How does a complex nervous system arise during development? Millions to billions of neurons, each one essentially unique, precisely interconnect to create a functional central nervous system (CNS) that drives animal behavior (Herculano-Houzel, 2009). Work over several decades shows that developmentally established layers of spatial and temporal organization underlie the genesis of a complex CNS. For example, during spinal cord development in vertebrates, different types of progenitor cells arise across the dorso-ventral axis and generate distinct neuronal lineages in a precise spatial and temporal order. The pMN progenitors are located in a narrow layer in the ventral spinal cord and generate all motor neurons (Tsuchida et al., 1994; Pfaff et al., 1996; Briscoe et al., 2000). Similarly, twelve distinct pools of progenitors that arise in distinct dorso-ventral domains generate at least 22 distinct interneuronal lineages. Within each lineage, neurons appear to acquire similar identities: they express similar sets of transcription factors, use the same neurotransmitter, extend processes in a similar manner and participate in circuits executing a specific behavior (Jessell, 2000; Alaynick et al., 2011; Lu et al., 2015).

The adult Drosophila ventral nerve cord (VNC), like the vertebrate spinal cord, also manifests a lineage-based organization. The cellular complexity of the VNC arises from a set of segmentally repeated set of 30 paired and one unpaired neural stem cells (Neuroblasts [NBs]), which arise at stereotypic locations during early development (Doe, 1992). These individually identifiable NBs undergo two major phases of proliferation: the embryonic phase generates the functional neurons of the larval CNS, some of which are remodeled to function in the adult, and the post-embryonic phase generates most of the adult neurons (Truman and Bate, 1988; Truman, 1990; Prokop and Technau, 1991; Bossing et al., 1996; Schmid et al., 1999; Consoulas et al., 2002). The division mode within NB lineages adds another layer to the lineage-based organization of the VNC. Each NB generates a secondary precursor cell, which divides via Notch-mediated asymmetric cell division to generate two neurons with distinct identities (Spana and Doe, 1996; Skeath and Doe, 1998). After many rounds of such cell divisions, each NB ends up producing two distinct hemilineages of neurons, termed Notch-ON or the ‘A’ and Notch-OFF or the ‘B’ hemilineage. In this paper, we focus only on postembryonic hemilineages, which from this point on in the paper we refer to as hemilineages for simplicity. Within a hemilineage, neurons acquire similar fates based on transcription factor expression, neurotransmitter usage, and axonal projection (Truman et al., 2010; Lacin et al., 2014; Lacin et al., 2019). Moreover, neurons of each hemilineage appear dedicated for specific behaviors. For example, artificial neuronal activation of the glutamatergic hemilineage 2A neurons elicit specifically high frequency wing beating, while the same treatment of the cholinergic hemilineage 7B neurons leads to a specific take-off behavior (Harris et al., 2015). Thus, hemilineages represent the fundamental developmental and functional unit of the VNC.

We previously mapped the embryonic origin, axonal projection pattern, transcription factor expression, and neurotransmitter usage of essentially all hemilineages in the adult Drosophila VNC (Truman et al., 2004; Truman et al., 2010; Lacin et al., 2014; Harris et al., 2015; Lacin and Truman, 2016; Shepherd et al., 2016; Lacin et al., 2019; Shepherd et al., 2019). Here, we leverage this information to elucidate how a specific transcription factor, Unc-4, acts within individual hemilineages during adult nervous system development to regulate neuronal connectivity and function, and animal behavior. Unc-4, an evolutionarily conserved transcriptional repressor, is expressed post-mitotically in seven of the 14 cholinergic hemilineages in the VNC: three ‘A’ -Notch-ON- hemilineages (11A, 12A, and 17A) and four ‘B’ -Notch-OFF- hemilineages (7B, 18B, 19B, and 23B) (Miller et al., 1992; Rovescalli et al., 1996; Lacin et al., 2014; Lacin et al., 2019; Nittoli et al., 2019). For four of the Unc-4⁺ hemilineages (7B, 17A, 18B, and 23B), the neurons of the sibling hemilineage undergo cell death (Truman et al., 2010). For the remaining three (11A, 12A, and 19B), the neurons of the sibling hemilineage are GABAergic (Lacin et al., 2019). Unc-4 expression in these hemilineages is restricted to postmitotic neurons and it appears to mark uniformly all neurons within a hemilineage during development and adult life (Lacin et al., 2014; Lacin and Truman, 2016).

We generated a set of precise genetic tools that allowed us to uncover lineage-specific functions for Unc-4: in the 11A hemilineage, Unc-4 drives the cholinergic identity and suppresses the GABAergic fate; in the 7B hemilineage, Unc-4 promotes correct axonal projection patterns and the ability of flies to execute a stereotyped flight take-off behavior. We also find that Unc-4 is expressed in the precursors of chordotonal sensory neurons and required for the development of these sensory organs, with functional data indicating Unc-4 functions in this lineage to promote climbing, walking, and grooming activities.

In the absence of extant mutant alleles of unc-4 gene, we used CRISPR/Cas9 technology to engineer both null and conditional null alleles of unc-4 as well as GAL4 and split-GAL4 reporter lines of unc-4 in order to assess unc-4 function in the postembryonic CNS (Figure 1; see also Materials and methods). First, we generated the unc-4^FRT fly line in which we inserted two Flippase Recognition Target (FRT) sites in the same orientation flanking the 2^nd and 3^rd unc-4 exons as well as an attP site immediately 5’ to the second exon. We used this parental unc-4^FRT stock to generate all mutant lines and the GAL4/split GAL4 reporter lines. To generate the unc-4^null mutant line, we used Flippase (FLP) to excise the 2^nd and 3^rd exons of unc-4 in the germline, generating an unc-4 allele that lacks the homeodomain (Figure 1A,B). Molecular analysis confirmed the presence of the expected deletion and immunostainings revealed no detectable protein expression in the unc-4^null mutant (Figure 1C).

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To generate GAL4 and split GAL4 lines reporter lines of unc-4, we used Trojan-GAL4 system (Diao et al., 2015) to insert three different GAL4 exons – GAL4, GAL4-AD, and GAL4-DBD into the attP site of the unc-4^FRT line (DBD: DNA binding domain of GAL4; AD: activation domain of GAL4). When crossed to appropriate transgenes, each of these alleles faithfully recapitulated Unc-4 expression in the VNC and brain in the larval and adult CNS (Figure 1D–F and not shown). Each of these lines is also a null allele of unc-4, as the Trojan exons truncate the Unc-4 protein after its first exon. This conclusion is confirmed by the absence of detectable levels of Unc-4 protein in larvae hemizygous for unc-4^GAL4, unc-4^AD, and unc-4^DBD (not shown). Leveraging these transgenes, we tracked Unc-4 expression in the peripheral nervous system (PNS) and found that Unc-4 is expressed in all progenitors of leg chordotonal neurons (also called sensory organ precursors [SOPs]) and head sense organs in the larvae as well as many adult sensory neurons including chordotonal and bristle sensory neurons in the leg and Johnston’s organ and olfactory neurons in the antenna (Figure 1G–H; Figure 1—figure supplement 1).

To determine if we can use the unc-4^FRT line to ablate Unc-4 function in a lineage-specific manner, we used NB7-4 -GAL4 to drive UAS-linked FLP expression specifically in NB7-4 during embryonic stages in unc-4^FRT homozygotes (Lacin and Truman, 2016). NB7-4 gives rise to the Unc-4⁺ 23B neurons (Lacin et al., 2014). This manipulation resulted in loss of Unc-4 expression specifically in 23B neurons, but no other lineage (Figure 1I), highlighting the ability of our system to ablate Unc-4 function in a lineage specific manner.

Loss of Unc-4 results in defined behavioral defects

All loss of function unc-4 alleles we generated (null, AD, DBD, and GAL4) exhibited semi-lethality in the hemizygous or homozygous state, with many pharate adults failing to eclose from the pupal case. Escaper unc-4 null flies emerged as adults with a frequency of 13% of the expected Mendelian rate. Adult unc-4 null mutant males were fertile, but females were infertile. Examination of unc-4 females revealed that they were impaired in egg laying and their ovaries were dramatically enlarged due to egg retention (not shown), a phenotype that may arise due to defective functioning of sensory neurons in the female reproductive tract, some of which express Unc-4 (Figure 1—figure supplement 1). unc-4 mutant adults also exhibited defects in VNC executed behaviors (Figure 2). All unc-4 mutant animals manifested erect wings, and none could fly or jump (Figure 2A,C,I; Video 1). unc-4 mutant adults could walk but only in an uncoordinated manner as most failed a climbing assay (see methods) due to falling or being extremely slow (Figure 2G,I; Videos 2 and 3). unc-4 mutants also showed impaired grooming due to lack of coordination among legs, and they completely failed in executing three-leg rubbing (Figure 2E,I; Video 4), a characteristic behavior which requires coordination of three legs for cleaning (Phillis et al., 1993).unc-4^DBD-tub^AD driven unc-4 expression rescued the observed behavioral defects as well as the semilethality (Figure 2B,D,F,H; Videos 2–6). These rescued animals, however, did not behave like wild type: they were still slow to take-off (Video 6) and still exhibited defects in three-leg rubbing (not shown). To ensure that all observed defects were the result of loss of Unc-4 function, we introduced a BAC transgene that contains the unc-4 locus (Venken et al., 2010) into unc-4 mutant flies and observed essentially 100% rescue of all observed phenotypes (Figure 2I; also compare Videos 6 and 7). Our findings, thus, clearly show that Unc-4 function is required for take-off, flight, climbing and grooming behaviors.

Figure 2

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Peripheral contributions to the unc-4 behavioral phenotypes

Next, we wanted to determine whether Unc-4 functions within specific CNS or PNS lineages to control specific behaviors. We used two GAL4 driver lines in conjunction with our unc4^FRT line to remove Unc-4 function in both the CNS and PNS or just the CNS. We used sca-GAL4 to remove Unc-4 function in both the CNS and PNS (Mlodzik et al., 1990) and dpn^EE- GAL4 to remove Unc-4 function only in the CNS (Yang et al., 2016; Figure 3A–H). sca-GAL4 removed most Unc-4 expression from the CNS, all of Unc-4 expression from the progenitors of chordotonal sense organs, and also Unc-4 expression in epithelial cells of the leg disc (Figure 3B,F). dpn^EE-GAL4 which is expressed specifically in CNS NBs (Emery and Bier, 1995; Yang et al., 2016) removed most Unc-4 expression from the CNS but left Unc-4 expression in leg discs largely intact (Figure 3C,G). sca-GAL4 mediated knock-out of Unc-4 function led to the same behavioral defects observed in unc-4 mutant animals: all had erect wings, failed to fly, and showed grooming and climbing defects (Table 1; Video 8). By contrast, the dpn^EE-GAL4 mediated removal from the CNS gave a partial phenotype with the flies having erect wings, an inability to fly, and mild grooming defects (Table 1), but they performed well on the climbing assay scoring at the same level as controls (Table 1; Video 9). These results suggest that Unc-4 related PNS deficits are responsible for the poor climbing performance, while both PNS and CNS deficits are responsible for grooming defects.

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Table 1

		Success in three-leg rubbing		Success in climbing, time to climb§ (in seconds)
Driver	Target cell types	Control	unc-4 removal	Control	unc-4 removal
sca-GAL4	All CNS and PNS neurons	88.9%, N = 18	0%*, N = 20	100%, 17.7 s. +/- 8, N = 22	9.1%*, N/A¶, N = 22
dpn-GAL4	All CNS neurons	81.8%, N = 22	28.5%*, N = 21	90%, 20.1 s. +/- 15, N = 30	80%†, 27.6 s.‡+/- 20.5, N = 28
ato-Gal4	Chordotonal neurons	90%, N = 33	14.8%*, N = 27	N/A**	N/A**

1. ^*Significant change, Fisher exact test P value < 0.001.

   ^†Non-significant change, Fisher exact test p value>0.1.

2. ^‡Non-significant change, Mann-Whitney U Test p value>0.1.

   ^§failed climbing attempts not included for quantification.

3. ^¶ not assessed since most animals failed to climb.

   ^**ato-GAL4 >FLP expression impaired the negative geotaxis in both control and experimental animals, thus climbing assay was not performed.

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Due to the known role of the chordotonal organs in leg-related behaviors (Tuthill and Wilson, 2016), we asked if Unc-4 function in the chordotonal neurons is required for wild-type climbing behavior. To do this, we used a chordotonal SOP-specific driver ato-GAL4 (Hassan et al., 2000) to remove Unc-4 function completely from chordotonal lineage (progenitors and neurons), while leaving Unc-4 expression in the CNS intact (Figure 3D,H). ato-GAL4 also unexpectedly removed Unc-4 expression in the epithelial cells of most leg discs (Figure 3H). ato-GAL4 mediated removal of Unc-4 function in chordotonal lineage and epithelial cells resulted in coordination defects in leg movements: we observed defects in three-leg rubbing (Table 1; Video 10) and walking (Videos 11 and 12). We did not assess climbing behavior of the resulting flies because ato-GAL4 driven FLP expression impaired the negative-geotaxis behavior of control animals (presumably due to ato-GAL4 expression in the gravity sensing Johnston organ or its progenitor cells). To assess walking, we challenged flies to walk horizontally in small-diameter tubes. Control animals walked in a coordinated manner without falling, but unc-4 null mutants and ato-GAL4 >unc4 ^FRT flies were slow and fell often (Videos 11 and 12; Figure 3I). Thus, our results indicate that loss of unc-4 function in the leg discs, presumably in the chordotonal lineage, is required to drive proper leg-related behaviors, including three-leg rubbing, walking, and likely climbing.

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To ascertain whether Unc-4 function during chordotonal organ development may underlie the observed behavioral defects, we assessed Unc-4 function on adult femoral chordotonal organ (feCO) neurons, the major chordotonal organ in the leg. We visualized them in control and unc-4 mutant animals via iav-GAL4, which marks all chordotonal neurons in the adult (Kwon et al., 2010). In control animals, feCO neuronal cell bodies are clustered and located proximally near the trochanter border; they extend dendrites in a stereotyped fashion (Figure 3J). In the mutants, the cell bodies of many of the neurons were displaced across the femur (Figure 3K,L) and their bundled dendritic projections appeared to be disrupted as compared to controls; all of these defects were rescued with restored Unc-4 function (Figure 3M). Of note, axonal projections of the FeCO neurons in the VNC of unc-4 mutant animals appeared like wild-type (not shown). Unc-4, therefore, appears to regulate the patterning and dendritic projections of chordotonal neurons in the leg, with this function likely underlying the observed defects in three-leg rubbing, walking, and climbing.

Unc-4 and neurotransmitter expression

The central expression of Unc-4 is striking because that seven hemilineages that express it are all cholinergic (Lacin et al., 2019). To determine if Unc-4 function is required for the survival or proper differentiation of these neurons, we combined unc-4^DBD line with the ubiquitous tub^AD line to visualize all Unc-4⁺ neurons in control and unc-4 mutant animals. As shown in Figure 4A, we observed no gross defects in the number and position of Unc-4⁺ neurons in the absence of Unc-4 function, indicating Unc-4 is not required for its own expression nor is it required for the survival of these neurons.

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We then determined the neurotransmitter profile of these neurons in control and unc-4 mutant background by intersecting unc-4 split GAL4 lines with neurotransmitter specific split-GAL4 lines expressed in cholinergic, glutamatergic, or GABAergic neurons (Diao et al., 2015; Lacin et al., 2019). We previously showed that all of the Unc-4⁺ postembryonic hemilineages are cholinergic and that half of all cholinergic hemilineages (7 of 14) express Unc-4 in the VNC (Lacin et al., 2019). To confirm this and visualize cholinergic Unc-4⁺ neurons, we intersected unc-4^AD expression with the cholinergic neuronal driver ChAT^DBD (Figure 4B). As expected, most Unc-4⁺ neurons including neurons of all seven postembryonic hemilineages (identified based on location and axonal projection) in the control VNC were marked with the split-GAL4 combination of unc-4^AD-ChAT^DBD, confirming the cholinergic identity of the Unc-4⁺ hemilineages. In the unc-4 mutant VNC, the expression of this split-GAL4 combination appeared grossly normal and marked all seven Unc-4⁺ postembryonic hemilineages, indicating that Unc-4 function is not necessary for the cholinergic fate of most (but not all, see below) neurons in these hemilineages.

The relationship of Unc-4 expression to the glutamatergic phenotype was determined by combining VGlut^DBD, which labels glutamatergic neurons, with unc-4^AD. Here, unc-4^AD-VGlut^DBD driven GFP expression identified several glutamatergic Unc-4⁺ motor neurons in thoracic and abdominal segments in addition to a few occasional interneurons in the thoracic segments (Figure 4C). Based on their axon projections, these motor neurons were the MN1-5 neurons, that innervate the indirect flight muscles, as well as motoneurons to neck and haltere muscles and the abdominal body wall (Figure 4—figure supplement 1). Most of these adult motoneurons come from remodeled larval motoneurons (Consoulas et al., 2002). Comparison of the adult cells with unc-4^AD-VGlut^DBD expression in the larval VNC indicated that these are remodeled versions of larval U/CQ motor neurons, from NB7-1 while others are likely embryonic-born neurons from NB2-4 lineage (Figure 4—figure supplement 2). In the VNC of unc-4 mutant adults, these motor neurons were still present and marked with unc-4^AD-VGlut^DBD, but they were disorganized and reduced in size (Figure 4C; the MN5 size: 283+/- 54 μm² in controls vs 94+/- 31 μm² in mutants; N = 10 and 14 cells, respectively). Unc-4, thus, is expressed in embryonic-born remodeled adult motor neurons and functions to promote their growth.

Finally, we intersected unc-4^DBD with gad1^AD, a line that is expressed in all GABAergic neurons, to assess whether Unc-4 regulates GABA fate. In control animals, we failed to detect any GABAergic Unc-4⁺ neurons in the VNC (Figure 4D). RNA in situ hybridization against gad1 and ChAT genes confirmed this and our previous published findings and showed that all Unc-4⁺ postembyonic hemilineages in the VNC express ChAT mRNA but not gad1 mRNA (Figure 4—figure supplement 2). In unc-4 mutants, however, unc-4^DBD-gad1^AD marked many neurons in the VNC (Figure 4D). Presence of a BAC clone containing unc-4 locus reversed this expression pattern, significantly reducing the number of ectopic GABAergic neurons (Figure 4E). We identified these cells as a subset of the cholinergic hemilineage 11A (Harris et al., 2015; Lacin et al., 2019; Shepherd et al., 2019) based on the following observations: (i) like 11A neurons, they reside in posterior and dorsal region of only T1 and T2 segments (Figure 4D), (ii) their axonal trajectories match that of the 11A neurons (Figure 4F–G), (iii) they express Nkx6, a marker for 11A neurons (Figure 4H), and (iv) they intermingle with Eve⁺ 11B neurons, which are the sibling neurons of 11A neurons (Lacin and Truman, 2016; Lacin et al., 2019; Figure 4I).

To test whether the transformed 11A neurons in the unc-4 mutants are purely GABAergic or exhibit mixed GABAergic/cholinergic identity, we assayed them for the presence of ChAT and gad1 mRNA (Figure 4J) and found that 92 +/- 5% expressed gad1 mRNA, 2 +/- 4% expressed ChAT mRNA, and none co-expressed both (N = 93 cells, 5 CNSs). Thus, most of these 11A neurons switch from a cholinergic to a GABAergic fate. Moreover, most of these converted neurons show GABA production as well as the gad1 mRNA (Figure 4K). Unc-4, thus, appears to act in 11A hemilineage to promote the cholinergic fate and repress the GABAergic fate. Importantly, misexpression of Unc-4 in immature neurons GABAergic VNC lineages, such as 13A and 19A, throughout development did not, however, alter their GABAergic fate. Consequently, Unc-4 is not a general repressor of the GABAergic fate (N = 5 animals; not shown).

11A neurons likely function in take-off behavior of flies (Harris et al., 2015; Kennedy and Broadie, 2018). We could not investigate if the ectopic GABA fate observed in 11A neurons lead to any behavioral defect because we lacked a driver to remove Unc-4 only from this hemilineage.

Unc-4 function is required for proper neuronal projections in 7b neurons

To determine if loss of Unc-4 function leads to defects in the anatomy of neurons in the Unc-4⁺ hemilineages, we visualized them in both mutant and control flies. Unc-4 is expressed in hemilineages 7B, 11A, 12A, 17A, 18B,19B, and 23B. We used lineage-specific GAL4 lines built for this study (see methods) together with NB intersected reporter immortalization technique (Awasaki et al., 2014) to individually visualize the 7B, 12A, 18B, 23B hemilineages (Figure 5A–D). We also used the immortalization technique for the 11A and 19B neurons, but the anatomy was complicated by the presence of their surviving sibling hemilineages (not shown). We could not evaluate 17A neurons due to lack of reagents. The loss of Unc-4 function had no gross defect on neuronal projections with the notable exception of hemilineage 7B. These neurons exhibited altered projections into the leg neuropils in the absence of Unc-4 function (Figure 5A; see below).

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Lineage 7B neurons are present in all thoracic segments, but only neurons of the T2 segment innervate the leg neuropils (Shepherd et al., 2019). Using multicolor flip-out clones in wild-type animals (Nern et al., 2015), we found that many 7B neurons in T2 send ipsilateral projection to the T2 leg neuropil, others project into all of the contralateral leg neuropils, and others do not innervate the leg neuropil at all (Figure 6A; not shown). Of note, we found that the projections of 7B neurons into the ipsilateral leg neuropil have synaptic output sites, indicating that they communicate to other neurons to execute leg-related behaviors (Figure 6B). We used two different reporter immortalization strategies as well as MARCM approach to follow axonal projections of hemilineage 7B neuron in the absence of Unc-4 (see methods). All three gave the same result: loss of unc-4 severely reduced 7B projections into the ipsilateral leg neuropils and also reduced contralateral leg projections (Figure 6C–I). Unc-4, therefore, functions cell autonomously in 7B neurons to support their proper innervation of the leg neuropil.

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Unc-4 promotes the development of flight take-off circuit

The anatomical changes in the 7B neurons with the loss of Unc-4 function is paralleled by a deficit in take-off behavior. Previous work showed that activation of 7B neurons elicits take-off behavior in decapitated flies (Harris et al., 2015). We used ey-^AD;dbx^DBD driven FLP expression in unc4^FRT flies to remove Unc-4 specifically in 7B neurons (Figure 7A,B; Figure 7—figure supplement 1). To test the take-off behavior of the resulting flies, we first assessed their response to banging the vial in which they are housed (Video 13). Control animals responded to this stimulus by exhibiting a take-off behavior. In contrast, 7B specific Unc-4 knock-out flies raised their wings in an apparent attempt to take off, but they failed to be airborne. Some were able to take off after an unusual duration and multiple rounds of wing raise. We then recorded the take-off behavior of these flies with a high-speed camera after eliciting escape response via visual stimuli (Williamson et al., 2018). This analysis revealed that 7B specific Unc-4 knock-out flies process the visual stimulus, initiate the take-off behavior by adjusting their posture and raising their wings, but fail to extend their middle legs to cause lift off (Figure 7B,C; Video 14). Interestingly, some of these flies initiated the flight by just wing flapping without any clear jump (Video 15). These results demonstrate that hemilineage 7B specific removal of Unc-4 impairs take-off behavior.

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The altered behavior seen in the flies that have lost Unc-4 function in their 7B neurons could result from the loss of function of a critical component in the take-off circuit or a mis-wiring of the cells to make novel connections that interfere with take-off. For example, in C. elegans, loss of Unc-4 function alters locomotion behavior due to ectopic synaptic connections of Unc-4⁺ neurons. We investigated whether the observed take-off phenotype was due to the gain of a novel function for the 7B neurons by ablating them during development via expressing the apoptotic gene, head involution defective (hid) in NB3-2 (Bergmann et al., 1998). The resulting animals behaved essentially identical as 7B specific unc-4 mutants (Video 16), indicating the impairment take-off behavior is due to the loss of 7B neuronal function. Based on our findings, we conclude that Unc-4 acts in 7B neurons to promote their leg neuropil innervation, connections that are required for flies to use their legs properly during take-off.

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Using precise genetic tools, we dissected the function of the Unc-4 transcription factor in a lineage-specific manner. We found that within the PNS, Unc-4 function is needed for the proper development of the leg chordotonal organ and walking behavior; whereas in the CNS, Unc-4 dictates neurotransmitter usage within lineage 11A and regulates axonal projection and flight take-off behavior in lineage 7B. Below, we discuss three themes arising from our work: lineage-specific functions of individual transcription factors, an association of Unc4+ lineages with flight, and the lineage-based functional organization of the CNS in flies and vertebrates.

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

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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, Data curation, Formal analysis, 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. W Ryan Williamson

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

   Contribution

   Resources, Visualization, Methodology

   Competing interests

   No competing interests declared

3. Gwyneth M Card

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

   Contribution

   Resources, Funding acquisition, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7679-3639

4. James B Skeath

   Department of Genetics, Washington University, Saint Louis, United States

   Contribution

   Resources, Funding acquisition, Writing - review and editing

   Competing interests

   No competing interests declared

5. 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

   Supervision, Funding acquisition, Visualization, Project administration, 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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