distal-less is required for neuronal differentiation and neurite outgrowth in the Drosophila olfactory system

Jessica Plavickia,b,1, Sara Maderb, Eric Pueschelb,c, Patrick Peeblesb, and Grace Boekhoff-Falka,b,c,d,2

aNeuroscience Training Program, bDepartment of Anatomy, cCellular and Molecular Biology Training Program, and dDepartment of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53706

Edited* by Barry Ganetzky, University of Wisconsin, Madison, WI, and approved December 21, 2011 (received for review November 10, 2010)

Vertebrate Dlx have been implicated in the differentiation (5, 6). From the LAL, projection neurons relay olfactory in- of multiple neuronal subtypes, including cortical GABAergic formation to the mushroom body (MB) and the lateral horn interneurons, and mutations in Dlx genes have been linked to (reviewed in refs. 3 and 4). In adults, the MB plays roles in the clinical conditions such as epilepsy and autism. Here we show regulation of sleep, locomotion, male courtship behavior, and that the single Drosophila Dlx homolog, distal-less,isrequired learning, including olfactory learning (7–12). both to specify chemosensory neurons and to regulate the mor- Both the DO and the LAL to which it sends olfactory in- phologies of their axons and dendrites. We establish that distal- formation arise from cells in the embryonic antennal segment. less is necessary for development of the mushroom body, a brain Fourteen sense organ precursors (SOPs) contribute to the DO region that processes olfactory information. These are important (13). Seven of these give rise to the olfactory portion of the distal-less DO, and seven are gustatory. Relatively few genes have been examples of function in an invertebrate nervous sys- fi tem and demonstrate that the Drosophila larval olfactory system identi ed to date that function during DO development. These include three basic helix–loop–helix (bHLH) transcription fac- is a powerful model in which to understand distal-less functions tor-encoding proneural genes: atonal (ato), absent multidendritic during neurogenesis.

neurons and olfactory sensilla (amos), and scute (sc) (13). Speci- BIOLOGY fication of four of the olfactory SOPs requires ato while three amos | antennal lobe | atonal | dorsal organ DEVELOPMENTAL require amos, and the DO gustatory SOPs are specified by sc (13). The cephalic gap genes orthodenticle (otd), empty spiracles lthough the distal-less (dll) gene is best known for its con- (ems), and buttonhead (btd), which are coexpressed in the an- Aserved role in limb patterning, its homologs play essential tennal head segment, also are required for DO development. roles in vertebrate neural development (reviewed in ref. 1). In- Individual otd, ems, and btd mutants lack both cuticular and deed, it has been proposed that the ancestral role for dll was in neuronal components of the DO (14). dll is downstream of otd, some aspect of neural development (2). Consistent with this ems, and btd (15), and the cuticular components of the DO and hypothesis, four of the six mammalian Dlx genes play essential terminal organ (TO) are missing in dll-null embryos (ref. 16 and roles in embryonic neural development, including development this work). Here, we show that the neuronal components of DO of multiple components of the olfactory system. Three Dlx genes also are defective in dll mutants and propose that dll is a key also are required for neurogenesis in regenerating components effector of cephalic gap gene function during DO development. of the adult olfactory epithelium and the olfactory bulb. How- The MB arises from the embryonic labral and ocular seg- ever, outside of the vertebrate lineage, the role of dll in olfac- ments. Four neuroblasts on either side of the brain serve as MB tion has not been examined. Given the biological importance of stem cells, proliferating throughout embryonic, larval, and pupal olfaction and its lengthy evolutionary history, we hypothesized life to give rise to the hundreds of larval and thousands of adult that dll also plays significant roles in the developing invertebrate MB neurons called Kenyon cells (17, 18). The dendrites and olfactory system and tested this idea in the genetically tractable axon collaterals of the Kenyon cells cluster in the MB calyces, model . along with afferents from the ORNs. Kenyon cell axons form Most previous studies of Drosophila olfaction have been carried a large tract called the peduncle (reviewed in ref. 19). Although out with the adult. However, the larval system represents a sub- the genes that specify MB precursors remain unknown, both an stantially simpler model in which to dissect neural development orphan nuclear encoded by the tailless (tll) gene and and wiring (reviewed in ref. 3). In addition, portions of the larval a encoded by the Drosophila Pax6 homolog olfactory system serve as templates for corresponding structures eyeless (ey) are required during the larval instars for proliferation in the adult, forming during embryogenesis, growing during the of MB neuroblasts (20, 21). In addition, ey and a transcription larval instars, and being remodeled during metamorphosis. Thus, fi factor encoded by dachshund (dac) are expressed in embryonic elucidating the earliest events in the speci cation and differen- MB neuroblasts and required to establish the axon tracts of the tiation of larval olfactory receptor neurons (ORNs) and olfactory MB (21–24). information processing centers is highly relevant to later devel- Here, we establish that dll is expressed during the initial stages opment. We therefore asked whether dll was required for the of olfactory system development and that dll mutants have strong differentiation of larval ORNs or other larval neurons required for relaying or processing of olfactory information. The larval Drosophila olfactory organ is called the dorsal organ (DO; Fig. 1A). The DO is of mixed modalities, mediating part of Author contributions: J.P. and G.B.-F. designed research; J.P. and S.M. performed research; E.P. and P.P. contributed new reagents/analytic tools; J.P., S.M., and G.B.-F. analyzed data; the gustatory response as well as all of the olfactory response and J.P. and G.B.-F. wrote the paper. (reviewed in refs. 3 and 4). The DO consists of 21 ORNs, 15 The authors declare no conflict of interest. gustatory receptor neurons (GRNs), and 42 support cells (reviewed in ref. 4). Each ORN possesses a single dendrite that *This Direct Submission article had a prearranged editor. innervates a domelike cuticular structure. The 21 ORN dendrites Freely available online through the PNAS open access option. are clustered into seven triplets. Each ORN also possesses 1Present address: School of Pharmacy, University of Wisconsin, Madison, WI 53705. a single axon that projects to a distinct glomerulus of the larval 2To whom correspondence should be addressed. E-mail: [email protected]. antennal lobe (LAL) (Fig. 1A). The LAL consists of 21 glomeruli This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and their associated local interneurons and projection neurons 1073/pnas.1016741109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1016741109 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 phenotypes in the Drosophila larval olfactory system. The DO phenotypes are more severe than those of other genes required for ORN development and include the loss of most ORNs. We also observe defects in MB neuron differentiation in dll mutants. Because of the severity of the defects and their early onset, it is likely that dll acts near the top of the genetic hierarchy governing DO development. In addition, dll continues to be expressed in neurons and support cells that constitute the DO, and behavioral assays indicate that dll functions in postmitotic ORNs to mediate larval olfactory behavior. The findings presented here provide support for a fundamental role for dll in invertebrate neural development and neuronal function. Importantly, because the defects are reminiscent of those observed in Dlx mutant mice, these results indicate that dll/Dlx may play similar roles in the invertebrate and vertebrate olfactory systems. Results dll Is Expressed During the Development of Peripheral and Central Components of the Larval Chemosensory System. Using immuno- histochemical staining and confocal microscopy, we have char- acterized dll expression during the development of the larval chemosensory system. The larval chemosensory system is speci- fied and differentiates during embryogenesis and is fully formed by hatching (13, 25, 26). dll is expressed in the developing larval chemosensory organs throughout embryogenesis (Fig. 1 B–E and Fig. S1 A–A′′) from the specification of sensory organ precursors (Fig. S1 A–A′′′) into the three larval instars (third instar, Fig. S1 B and C). During sense organ specification, dll is coexpressed with the proneural gene ato and the zinc-finger transcription factor senseless (sens) in the antennal, maxillary, and labial head segments (Fig. S1 A–A′′′). The larval Drosophila chemosensory system consists of three paired external cephalic chemosensory organs (reviewed in refs. 3 and 4) and three pairs of internal gustatory pharyngeal sensilla: the dorsal, ventral, and posterior pharyngeal sense organs (DPS, VPS, and PPS, respectively, in Fig. 1A). dll is expressed in all of these (e.g., Fig. 1 B–E). In the cephalic chemosensory organs, dll is expressed in neurons, support cells (sheath, socket, and shaft cells), and associated epidermal cells (Fig. 1 B–E). We also found that dll is expressed in the cephalic neuroectoderm early in embryonic development in precursors of the protocerebrum and deutocerebrum, which give rise to the MB and antennal lobe, respectively (Fig. S1 A–A′′′).

dll Function Is Necessary for Normal Larval Olfactory Behavior. The expression of dll in chemosensory structures suggested that dll may be required for normal olfaction. However, dll-null animals die at the end of embryogenesis (16), before their olfactory re- sponses can be assayed. We therefore tested third-instar larvae carrying multiple hypomorphic combinations of dll alleles in an

Fig. 1. dll is expressed in neurons and associated support cells during larval cells and epidermal cells. (D and E) Dorsal views at low (D) and high (E) olfactory system development and is required for neuronal development. magnification of a stage 16 elav-GAL4;UAS-mCD8-GFP Drosophila embryo (A) Schematic of the larval chemosensory system (adapted from ref. 3). stained for Dll (red) and Pros (blue). Dll is detected in DO neurons (white Neurons in the DOG, TOG, and VO ganglion (VOG) and the dorsal, poste- arrowheads in D and E) as well as the socket, sheath, and shaft support cells rior, and ventral pharyngeal sense organs (DPS, PPS, and VPS) respond to (brackets in D). Dll and Pros colocalize in sheath cells (purple; asterisks in E). chemical compounds encountered in the environment and relay chemo- (F–I) Dorsal views of stage 16 wild-type (F and G)anddll-null (H and I) sensory information to target areas in the central nervous system. ORNs in embryos stained for sensory neurons (monoclonal antibody 22C10; green), the DOG (white arrowheads in B–D) send afferent projections via the an- Dll (red), and Elav (blue). The wild-type DOG (arrowheads in F and G) tennal nerve (AN; arrows in B and D) to the LAL where they form glomeruli consists of ∼36 neurons (21 ORNs plus ∼15 GRNs), whereas dll-null embryos connected by local interneurons (LNs). Projection neurons (PNs) of the LAL (arrowheads in H and I) have an average of 7 DOG neurons. Orthogonal relay information via the inner antennocerebral tract (iACT) to brain areas slices through the corresponding confocal Z-series are shown in G and I.The associated with learning and memory: the lateral horn (LH) and MB. MN, horizontal green lines in F and H represent the planes of section for G and I. maxillary nerve. (B and C) Lateral views at low (B) and high (C) magnifica- The horizontal blue lines in G and I represent the depths within each Z- tion of a stage 16 elav-GAL4;UAS-mCD8-GFP Drosophila embryo stained for series of the images shown F and H. The asterisk in H marks neurons that are Dll (red) and the neuronal marker for embryonic lethal, abnormal vision not part of the DO. Absence of Dll staining was used to identify dll-null (Elav; blue). GFP is localized to the membranes of neurons. Dll expression is embryos. Anterior is to the left in all images. (B–F and H) Optical sections detected in the DOG (white arrowheads in B and C), the TOG (open (0.4 μm) were collected with a 40× objective. (C, E, F,andH)A1.25× digital arrowheads in B and C), and the VOG (asterisk in C) as well as in support zoom was used. (Scale bars: 50 mm.)

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1016741109 Plavicki et al. Downloaded by guest on October 1, 2021 Fig. 2. dll mutants exhibit olfactory deficits in larval behavioral assays. (A) Olfactory RIs of dll mutants and dll-RNAi animals. See SI Materials and Methods for details of the assay. Data are plotted as mean ± SEM. With Student’s two-tailed t test, P values <0.001 were obtained for dllSA1/dll3 (red histogram) and dllSA1/ dll+ (yellow histogram) compared with wild type (gray histogram). Anosmic (sbl; black histogram) larvae served as a positive control. We also tested the responses of third-instar larvae in which UAS-dll-RNAi was driven by a pan-neural GAL4 driver (elav-GAL4; light purple histogram), an ORN-specific driver (OR83b-GAL4; light green histogram), or the dll-GAL4 driver (light blue histogram). Animals carrying any of the GAL4 drivers exhibited significant (P < 0.001) impairments in their olfactory behavioral responses even without the dll-RNAi construct present [dll-GAL4 (dark blue histogram), RI = 0.52 ± 0.08; elav-GAL4 (dark purple histogram), RI = 0.49 ± 0.02; and OR83b-GAL4 (dark green histogram), RI = 0.58 ± 0.04]. Nonetheless, when the dll-RNAi construct was present, the responses were significantly worse than with the respective GAL4 drivers alone (P < 0.05 for elav-GAL4 driving dll-RNAi and P < 0.001 for either OR83b- GAL4 or dll-GAL4 driving dll-RNAi). dll-GAL4 driving dll-RNAi (light blue histogram), RI = −0.05 ± 0.05; elav-GAL4 driving dll-RNAi (light purple histogram), RI = ± ± – × 0.15 0.08; and OR83b-GAL4 driving dll-RNAi (light green histogram), RI = 0.25 0.05. (B E) Scanning electron micrographs (1,200 ) of third-instar larval DO BIOLOGY (red arrowheads) and TO (black arrowheads) from wild-type (B), dllSA1/dll3 (C), dll1/dll3 (D), and dllSA1/dll+ (E) heads. dllSA1/dll3 larvae have malformations in DEVELOPMENTAL the DO dome (C) and display significant impairments in behavioral assays (RI = 0.27 ± 0.03; red histogram in A). The DO dome is also malformed in dll1/dll3 larvae (C), but larvae still respond well to the attractive odorant (RI = 0.72 ± 0.09; orange histogram in A). In contrast, olfaction is significantly impaired in dllSA1/dll+ larvae (RI = −0.08 ± 0.16; yellow histogram in A) despite the proper formation of the cuticular components of the DO and TO (E). Anterior is at the top, and ventral midline is to the right. (Scale bar: 50 mm.)

olfactory behavioral assay (Fig. 2A and Fig. S2). The behavioral we have discovered defects in both the function and the de- responses of dll mutants to an attractive odorant [1 μL of un- velopment of olfactory system neurons in dll mutants. diluted ethyl acetate (EtOAc)] were compared with those of For instance, when a dll-RNAi construct is used to knock down wild-type and anosmic [smellblind (sbl)] larvae (27, 28). The se- dll activity, specifically in neurons, there are severe deficits in verity of the behavioral phenotype varied depending on the al- olfactory behaviors. Both elav-GAL4;UAS-dll-RNAi larvae, in lelic combination, and not all hypomorphic combinations which the dll-RNAi construct is expressed in both central and resulted in behavioral deficits. Data for two of the six allelic peripheral neurons (Fig. 2A, purple histograms), and OR83b- combinations that demonstrated statistically significant impair- GAL4;UAS-dll-RNAi larvae, in which the dll-RNAi construct is fi ments (P < 0.001) are shown (Fig. 2A). We note that all of these expressed in ORNs (Fig. 2A, green histograms), show signi cant combinations exhibited relatively normal motor activity. Thus, impairments in olfactory behavioral responses compared with the impairments are not caused by the inability to move. Con- each GAL4 driver alone. We also used a dll-GAL4 driver to sistent with this finding, allowing more time for the animals to express the dll-RNAi construct earlier and more broadly in the reach the target zone (10 min instead of 5 min) did not change larval olfactory system. The dll-GAL4;UAS-dll-RNAi larvae (Fig. the response indices (RIs). The DO dome is a perforated cu- 2A, blue histograms) exhibit a greater impairment in our assays ticular structure filled with dendritic arborizations from un- than either elav-GAL4;UAS-dll-RNAi or OR83b-GAL4;UAS-dll- derlying ORNs. ORNs provide afferent input to CNS structures, RNAi animals do. Because dll-GAL4 drives expression in support cells associated with the DO, loss of dll function in these cells which in turn direct behavioral responses to chemical cues. may contribute to the stronger behavioral impairment. Alterna- Consequently, cuticular malformations would be likely to impair tively, and not mutually exclusively, because the dll-GAL4 driver behavioral responses to odorants in larval behavioral assays. is active in DO precursors and in developing brain regions, in- Consistent with this idea, dllSA1/dll3 larvae have DO cuticular fi cluding the MB anlagen, the dll-GAL4;UAS-dll-RNAi behavioral malformations (Fig. 2 B and C) and display signi cant impair- phenotype may reflect a requirement for dll function during early ments in behavioral assays (Fig. 2A, red histogram). However, developmental events such as the specification and differentia- comparison of scanning electron micrographs and behavioral tion of DO and/or MB precursors. data revealed that normal behavioral responses occur in some dll hypomorphs despite cuticular malformations, whereas impaired Loss of dll Function Disrupts the Development of the DO Ganglion responses can occur in the absence of cuticular malformations. – 1 3 (DOG). dll-null embryos have fewer neurons (Fig. 1 F I) and fewer For instance, in dll /dll larvae, the DO dome is malformed (Fig. support cells (Fig. 3 A and B) than wild-type animals do in both 2D), but larvae still respond to an attractive odorant (Fig. 2A, the DO and the TO. The wild-type DO contains 36 neurons, orange histogram). In contrast, olfaction is significantly impaired comprising 21 ORNs plus 15 GRNs. However, an average of SA1 + in dll /dll larvae (Fig. 2A, yellow histogram) despite the only seven neurons is found in dll-null embryos (n = 10). Thus, proper formation of cuticular components of the DO (Fig. 2E). both ORNs and GRNs are compromised. However, no ectopic The DO dome also appears normal in dll-GAL4;UAS-dll-RNAi TUNEL staining is observed in these embryos, suggesting that larvae, which show olfactory deficits (Fig. 2A, blue histograms), the reduction in neuron number is occurring via reduced speci- indicating that reduced dll function might affect the neural fication or proliferation of precursors. In addition, the remain- components of the olfactory system. Indeed, as described below, ing neurons exhibit aberrant morphologies. Specifically, the DO

Plavicki et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 Fig. 3. Dendrite malformations, axon projection defects, and support cell loss in dll-null embryos. Lateral views of stage 16 wild-type (A and A’) and dll-null (B) embryos stained for Cut (blue), Dll (red), and sensory neurons (monoclonal antibody 22C10; green). Cut is coexpressed with Dll in the majority of DOG support cells (white arrowheads), TOG support cells (open arrowheads), and VOG support cells and is weakly coexpressed in a few neurons in all three ganglia. In dll-null embryos, the number of support cells is decreased and the remaining support cells are disorganized (compare B with A and A’). Lateral views of stage 16 wild-type (C and C′) and dll-null (D) embryonic heads stained for neurons (monoclonal antibody 22C10; green) and Dll (red). Dendrites Fig. 4. Loss of dll function disrupts the MBs. (A and A′) Lateral view of an (white arrows) of the DOG (white arrowheads) are malformed in dll-null embryonic stage 12 brain stained for Dac (green), Dll (red), and Elav (blue). embryos. Dendrites (open arrows) of the TOG (open arrowheads) are also Arrows point to young Kenyon cells, a subset of which coexpress Dll and Dac. abnormal. The axons from both olfactory and GRNs of the DOG initially project (B–E) Frontal views of late third-instar MBs of wild-type (B) and dll3/dll7 (C–E) via the antennal nerve (white tracing in C). However, before reaching the larvae carrying OK107-GAL4 driving UAS-mCD8-GFP (green) and stained brain, ORN and GRN axons normally defasciculate, with ORNs innervating the with Fas2 antibody (red). The midline is toward the left. Arrows indicate the LAL and GRNs projecting to the SOG. Axons from the TOG and the VOG form Kenyon cell clusters (Kcs), which are greatly reduced in the dll mutants. the maxillary nerve (blue tracing in C), which also normally targets the SOG. In Arrowheads indicate the calyces, which also are reduced in the dll mutants. dll-null animals, afferent projections from the DOG and TOG form a single d, dorsal lobe; m, medial lobe; p, peduncle. The lobes and peduncles are fascicle directed toward the SOG (asterisks in B and D). Absence of Dll staining thinner in the dll mutants, and the relative position of the peduncle and – was used to identify dll-null embryos in B and D.(A B) Three-dimensional dorsal lobe is abnormal in the dll mutants. (Scale bars: 50 mm.) reconstructions of optical sections (0.4 μm) were collected with a 40× objective and a 1.4× digital zoom. (C–D) Optical sections (0.4 μm) were collected with × a40 objective. Anterior is to the left in all images. (Scale bars: 50 mm.) mary neural clusters in the protocerebrum and the anterior deutocerebrum, including all four of the late embryonic primary neuronal clusters that contribute to embryonic and larval MB dendrites are loosely associated rather than tightly bundled (Fig. tracts. Axons from these clusters create connections both within C D A–B′ 3 and and Fig. S3 ), and the axons of the remaining DO and between the protocerebral and deutocerebral neuromeres as neurons project toward the subesophageal ganglion (SOG) (Fig. fi well as provide a scaffold for fasciculation of axonal projections 3 C and D). This nding is consistent with GRN identity in that from late embryonic- and larval-born neurons (29–31). Here, we DO GRNs initially project along the antennal nerve, before show Dll expression in the nascent MB Kenyon cells of branching ventrally to target the SOG. Both the neuron loss and stage 12 embryos (Fig. 4 A and A′) that persists into later em- the dendritic malformation phenotypes also are observed in SA1 + 7 bryonic stages 15 and 16 (Fig. S3 A and C). Consistent with this other dll mutant genotypes, including dll /dll and dll homo- expression pattern, loss of dll activity leads to loss of a primary zygotes (e.g., Fig. S4 A and B). axon bundle emanating from the MBs and projecting along the dorsal and ventral tracts of the supraesophageal commissure (Fig. dll ato1 amos1 Expression Persists in and Mutants. ato and amos are S3 A–D′). Reducing dll activity via either RNAi or mutation also necessary for the specification of DO ORNs, whereas members – – – leads to disruption of the MB peduncles (Fig. 4 B E), indicating of the achaete scute (AS C) complex are needed for DO GRN that dll is required for axon pathfinding and/or viability in the MB fi 1 1 19 speci cation (13). ato , amos , and sc mutant embryos have Kenyon cells. In dll3/dll7 hypomorphs, the Kenyon cell clusters are 1 ∼ 1 decreased numbers of DO neurons (ato : 23 neurons; amos : reduced in size by at least 50%, the calyces are markedly smaller, ∼ 19 ∼ 27 neurons; and sc : 29 neurons) (13). dll and ato are coex- the dorsal and medial lobes are thinned, and the relative posi- pressed in clusters of cells in the antennal and maxillary seg- tions of the peduncle and the dorsal lobe are abnormal. Even ments (Fig. S1 A and A′′); however, the loss-of-neuron stronger phenotypes were observed with elav-GAL4 driving a dll- phenotype observed in dll-null embryos is more severe with only RNAi construct. In this case, Fasciclin II (Fas2) was undetectable seven DO neurons remaining, suggesting that dll functions either in the MBs. However, when a UAS-mCD8-GFP transgene was upstream of or in parallel to these proneural genes. Consistent included to permit visualization of the Kenyon cells, the pheno- 1 1 with this idea, dll expression is not lost in ato or amos mutants, type became much milder, probably because of titration of the and dll continues to be expressed in the remaining DO, TO, and GAL4 protein by UAS-mCD8-GFP. Similar titration effects have ventral organ (VO) neurons and support cells as well as in ec- been reported previously (e.g., ref. 32). When UAS-dicer was todermal cells associated with the sense organs (Fig. S3 E and F). added to enhance the dll-RNAi activity (genotype was w;UAS- dll-RNAi UAS-mCD8-GFP/+;UAS-dicer/+;OK107-GAL4/+), the dll Is Necessary for the Development of Central Components of the phenotype was stronger, nearly identical to that observed for Larval Olfactory System. Based on lacZ enhancer trap analysis, the dll3/dll7 hypomorphs, but still not as strong as dll-RNAi in Sprecher et al. (29) reported dll expression in a number of pri- the absence of other UAS-containing transgenes. Parallel experi-

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1016741109 Plavicki et al. Downloaded by guest on October 1, 2021 ments carried out with OK107-GAL4 yielded phenotypes similar therefore is possible that some of the defects we observed in dll to those observed with elav-GAL4, suggesting that the pheno- mutant MB lobes (which consist of axon tracts) and calyces types seen in the dll mutants are intrinsic to the MB. (which contain Kenyon cell dendrites) are caused by mis- However, we do observe additional brain defects in dll regulation of futsch. Other likely effectors of dll function during mutants. Specifically, in dll-null embryos, the brain commissures axon pathfinding in both DO and MB are Pak3 (42) and Down are defective (Fig. S3 A–D′) and the microtubule-associated syndrome cell adhesion molecule (Dscam) (43–45). Both play Futsch protein recognized by monoclonal antibody 22C10 is important roles in axon guidance, including the targeting of adult consistently mislocalized (compare Fig. 3 D and C′ and Fig. S3 B Drosophila ORNs (42, 43). Both also have been identified as and B′ with Fig. S3 A and A′). The supraesophageal commissure putative downstream targets of vertebrate Dlx1/2 (46). The loss- normally consists of three primary axon tracts (31). One of these of-function Drosophila dll phenotypes in both DOG and MB is absent in the dll-null embryos (compare Fig. S3 C′ and D′), are reminiscent of DSCAM phenotypes and consistent with dll most likely the one emanating from the MBs. The subesophageal regulation of DSCAM in multiple neuronal subtypes. commissure consists of fascicles from at least two primary axon Given the dramatic reduction in Kenyon cell number in the dll tracts (ref. 31, but see also ref. 33). Both of these are missing in hypomorphs, we might have expected the peduncles and lobes to dll-null embryos (Fig. S3 C–D′). These axon scaffold phenotypes be even thinner than observed. However, Noveen et al. (21) are likely to reflect the effects of disrupting the development of observed a similar reduction in Kenyon cell number without multiple dll-expressing primary neural clusters. concomitant thinning of the lobes in eyR mutants. In this case, ablation studies were used to demonstrate that, at late third in- Discussion star, recently born Kenyon cells have not yet contributed to MB dll in DO Specification and Differentiation. The DO phenotype lobes. We therefore anticipate that MB defects may be more exhibited by dll-null embryos is more severe than that of sc, pronounced in dll mutant adults. amos,orato single mutants or amos;ato double mutants, most closely resembling that of the sc;amos;ato triple mutants (13), Drosophila dll and Vertebrate Dlx Function. This work constitutes an indicating that dll might lie upstream of the proneural genes and important description of dll function in the invertebrate nervous regulate their expression in DO precursors. However, ato is system. We have demonstrated requirements for dll in multiple expressed in the antennal segments of dll-null embryos, and dll is neuronal subtypes, including the larval Drosophila ORNs, GRNs, expressed in ato1 and amos1 mutants. Altogether, these data and Kenyon cells. Vertebrate Dlx genes are required for the specification, differentiation, and migration of neural progenitor indicate that dll is likely to act in parallel with the proneural BIOLOGY genes during DO development. cells that both build and regenerate parts of the olfactory system prospero (pros) also is required for DO development. pros (reviewed in ref. 1). The reagents and techniques available in DEVELOPMENTAL encodes a homeodomain transcription factor that is asymmetri- Drosophila, together with the existence of a single dll gene, make cally distributed during SOP division (reviewed in ref. 34). In it a powerful model for investigation of dll function and have the other contexts, pros represses neuronal stem-cell proliferation potential to provide insight into how disruptions in Dlx function while promoting neuronal differentiation (35). Mutations in pros result in aberrant neural development in vertebrates and con- result in gustatory behavioral deficits (36) and disrupt axon and tribute to neuropathologies such as epilepsy, Rett syndrome, and dendrite outgrowth from both DO and TO neurons (37). The autism as well as to the neurological defects associated with – axon pathfinding defects exhibited by pros mutants resemble Down syndrome (47 51). those seen in dll-null embryos, although it is unclear whether the The functions of the cephalic gap genes in Drosophila and same subsets of neurons are affected. vertebrate brains are proposed to represent conserved genetic programs in the development of the central nervous systems of – dll in the Brain. The axon scaffolding associated with the MBs insects and mammals (52 54). Nonetheless, because of differ- fi in the embryonic brain is disrupted in dll-null embryos. Specifi- ences in ORN speci cation and targeting, it is not thought that the cally, projections from the MB Kenyon cells across the supra- olfactory systems of vertebrates and invertebrates share a com- esophageal commissure appear to be missing in late-stage mon origin (e.g., refs. 3 and 55). However, the growing number of embryos. MB defects also were observed in the brains of larvae developmental genetic parallels, including the requirements for in which postmitotic drivers such as elav-GAL4 and OR83b- dll and the Dlx genes part of the invertebrate and vertebrate ol- GAL4 were used in conjunction with dll-RNAi to knock down factory systems, in conjunction with the similar wiring of these activity. This finding indicates that dll also may be necessary for systems, suggests that this question should be revisited. Even if the the later specification and/or differentiation of larval-born vertebrate and invertebrate olfactory systems have evolved in- fi Kenyon cells. Because elav-GAL4 is not active until neurons are dependently toward similar architectures, there is now suf cient specified, the disruptions in the larval MBs observed in elav- precedent from other tissue and organ systems that the genetic GAL4;UAS-dll-RNAi animals are consistent with a role for dll in tool kit is likely to be conserved and that further studies in Dro- either axon guidance or viability of the postmitotic neurons. sophila will provide insights into the mechanisms regulating the The brain phenotypes we have detected in dll mutants resemble differentiation of olfactory system components (56). those of cephalic gap gene mutants. Specifically, loss of otd, ems, or btd results in embryonic brain segmentation defects and dis- Materials and Methods rupts the formation of brain commissures and axon tracts (38, Embryo collection and fixation were performed as described (57) with staging 39). In otd mutants, protocerebral neuroblasts, including the MB of embryos according to Campos-Ortega and Hartenstein (58). For Xgal precursors, are missing. In ems and/or btd mutants, subsets of stainings, third instar larvae were dissected in PBS and larval heads were neuroblasts are lacking in the deutocerebral neuromere, which fixed and stained according to (59). Confocal images were collected on a Zeiss LSM 510 microscope. 3D reconstructions were made using Zeiss LSM harbors the LAL, and the tritocerebral neuromere, which receives fi gustatory inputs. (40). It therefore is possible that dll is a key software and images were processed using combinations of Zeiss LSM l- effector of cephalic gap gene function during brain development. tering functions, Image J and Adobe Photoshop. For scanning electron micrographs, third instar samples were fixed overnight at 4 °C according to dll Gerber et al. (60). Following a graded EtOH series, samples were covered in and Axons. As described above, pros mutants exhibit DO axon gold palladium using an Auto Conductavac IV Sputter Coater, and a Hitachi defects similar to those of dll. It therefore is possible that Dll and S570 scanning electron microscope with a Gatan digital capture system was Pros collaborate to regulate other genes needed for axon path- used to collect the images. Larval behavioral assays were performed finding by DOG neurons. In the brain, but not in the DOG or according to Rodrigues and Siddiqi (61). For a detailed description of TO ganglion (TOG), we have observed mislocalization of Futsch materials and methods including Drosophila strains, immunohistochemistry protein in dll mutants. Futsch is a microtubule-associated protein procedures, confocal and scanning electron microscopy, and behavioral with functions in both axonogenesis and dendritogenesis (41). It assays, see SI Materials and Methods.

Plavicki et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 ACKNOWLEDGMENTS. We thank Ralph Albrecht and Joseph Heinz for of the National Institute of Child Health and Human Development, and scanning electron microscopy training and access; Hugo Bellen for sens and maintained by the University of Iowa Department of Biological Sciences. ato antibodies; Dan Eberl for ato1/ato1 flies; Andrew Jarman and Petra zur We thank the Transgenic RNAi Project (TRiP) at Harvard Medical School Lage for ato and amos antibodies and mutants; and Janie Yang for technical (National Institutes of Health/National Institute of General Medical Sciences assistance. We also thank Drs. Margaret McFall-Ngai and Ned Ruby for access Grant R01-GM084947) for providing transgenic RNAi fly stocks and/or plas- to the Zeiss LSM confocal microscope in the University of Wisconsin Depart- mid vectors used in this study. The authors are grateful for the thoughtful ment of Medical Microbiology and Immunology (which is supported by Na- comments of two anonymous reviewers. J.P. was supported by National tional Institutes of Health Grant RR12294). The monoclonal, Cut Elav, Fas2, Institutes of Health Training Grant T32 GM-7507 and a grant from the Uni- Pros, Repo, and 22C10 antibodies used in these studies were obtained from versity of Wisconsin Graduate School. This work also was supported by Na- the Developmental Studies Hybridoma Bank, developed under the auspices tional Institutes of Health Grant R01 GM59871 (to G.B.-F.).

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