD(this study))and GABA specific antibodies. In total, we identified 12 GABAergic lineages: 0A, 1B, 3B, 5B, 6A, 6B, 9A, 11B, 12B, 13A, 13B, and 19A (Figure 5K).

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The local interneurons produced by the median NB (MNB) are reported to be GABAergic throughout insects (Goodman and Spitzer, 1979; Goodman et al., 1980; O'Dell and Watkins, 1988; Thompson and Siegler, 1991; Witten and Truman, 1991; Thompson and Siegler, 1993; Bossing and Technau, 1994; Witten and Truman, 1998; Schmid et al., 1999; Shepherd et al., 2016). The 0A neurons, which derive from the MNB, in Drosophila were cleanly labeled by NB intersected reporter immortalization and Engrailed/Invected expression (Table 1; Figure 5—figure supplement 1A). We detected 50 ± 3 (n = 4) 0A neurons in the T1 and T2 hemisegments and 30 neurons (n = 1) in the T3 hemisegment. These neurons were immunopositive for GABA and immunonegative for ChAT (Figure 5A; Figure 5—figure supplement 1A). Consequently, as in other insects, postembryonic progeny of the MNB in Drosophila are also GABAergic.

The 1B neurons are generated in the anterior border of the T2, T3, and A1 segments (Truman et al., 2004), but their cell bodies are pulled anteriorly during metamorphosis and become situated on the posterior border of the next anterior segment in the adult (Harris et al., 2015). 1B neurons are local interneurons that innervate the leg neuropil. We immortalized the reporter expression in the progeny of NB1-2 and visualized the 1A and 1B neurons (Table 1). The 1B hemilineage can easily be distinguished from the 1A hemilineage in the adult due to their segmental separation. The 1B cluster contained 56 ± 2 neurons (n = 5 hemisegments), and 37 ± 2 (n = 5) of them express Nmr1 (Figure 5—figure supplement 1B). We found that GABA, but never ChAT, immunostaining marks 1B neurons (Figure 5B; Figure 5—figure supplement 1B). Thus, our results show that the 1B neurons are GABAergic.

We visualized the entire progeny of NB7-1, which are the 3A and 3B neurons, via NB intersected reporter immortalization (Table 1). The 3A neurons express Nkx6 during both larval and adult stages (Lacin et al., 2014). Many of the 3B neurons express Dbx during larval development, however after pupa formation their Dbx expression disappears (Lacin et al., 2014). Within the NB7-1 lineage we assumed that Nkx6 expressing cells are the 3A neurons and that Nkx6 negative cells are the 3B neurons. We found that the Nkx6-negative 3B neurons express GABA, while the Nkx6-positive 3A neurons express ChAT (Figure 5—figure supplement 1C; Figure 6B). We detected 41 ± 3 (n = 3) 3B neurons per T1 cluster. We expect a similar number of 3B neurons for T2 segments, but did not obtain quantifiable clones. T3 segments did not appear to have any 3B neurons as previously suggested (Harris et al., 2015). To confirm that the 3B neurons are GABAergic, we stained larval nerve cord expressing gad1-LexA reporter with the Dbx antibody. As expected, we found that gad1-LexA reporter marks immature Dbx⁺ 3B neurons during larval stages (Figure 5C). Additionally, we used a GAL4 driver, SS20872, which marks a subset of 3B neurons and showed that gad1-LexA labels these neurons (Figure 5—figure supplement 1C). In summary, our results indicate that 3B neurons are GABAergic.

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Neurons of the 5B cluster are located in the ventral side of the nerve cord and have complex projections (Harris et al., 2015). We visualized 5B adult neurons via NB intersected reporter immortalization (Table 1). The 5B cluster is composed of 33 ± 3 neurons (n = 6) in adult flies. The GABA antibody stains all of these neurons, while the ChAT antibody fails to do so (Figure 5D; Figure 5—figure supplement 1D). Thus, 5B neurons are GABAergic.

We immortalized R45D04-GAL4 reporter expression in the NB5-2 progeny to visualize the 6A and 6B clusters. Since 6A and 6B neurons intermingle, we failed to separate them. Nevertheless, we found that the entire postembryonic progeny of NB5-2, 6A and 6B neurons, are GABAergic (Figure 5E). We further confirmed these by findings 6A neurons with 96A08-GAL4 and 6B neurons with Vestigial (Vg) or Engrailed (En) expression and showed that both 6A and 6B neurons are GABAergic (Figure 5—figure supplement 1E and not shown). Based on one quantifiable sample obtained from NB intersected reporter immortalization, we detected 51 6A neurons (En negative) and 42 6B neurons (En positive) in a T1 hemisegment.

The 9A neurons are the major neuronal cluster that arises from NB3-5 and innervate the leg neuropil (Harris et al., 2015; Lacin and Truman, 2016). We identified 9A neurons based on Msh expression. We also used msh^AD-tub^DBD reporter to visualize these neurons. We counted 73 ± 3 (n = 4) 9A neurons per cluster and all appeared to be immunopositive for GABA but not ChAT (Figure 5F; Figure 5—figure supplement 1F). Moreover, we failed to detect any 9A neurons with split GAL4 combination of msh^AD-ChAT^DBD or msh^AD-VGlut^DBD indicating the absence of cholinergic or glutamatergic neurons in 9A hemilineage (data not shown). Thus, the 9A neurons use GABA as their neurotransmitter.

Neurons of the 11B cluster are located on the dorsal surface of the nerve cord and marked by Eve expression (Figure 5G). In addition to Eve, we used reporter immortalization in NB7-2 progeny to identify 11B neurons (Table 1). We detected 68 ± 4 (n = 3) 11B neurons in T1 hemisegments. In T2 and T3, the number of 11B neurons was dramatically reduced to 7 and 2 neurons, respectively (n = 2 for each). Both GABA immunostaining and gad1-lexA reporter mark 11B neurons while ChAT-LexA fails to mark any 11B neurons. (Figure 5G; Figure 5—figure supplement 1G). Thus, 11B neurons are GABAergic.

The 12B neurons innervate the leg neuropil, and they are located medially relative to their sibling 12A lineage (Harris et al., 2015). We visualized 12A and 12B adult neurons via reporter immortalization in the progeny of NB6-1 (Table 1) and identified 12B neurons based on their lateral location. We also used the expression of Nkx6 and Nmr1, which are expressed in 12B neurons in a partially overlapping manner (Figure 5—figure supplement 1H). We detected 67 ± 7 (n = 4) 12B neurons per cluster and found that 12B neurons were marked with GABA antibody but none showed ChAT expression (Figure 5H; Figure 5—figure supplement 1H). Thus, our results indicate that the 12B neurons are GABAergic.

The 13A and 13B clusters arise from NB4-2 and innervate the leg neuropil ipsilaterally and contralaterally, respectively (Harris et al., 2015; Lacin and Truman, 2016). We visualized these sibling lineages via NB intersected reporter immortalization (Table 1). The 13A neurons are located laterally compared to the medially located 13B neurons (Figure 5I). The 13A cluster is composed of 67 ± 6 neurons (n = 3) and around half of them express Dbx; the 13B cluster is composed of 47 ± 1 neurons (n = 4) and most express Dichaete (Figure 5—figure supplement 1I). We found that both the 13A and 13B clusters were marked with GABA, but not ChAT (Figure 5I; Figure 5—figure supplement 1I). Of note, we found that 13B neurons exhibit higher GABA levels than 13A neurons across several animals (n = 4) (Figure 5I; Figure 5—figure supplement 1I). In conclusion, both the 13A and 13B hemilineages are GABAergic.

Neurons of the 19A cluster are located dorso-laterally in the nerve cord and innervate the leg neuropil (Harris et al., 2015). We visualized them via reporter immortalization in the NB6-2 progeny (Table 1) and identified them based on their more lateral location compared to 19B neurons. We detected 62 ± 4 (n = 4) 19A neurons per cluster and most express Dbx (Figure 5—figure supplement 1J). Based on immunostainings, we found that 19A neurons were marked by GABA, and we did not detect any 19A neuron expressing ChAT, indicating that the 19A neurons are GABAergic (Figure 5J; Figure 5—figure supplement 1J).

In conclusion, we identified 12 GABAergic hemilineages. Half of them (1B, 9A, 12A, 13A, 13B, and 19A) are composed of interneurons innervating the leg neuropil. The other half either exhibit complex projection patterns across the VNC or innervate the flight related neuropils. Like glutamatergic lineages, GABAergic lineages appeared to be composed of purely GABAergic neurons. In some lineages (e.g. 9A, 12B, 13A, 13B, and 19A), we detected occasional GABA negative cells. We think this is mainly due to the inability of GABA antibody to work robustly.

Co-expression of neurotransmitter

Our observation that ChAT reporters mark noncholinergic neurons (e.g. 14A neurons, Figure 1B) made us investigate in more detail if these and other neurons use more than one fast acting neurotransmitter. First, we used the split GAL4 combination of gad1^AD and VGlut^DBD to test if any neuron co-expresses GABA and glutamate. We did not find any neuron in either the VNC or brain that reproducibly expressed UAS-linked reporter gene with this combination, indicating the absence of such neurons (Figure 7A,B). Moreover, antibody staining revealed that gad1 and VGlut reporters marked only GABA⁺ or VGlut⁺ neurons, respectively (Figure 7—figure supplement 1A,B). Thus, our findings indicate that no neurons use both GABA and glutamate neurotransmitters.

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Next, we tested if ChAT is expressed by GABAergic and glutamatergic neurons, which were visualized by gad1 and VGlut reporters, respectively. We failed to and detect any significant ChAT staining in these neurons. Since ChAT antibody does not produce robust signals reproducibly, we utilized genetic tools reporting ChAT expression (Diao et al., 2015) to test if cholinergic neurons use other major neurotransmitters. To our surprise, we found that ChAT-GAL4 marked many neurons in both the VNC and brain that were also labeled by GABA or VGLUT antibodies (Figure 7—figure supplement 1C). We focused on the GABAergic set of ChAT-GAL4 marked neurons by visualizing them via gad1^AD-ChAT^DBD (Figure 7B). We found that most of these neurons are members of the 5B and 11B hemilineages of the VNC and other lineages in the brain (Figure 7C–E). Antibody staining showed that GABA marks these neurons strongly while the ChAT antibody did not label any of them. To see if these neurons express ChAT transcript, we performed a modified version of RNA in situ hybridization, which employs a chain reaction and can reveal transcripts with low copy numbers (Choi et al., 2018).We found that these ChAT-immunonegative neurons indeed express ChAT transcript, but at very low levels compared to neurons in clusters that showed ChAT immunostaining (Figure F). Most of the detected transcripts were in the nucleus, typically appearing as two transcription dots per nucleus. We interpret these as being transcription spots formed by the nascent transcripts associated with the two ChAT genes per nucleus. We rarely detected small amounts of ChAT signal outside the nucleus (not shown). We expect that the transcripts are likely actively degraded and do not result in transcript accumulation in the cytoplasm or in ChAT protein. We also observed low levels of ChAT transcripts in glutamatergic neurons of the clusters of 14A interneurons and 15B motor neurons (Figure G, H and not shown). These neurons also showed up using the ChAT genetic reporters, but failed to show ChAT immunostaining. In conclusion, our results indicate that the ChAT gene is transcribed in glutamatergic and GABAergic cells of certain lineages and suggests that these transcripts do not result in functional enzyme.

The Drosophila VNC is a great model system to investigate how neurons acquire different fates, form functional circuits, and direct specific vital behaviors. To study the functional assembly of this complex tissue, we have taken a lineage-based approach to divide the VNC into developmentally and functionally related units, which are the hemilineages.

The adult VNC is made up primarily from 34 hemilineages - clusters of lineally related, segmentally repeated, postembryonic-born neurons. Previous work systematically characterized the development and neuronal morphologies of these hemilineages, and showed that neurons within a hemilineage adopt similar fates, evident from their immature axonal projection and transcription factor expression (Truman et al., 2004; Truman et al., 2010; Lacin et al., 2014; Harris et al., 2015; Shepherd et al., 2016). Here, we systematically mapped the neurotransmitter choice of most VNC neurons by studying all of the postembryonic hemilineages. Surprisingly, we found that neurotransmitter code in the VNC is simple in that all neurons within a hemilineage use the same neurotransmitter, thus transmitter identity is determined at the stem cell level. These results further support our earlier findings that hemilineages represent functional units, both in terms of anatomy and now neurotransmitter chemistry.

Our statement that all neurons within a hemilineage use the same neurotransmitter excludes the neurons that are born during early embryonic neurogenesis. As mentioned earlier, neurons born during this time are highly diverse and might use a different neurotransmitter than the rest of the neurons in the hemilineage. For example, both NB4-2 and NB5-2 generate glutamatergic motor neurons from their early embryonic divisions (Landgraf et al., 1997; Schmid et al., 1999; Lacin and Truman, 2016), but their postembryonic progenies (both A and B hemilineages) are purely GABAergic. Similarly, glutamatergic U/CQ motor neurons, which are born in early embryonic stages share the same hemilineage with the cholinergic 3A neurons (Landgraf et al., 1997; Schmid et al., 1999; Lacin et al., 2009). Thus, neuronal fates within a hemilineage can be dramatically different when embryonic and postembryonic neurons are compared. We believe the reason why postembryonic hemilineages in the VNC are homogenous in terms of neuronal fate is due to the expansion of particular, later-born neuronal classes as neuronal lineages became larger during evolution of more derived insects to accommodate more complex behaviors such as flight. Since all insect species have similar sets of NBs, new behaviors (e.g. flight) appear to have evolved via changes in the number of neurons generated by each NB, but not changes in the number of NBs (Truman and Bate, 1988). Moreover, the Notch mediated asymmetric division enabled the insect to have two distinct clonal populations of neurons (hemilineages) from a single NB. Interestingly, only 34 of 50 potential hemilineages are used in the adult fly VNC while 16 of them are eliminated by apoptosis. Thus, flies have the potential to acquire novel behaviors by simply resurrecting hemilineages that are fated to die.

At least within the thorax, the hemilineages express the same transmitter regardless of their segment of residence. This conservation was expected for the hemilineages that contribute to the leg neuropils, since neuron numbers and the projections of these cells appear similar across the different thoracic segments. On the other hand, the hemilineages innervating the dorsal, flight-related neuropils have segment specific organization and show dramatically different axonal projections depending on their segmental location. For example, 7B neurons in each segment have unique projection and appear to execute distinct behaviors (Harris et al., 2015). Despite these differences, 7B neurons use acetylcholine in every segment. These results show that the neurotransmitter fate is tightly linked to the lineage origin and that the segmental diversification of the 7B neurons with the evolution of the derived flight system of flies may have had to occur with this transmitter constraint.

We expect that most hemilineages in the fly brain are also homogenous in terms of neurotransmitter expression as we observed large neuronal clusters expressing the same neurotransmitter. However, some complex brain hemilineages that have diverse neuronal populations might have different neurotransmitter expression as it was shown for the lAL lineage (Lai et al., 2008; Lin et al., 2010).

Here, we also extended our earlier transcription factor expression studies in immature neurons of larval stages into the mature neurons of the adult. We found that the expression of many transcription factors is maintained into adult stages and can be used to mark specific hemilineages in the adult. However, some transcription factors are expressed transiently during development. For example, Dbx marks many immature 3B neurons in the larva (Lacin et al., 2014), but its expression disappears in these neurons after pupa formation (unpublished data). From the expression analysis of the limited number of transcription factors studied here, we did not find any factor that specifically marked all neurons of a specific neurotransmitter type. However, we identified a few transcription factors, whose expression tightly correlated with the neurotransmitter fate. For example, Dbx expression is restricted to GABAergic neurons, even though Dbx does not appear to promote the GABA fate by itself as GABAergic fate is unaltered in response to Dbx loss or misexpression (unpublished data). Similarly, we found that Unc-4 expression is restricted to cholinergic lineages among the postembryonic lineages in the VNC; however, in the brain Unc-4 is expressed in glutamatergic lineages in addition to cholinergic lineages, suggesting that different parts of the CNS might use the same transcription factor for different fates (unpublished data) via utilizing different cofactors. Supporting this, we found that none of the GABAergic lineages in the VNC are marked with Lim3, which was shown to be expressed in most GABAergic neurons of the fly optic lobe and required for their GABAergic identity (Konstantinides et al., 2018). The reverse scenario where neurons acquire the same neurotransmitter identity via different transcriptional regulatory networks is also commonly observed. For example in the C. elegans nervous system, distinct combinations of 13 transcription factors are responsible for VGlut expression in 25 different glutamatergic neuron classes (Serrano-Saiz et al., 2013). Thus, transcription factors act together combinatorially rather than individually to specify neurotransmitter fate.

Our study did not find any correlation between the Notch state of the neurons and neurotransmitter identity, an observation made in the optic lobe (Konstantinides et al., 2018). We found that any neurotransmitter type can be observed in both ‘A’ and ‘B’ hemilineages. We also noted that NB4-2 (progenitor of 13A/B) and NB5-2 (progenitor of 6A/B) are the only two NBs in which both the ‘A’ and ‘B’ hemilineages use the same neurotransmitter, GABA.

Interestingly, the neurotransmitter pattern of Drosophila hemilineages appears to be conserved in other insect species. For example, based on location and morphology, we deduced that sibling ‘K_l’ and ‘K_m’ GABAergic clusters of the moth, Manduca Sexta, are homologous to the 13A and 13B hemilineages, respectively, and the ‘M’ cluster is likely homologous to the 6A, 6B, and 5B neurons, which form a large cluster GABAergic in the posterior thoracic ganglia of the fly (Witten and Truman, 1991). Similar GABAergic clusters were also observed in the nerve cords of grasshopper (O'Dell and Watkins, 1988) and silverfish (unpublished data, HL and JWT). Interestingly, like the Drosophila VNC, the grasshopper nerve cord contains two clusters of En⁺GABA⁺ neurons, named ‘A’ and ‘B’ groups, which are likely homologous to 0A and 6B neurons, respectively (Siegler et al., 2001).

Unexpectedly, we detected ChAT transcripts in many GABAergic and glutamatergic neurons, most of which are members of lineages 5B and 11B (GABAergic) and 14A and 15B (glutamatergic). The low levels of ChAT transcripts and the lack of ChAT immunostainings in these cells suggested that ChAT transcripts are actively degraded and not translated. It is possible that these neurons produce acetylcholine but only in certain conditions for example during development or under stress. Indeed, neurotransmitter switching has been observed in many neurons of vertebrates, however, most of these switches involve aminergic neurotransmitters (Spitzer, 2015).

Another possibility for the presence of ChAT transcripts in noncholinergic neurons is that it is a remnant of a neurotransmitter switch that might have happened during evolution. 15B neurons are a good candidate for such a possibility. 15B motor neurons are glutamatergic like all other fly motor neurons, while all vertebrate and some invertebrate (e.g, C. elegans and Aplysia) motor neurons are cholinergic (reviewed in Fieber, 2017), raising the possibility that motor neurons of the common ancestor used acetylcholine. Thus, the ChAT expression in 15B motor neurons might be a vestige from the cholinergic ancestry of motor neurons.

All data generated or analysed during this study are included in the manuscript and supporting files.

1. 1. Adachi Y
   2. Hauck B
   3. Clements J
   4. Kawauchi H
   5. Kurusu M
   6. Totani Y
   7. Kang YY
   8. Eggert T
   9. Walldorf U
   10. Furukubo-Tokunaga K
   11. Callaerts P

   (2003) Conserved cis-regulatory modules mediate complex neural expression patterns of the eyeless gene in the Drosophila brain

   Mechanisms of Development 120:1113–1126.

   https://doi.org/10.1016/j.mod.2003.08.007

   • Google Scholar
2. 1. Almeida MS
   2. Bray SJ

   (2005) Regulation of post-embryonic neuroblasts by Drosophila grainyhead

   Mechanisms of Development 122:1282–1293.

   https://doi.org/10.1016/j.mod.2005.08.004

   • PubMed
   • Google Scholar
3. 1. Arber S

   (2012) Motor circuits in action: specification, connectivity, and function

   Neuron 74:975–989.

   https://doi.org/10.1016/j.neuron.2012.05.011

   • Google Scholar
4. 1. Awasaki T
   2. Kao CF
   3. Lee YJ
   4. Yang CP
   5. Huang Y
   6. Pfeiffer BD
   7. Luan H
   8. Jing X
   9. Huang YF
   10. He Y
   11. Schroeder MD
   12. Kuzin A
   13. Brody T
   14. Zugates CT
   15. Odenwald WF
   16. Lee T

   (2014) Making Drosophila lineage-restricted drivers via patterned recombination in neuroblasts

   Nature Neuroscience 17:631–637.

   https://doi.org/10.1038/nn.3654

   • PubMed
   • Google Scholar
5. 1. Baek M
   2. Mann RS

   (2009) Lineage and birth date specify motor neuron targeting and dendritic architecture in adult Drosophila

   Journal of Neuroscience 29:6904–6916.

   https://doi.org/10.1523/JNEUROSCI.1585-09.2009

   • PubMed
   • Google Scholar
6. 1. Birkholz O
   2. Rickert C
   3. Nowak J
   4. Coban IC
   5. Technau GM

   (2015) Bridging the gap between postembryonic cell lineages and identified embryonic neuroblasts in the ventral nerve cord of Drosophila melanogaster

   Biology Open 4:420–434.

   https://doi.org/10.1242/bio.201411072

   • PubMed
   • Google Scholar
7. 1. Bossing T
   2. Udolph G
   3. Doe CQ
   4. Technau GM

   (1996) The embryonic central nervous system lineages of Drosophila melanogaster. I. neuroblast lineages derived from the ventral half of the neuroectoderm

   Developmental Biology 179:41–64.

   https://doi.org/10.1006/dbio.1996.0240

   • PubMed
   • Google Scholar
8. 1. Bossing T
   2. Technau GM

   (1994)

   The fate of the CNS midline progenitors in Drosophila as revealed by a new method for single cell labelling

   Development 120:1895–1906.

   • PubMed
   • Google Scholar
9. 1. Briscoe J
   2. Pierani A
   3. Jessell TM
   4. Ericson J

   (2000) A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube

   Cell 101:435–445.

   https://doi.org/10.1016/S0092-8674(00)80853-3

   • PubMed
   • Google Scholar
10. 1. Chen HM
    2. Huang Y
    3. Pfeiffer BD
    4. Yao X
    5. Lee T

    (2015) An enhanced gene targeting toolkit for Drosophila: Golic+

    Genetics 199:683–694.

    https://doi.org/10.1534/genetics.114.173716

    • PubMed
    • Google Scholar
11. 1. Choi HMT
    2. Schwarzkopf M
    3. Fornace ME
    4. Acharya A
    5. Artavanis G
    6. Stegmaier J
    7. Cunha A
    8. Pierce NA

    (2018) Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust

    Development 145:dev165753.

    https://doi.org/10.1242/dev.165753

    • PubMed
    • Google Scholar
12. 1. Croset V
    2. Treiber CD
    3. Waddell S

    (2018) Cellular diversity in the Drosophila midbrain revealed by single-cell transcriptomics

    eLife 7:e34550.

    https://doi.org/10.7554/eLife.34550

    • PubMed
    • Google Scholar
13. 1. Daniels RW
    2. Collins CA
    3. Gelfand MV
    4. Dant J
    5. Brooks ES
    6. Krantz DE
    7. DiAntonio A

    (2004) Increased expression of the Drosophila vesicular glutamate transporter leads to excess glutamate release and a compensatory decrease in quantal content

    Journal of Neuroscience 24:10466–10474.

    https://doi.org/10.1523/JNEUROSCI.3001-04.2004

    • PubMed
    • Google Scholar
14. 1. Diao F
    2. Ironfield H
    3. Luan H
    4. Diao F
    5. Shropshire WC
    6. Ewer J
    7. Marr E
    8. Potter CJ
    9. Landgraf M
    10. White BH

    (2015) Plug-and-play genetic access to Drosophila cell types using exchangeable exon cassettes

    Cell Reports 10:1410–1421.

    https://doi.org/10.1016/j.celrep.2015.01.059

    • PubMed
    • Google Scholar
15. 1. Enriquez J
    2. Venkatasubramanian L
    3. Baek M
    4. Peterson M
    5. Aghayeva U
    6. Mann RS

    (2015) Specification of individual adult motor neuron morphologies by combinatorial transcription factor codes

    Neuron 86:955–970.

    https://doi.org/10.1016/j.neuron.2015.04.011

    • PubMed
    • Google Scholar
16. Book

    1. Fieber LA

    (2017) Neurotransmitters and Neuropeptides of Invertebrates

    The Oxford Handbook of Invertebrate Neurobiology.

    https://doi.org/10.1093/oxfordhb/9780190456757.013.10

    • Google Scholar
17. 1. Gendrel M
    2. Atlas EG
    3. Hobert O

    (2016) A cellular and regulatory map of the GABAergic nervous system of C. elegans

    eLife 5:e17686.

    https://doi.org/10.7554/eLife.17686

    • PubMed
    • Google Scholar
18. 1. Goodman CS
    2. Pearson KG
    3. Spitzer NC

    (1980) Electrical excitability: a spectrum of properties in the progeny of a single embryonic neuroblast

    PNAS 77:1676–1680.

    https://doi.org/10.1073/pnas.77.3.1676

    • PubMed
    • Google Scholar
19. 1. Goodman CS
    2. Spitzer NC

    (1979) Embryonic development of identified neurones: differentiation from neuroblast to neurone

    Nature 280:208–214.

    https://doi.org/10.1038/280208a0

    • Google Scholar
20. 1. Gowda SBM
    2. Paranjpe PD
    3. Reddy OV
    4. Thiagarajan D
    5. Palliyil S
    6. Reichert H
    7. VijayRaghavan K

    (2018) GABAergic inhibition of leg motoneurons is required for normal walking behavior in freely moving Drosophila

    PNAS 115:E2115–E2124.

    https://doi.org/10.1073/pnas.1713869115

    • PubMed
    • Google Scholar
21. Thesis

    1. Harris R

    (2013) Form and Function of the Secondary Hemilineages in the Adult Drosophila Thoracic Nervous System

    University of Washington.

    https://digital.lib.washington.edu/researchworks/handle/1773/22066

    • Google Scholar
22. 1. Harris RM
    2. Pfeiffer BD
    3. Rubin GM
    4. Truman JW

    (2015) Neuron hemilineages provide the functional ground plan for the Drosophila ventral nervous system

    eLife 4:e04493.

    https://doi.org/10.7554/eLife.04493

    • Google Scholar
23. 1. Hartenstein V
    2. Wodarz A

    (2013) Initial neurogenesis in Drosophila

    Wiley Interdisciplinary Reviews. Developmental Biology 2:701–721.

    https://doi.org/10.1002/wdev.111

    • PubMed
    • Google Scholar
24. 1. Isshiki T
    2. Pearson B
    3. Holbrook S
    4. Doe CQ

    (2001) Drosophila neuroblasts sequentially express transcription factors which specify the temporal identity of their neuronal progeny

    Cell 106:511–521.

    https://doi.org/10.1016/S0092-8674(01)00465-2

    • PubMed
    • Google Scholar
25. 1. Jenett A
    2. Rubin GM
    3. Ngo TT
    4. Shepherd D
    5. Murphy C
    6. Dionne H
    7. Pfeiffer BD
    8. Cavallaro A
    9. Hall D
    10. Jeter J
    11. Iyer N
    12. Fetter D
    13. Hausenfluck JH
    14. Peng H
    15. Trautman ET
    16. Svirskas RR
    17. Myers EW
    18. Iwinski ZR
    19. Aso Y
    20. DePasquale GM
    21. Enos A
    22. Hulamm P
    23. Lam SC
    24. Li HH
    25. Laverty TR
    26. Long F
    27. Qu L
    28. Murphy SD
    29. Rokicki K
    30. Safford T
    31. Shaw K
    32. Simpson JH
    33. Sowell A
    34. Tae S
    35. Yu Y
    36. Zugates CT

    (2012) A GAL4-driver line resource for Drosophila neurobiology

    Cell Reports 2:991–1001.

    https://doi.org/10.1016/j.celrep.2012.09.011

    • PubMed
    • Google Scholar
26. 1. Jessell TM

    (2000) Neuronal specification in the spinal cord: inductive signals and transcriptional codes

    Nature Reviews Genetics 1:20–29.

    https://doi.org/10.1038/35049541

    • PubMed
    • Google Scholar
27. 1. Johansen J
    2. Halpern ME
    3. Johansen KM
    4. Keshishian H

    (1989) Stereotypic morphology of glutamatergic synapses on identified muscle cells of Drosophila larvae

    The Journal of Neuroscience 9:710–725.

    https://doi.org/10.1523/JNEUROSCI.09-02-00710.1989

    • PubMed
    • Google Scholar
28. 1. Kambadur R
    2. Koizumi K
    3. Stivers C
    4. Nagle J
    5. Poole SJ
    6. Odenwald WF

    (1998) Regulation of POU genes by castor and hunchback establishes layered compartments in the Drosophila CNS

    Genes & Development 12:246–260.

    https://doi.org/10.1101/gad.12.2.246

    • PubMed
    • Google Scholar
29. 1. Konstantinides N
    2. Kapuralin K
    3. Fadil C
    4. Barboza L
    5. Satija R
    6. Desplan C

    (2018) Phenotypic convergence: distinct transcription factors regulate common terminal features

    Cell 174:622–635.

    https://doi.org/10.1016/j.cell.2018.05.021

    • PubMed
    • Google Scholar
30. 1. Kulkarni SJ
    2. Newby LM
    3. Jackson FR

    (1994) Drosophila GABAergic systems. II. mutational analysis of chromosomal segment 64ab, a region containing the glutamic acid decarboxylase gene

    MGG Molecular & General Genetics 243:555–564.

    https://doi.org/10.1007/BF00284204

    • PubMed
    • Google Scholar
31. 1. Kvon EZ
    2. Kazmar T
    3. Stampfel G
    4. Yáñez-Cuna JO
    5. Pagani M
    6. Schernhuber K
    7. Dickson BJ
    8. Stark A

    (2014) Genome-scale functional characterization of Drosophila developmental enhancers in vivo

    Nature 512:91–95.

    https://doi.org/10.1038/nature13395

    • Google Scholar
32. 1. Lacin H
    2. Zhu Y
    3. Wilson BA
    4. Skeath JB

    (2009) Dbx mediates neuronal specification and differentiation through cross-repressive, lineage-specific interactions with eve and hb9

    Development 136:3257–3266.

    https://doi.org/10.1242/dev.037242

    • PubMed
    • Google Scholar
33. 1. Lacin H
    2. Zhu Y
    3. Wilson BA
    4. Skeath JB

    (2014) Transcription factor expression uniquely identifies most postembryonic neuronal lineages in the Drosophila thoracic central nervous system

    Development 141:1011–1021.

    https://doi.org/10.1242/dev.102178

    • PubMed
    • Google Scholar
34. 1. Lacin H
    2. Truman JW

    (2016) Lineage mapping identifies molecular and architectural similarities between the larval and adult Drosophila central nervous system

    eLife 5:e13399.

    https://doi.org/10.7554/eLife.13399

    • PubMed
    • Google Scholar
35. 1. Lai SL
    2. Awasaki T
    3. Ito K
    4. Lee T

    (2008) Clonal analysis of Drosophila antennal lobe neurons: diverse neuronal architectures in the lateral neuroblast lineage

    Development 135:2883–2893.

    https://doi.org/10.1242/dev.024380

    • PubMed
    • Google Scholar
36. 1. Landgraf M
    2. Bossing T
    3. Technau GM
    4. Bate M

    (1997) The origin, location, and projections of the embryonic abdominal motorneurons of Drosophila

    The Journal of Neuroscience 17:9642–9655.

    https://doi.org/10.1523/JNEUROSCI.17-24-09642.1997

    • PubMed
    • Google Scholar
37. 1. Lee D
    2. O'Dowd DK

    (1999) Fast excitatory synaptic transmission mediated by nicotinic acetylcholine receptors in Drosophila neurons

    The Journal of Neuroscience 19:5311–5321.

    https://doi.org/10.1523/JNEUROSCI.19-13-05311.1999

    • PubMed
    • Google Scholar
38. 1. Lin S
    2. Lai SL
    3. Yu HH
    4. Chihara T
    5. Luo L
    6. Lee T

    (2010) Lineage-specific effects of notch/Numb signaling in post-embryonic development of the Drosophila brain

    Development 137:43–51.

    https://doi.org/10.1242/dev.041699

    • PubMed
    • Google Scholar
39. 1. Liu WW
    2. Wilson RI

    (2013) Glutamate is an inhibitory neurotransmitter in the Drosophila olfactory system

    PNAS 110:10294–10299.

    https://doi.org/10.1073/pnas.1220560110

    • PubMed
    • Google Scholar
40. 1. Long X
    2. Colonell J
    3. Wong AM
    4. Singer RH
    5. Lionnet T

    (2017) Quantitative mRNA imaging throughout the entire Drosophila brain

    Nature Methods 14:703–706.

    https://doi.org/10.1038/nmeth.4309

    • PubMed
    • Google Scholar
41. 1. Lu DC
    2. Niu T
    3. Alaynick WA

    (2015) Molecular and cellular development of spinal cord locomotor circuitry

    Frontiers in Molecular Neuroscience 8:25.

    https://doi.org/10.3389/fnmol.2015.00025

    • PubMed
    • Google Scholar
42. 1. Maurange C
    2. Cheng L
    3. Gould AP

    (2008) Temporal transcription factors and their targets schedule the end of neural proliferation in Drosophila

    Cell 133:891–902.

    https://doi.org/10.1016/j.cell.2008.03.034

    • PubMed
    • Google Scholar
43. 1. Nagarkar-Jaiswal S
    2. Lee P-T
    3. Campbell ME
    4. Chen K
    5. Anguiano-Zarate S
    6. Cantu Gutierrez M
    7. Busby T
    8. Lin W-W
    9. He Y
    10. Schulze KL
    11. Booth BW
    12. Evans-Holm M
    13. Venken KJT
    14. Levis RW
    15. Spradling AC
    16. Hoskins RA
    17. Bellen HJ

    (2015) A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

    eLife 4:e05338.

    https://doi.org/10.7554/eLife.05338

    • Google Scholar
44. 1. Nern A
    2. Pfeiffer BD
    3. Rubin GM

    (2015) Optimized tools for multicolor stochastic labeling reveal diverse stereotyped cell arrangements in the fly visual system

    PNAS 112:E2967–E2976.

    https://doi.org/10.1073/pnas.1506763112

    • PubMed
    • Google Scholar
45. 1. O'Dell DA
    2. Watkins BL

    (1988) The development of GABA-like immunoreactivity in the thoracic ganglia of the locust schistocerca gregaria

    Cell and Tissue Research 254:635–646.

    https://doi.org/10.1007/BF00226514

    • PubMed
    • Google Scholar
46. 1. Pereira L
    2. Kratsios P
    3. Serrano-Saiz E
    4. Sheftel H
    5. Mayo AE
    6. Hall DH
    7. White JG
    8. LeBoeuf B
    9. Garcia LR
    10. Alon U
    11. Hobert O

    (2015) A cellular and regulatory map of the cholinergic nervous system of C. elegans

    eLife 4:e12432.

    https://doi.org/10.7554/eLife.12432

    • PubMed
    • Google Scholar
47. 1. Pfeiffer BD
    2. Ngo TT
    3. Hibbard KL
    4. Murphy C
    5. Jenett A
    6. Truman JW
    7. Rubin GM

    (2010) Refinement of tools for targeted gene expression in Drosophila

    Genetics 186:735–755.

    https://doi.org/10.1534/genetics.110.119917

    • PubMed
    • Google Scholar
48. 1. Salvaterra PM
    2. Kitamoto T

    (2001) Drosophila cholinergic neurons and processes visualized with Gal4/UAS-GFP

    Gene Expression Patterns 1:73–82.

    https://doi.org/10.1016/S1567-133X(01)00011-4

    • PubMed
    • Google Scholar
49. 1. Schmid A
    2. Chiba A
    3. Doe CQ

    (1999)

    Clonal analysis of Drosophila embryonic neuroblasts: neural cell types, axon projections and muscle targets

    Development 126:4653–4689.

    • PubMed
    • Google Scholar
50. 1. Serrano-Saiz E
    2. Poole RJ
    3. Felton T
    4. Zhang F
    5. De La Cruz ED
    6. Hobert O

    (2013) Modular control of glutamatergic neuronal identity in C. elegans by distinct homeodomain proteins

    Cell 155:659–673.

    https://doi.org/10.1016/j.cell.2013.09.052

    • PubMed
    • Google Scholar
51. 1. Serrano-Saiz E
    2. Pereira L
    3. Gendrel M
    4. Aghayeva U
    5. Bhattacharya A
    6. Howell K
    7. Garcia LR
    8. Hobert O

    (2017) A neurotransmitter atlas of the Caenorhabditis elegans Male Nervous System Reveals Sexually Dimorphic Neurotransmitter Usage

    Genetics 206:1251–1269.

    https://doi.org/10.1534/genetics.117.202127

    • PubMed
    • Google Scholar
52. 1. Shepherd D
    2. Harris R
    3. Williams DW
    4. Truman JW

    (2016) Postembryonic lineages of the Drosophila ventral nervous system: neuroglian expression reveals the adult hemilineage associated fiber tracts in the adult thoracic neuromeres

    Journal of Comparative Neurology 524:2677–2695.

    https://doi.org/10.1002/cne.23988

    • PubMed
    • Google Scholar
53. 1. Siegler MV
    2. Pankhaniya RR
    3. Jia XX

    (2001) Pattern of expression of engrailed in relation to gamma-aminobutyric acid immunoreactivity in the central nervous system of the adult grasshopper

    The Journal of Comparative Neurology 440:85–96.

    https://doi.org/10.1002/cne.1371

    • PubMed
    • Google Scholar
54. 1. Skeath JB
    2. Doe CQ

    (1998)

    Sanpodo and notch act in opposition to numb to distinguish sibling neuron fates in the Drosophila CNS

    Development 125:1857–1865.

    • PubMed
    • Google Scholar
55. 1. Slemmon JR
    2. Campbell GA
    3. Selski DJ
    4. Bramson HN

    (1991) The amino terminus of the putative Drosophila choline acetyltransferase precursor is cleaved to yield the 67 kDa enzyme

    Molecular Brain Research 9:245–252.

    https://doi.org/10.1016/0169-328X(91)90008-L

    • PubMed
    • Google Scholar
56. 1. Spana EP
    2. Doe CQ

    (1996) Numb antagonizes notch signaling to specify sibling neuron cell fates

    Neuron 17:21–26.

    https://doi.org/10.1016/S0896-6273(00)80277-9

    • PubMed
    • Google Scholar
57. 1. Spitzer NC

    (2015) Neurotransmitter switching? no surprise

    Neuron 86:1131–1144.

    https://doi.org/10.1016/j.neuron.2015.05.028

    • PubMed
    • Google Scholar
58. 1. Takagawa K
    2. Salvaterra P

    (1996) Analysis of choline acetyltransferase protein in temperature sensitive mutant flies using newly generated monoclonal antibody

    Neuroscience Research 24:237–243.

    https://doi.org/10.1016/0168-0102(95)00999-X

    • PubMed
    • Google Scholar
59. 1. Thompson KJ
    2. Siegler MV

    (1991) Anatomy and physiology of spiking local and intersegmental interneurons in the median neuroblast lineage of the grasshopper

    The Journal of Comparative Neurology 305:659–675.

    https://doi.org/10.1002/cne.903050409

    • PubMed
    • Google Scholar
60. 1. Thompson KJ
    2. Siegler MV

    (1993) Development of segment specificity in identified lineages of the grasshopper CNS

    The Journal of Neuroscience 13:3309–3318.

    https://doi.org/10.1523/JNEUROSCI.13-08-03309.1993

    • PubMed
    • Google Scholar
61. 1. Thor S
    2. Thomas JB

    (1997) The Drosophila islet gene governs axon pathfinding and neurotransmitter identity

    Neuron 18:397–409.

    https://doi.org/10.1016/S0896-6273(00)81241-6

    • PubMed
    • Google Scholar
62. Preprint

    1. Tirian L
    2. Dickson B

    (2017) The VT GAL4, LexA and split-GAL4 driver line collections for targeted expression in the Drosophila nervous system

    bioRxiv.

    https://doi.org/10.1101/198648

    • Google Scholar
63. 1. Truman JW

    (1990) Metamorphosis of the central nervous system of Drosophila

    Journal of Neurobiology 21:1072–1084.

    https://doi.org/10.1002/neu.480210711

    • PubMed
    • Google Scholar
64. 1. Truman JW
    2. Schuppe H
    3. Shepherd D
    4. Williams DW

    (2004) Developmental architecture of adult-specific lineages in the ventral CNS of Drosophila

    Development 131:5167–5184.

    https://doi.org/10.1242/dev.01371

    • PubMed
    • Google Scholar
65. 1. Truman JW
    2. Moats W
    3. Altman J
    4. Marin EC
    5. Williams DW

    (2010) Role of Notch signaling in establishing the hemilineages of secondary neurons in Drosophila melanogaster

    Development 137:53–61.

    https://doi.org/10.1242/dev.041749

    • PubMed
    • Google Scholar
66. 1. Truman JW
    2. Bate M

    (1988) Spatial and temporal patterns of neurogenesis in the central nervous system of Drosophila melanogaster

    Developmental Biology 125:145–157.

    https://doi.org/10.1016/0012-1606(88)90067-X

    • PubMed
    • Google Scholar
67. 1. Tsuchida T
    2. Ensini M
    3. Morton SB
    4. Baldassare M
    5. Edlund T
    6. Jessell TM
    7. Pfaff SL

    (1994) Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes

    Cell 79:957–970.

    https://doi.org/10.1016/0092-8674(94)90027-2

    • PubMed
    • Google Scholar
68. 1. Venken KJ
    2. Schulze KL
    3. Haelterman NA
    4. Pan H
    5. He Y
    6. Evans-Holm M
    7. Carlson JW
    8. Levis RW
    9. Spradling AC
    10. Hoskins RA
    11. Bellen HJ

    (2011) MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes

    Nature Methods 8:737–743.

    https://doi.org/10.1038/nmeth.1662

    • PubMed
    • Google Scholar
69. 1. Wilson RI
    2. Laurent G

    (2005) Role of GABAergic inhibition in shaping odor-evoked spatiotemporal patterns in the Drosophila antennal lobe

    Journal of Neuroscience 25:9069–9079.

    https://doi.org/10.1523/JNEUROSCI.2070-05.2005

    • PubMed
    • Google Scholar
70. 1. Witten JL
    2. Truman JW

    (1991) The regulation of transmitter expression in Postembryonic lineages in the moth manduca sexta. II. role of cell lineage and birth order

    The Journal of Neuroscience 11:1990–1997.

    https://doi.org/10.1523/JNEUROSCI.11-07-01990.1991

    • PubMed
    • Google Scholar
71. 1. Witten JL
    2. Truman JW

    (1998) Distribution of GABA-like immunoreactive neurons in insects suggests lineage homology

    The Journal of Comparative Neurology 398:515–528.

    https://doi.org/10.1002/(SICI)1096-9861(19980907)398:4<515::AID-CNE4>3.0.CO;2-5

    • PubMed
    • Google Scholar
72. 1. Zhang Y
    2. Narayan S
    3. Geiman E
    4. Lanuza GM
    5. Velasquez T
    6. Shanks B
    7. Akay T
    8. Dyck J
    9. Pearson K
    10. Gosgnach S
    11. Fan CM
    12. Goulding M

    (2008) V3 spinal neurons establish a robust and balanced locomotor rhythm during walking

    Neuron 60:84–96.

    https://doi.org/10.1016/j.neuron.2008.09.027

    • PubMed
    • Google Scholar

Author details

1. Haluk Lacin

   1. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
   2. Department of Genetics, Washington University, Saint Louis, United States

   Contribution

   Conceptualization, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   For correspondence

   lacinhaluk@gmail.com

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2468-9618

2. Hui-Min Chen

   Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States

   Contribution

   Resources, Methodology

   Competing interests

   No competing interests declared

3. Xi Long

   Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States

   Contribution

   Resources, Methodology

   Competing interests

   No competing interests declared

4. Robert H Singer

   1. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
   2. Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, United States

   Contribution

   Resources, Methodology

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0002-6725-0093

5. Tzumin Lee

   Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States

   Contribution

   Resources, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

6. James W Truman

   1. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
   2. Friday Harbor Laboratories, University of Washington, Friday Harbor, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9209-5435

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