The Zinc Finger Transcription Factor Jing Is Required for Dendrite/Axonal

Developmental Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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The zinc ﬁnger transcription factor Jing is required for dendrite/axonal targeting in Drosophila antennal lobe development

Indu. S. Nair a,b, Veronica Rodrigues a,b,1, Heinrich Reichert c, K. VijayRaghavan b,n a Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai 400005, India b National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India c Biozentrum, University of Basel, Basel, Switzerland article info abstract

Article history: An important role in olfactory system development is played by transcription factors which act in Received 5 March 2013 sensory neurons or in their interneuron targets as cell autonomous regulators of downstream effectors Received in revised form such as cell surface molecules and signalling systems that control neuronal identity and process 11 June 2013 guidance. Some of these transcriptional regulators have been characterized in detail in the development Accepted 13 June 2013 of the neural elements that innervate the antennal lobe in the olfactory system of Drosophila. Here we identify the zinc finger transcription factor Jing as a cell autonomously acting transcriptional regulator Keywords: that is required both for dendrite targeting of projection neurons and local interneurons as well as for jing axonal targeting of olfactory sensory neurons in Drosophila olfactory system development. Immunocy- Olfactory system tochemical analysis shows that Jing is widely expressed in the neural cells during postembryonic Dendrite targeting development. MARCM-based clonal analysis of projection neuron and local interneuron lineages reveals Axonal targeting a requirement for Jing in dendrite targeting; Jing loss-of-function results in loss of innervation in specific glomeruli, ectopic innervation of inappropriate glomeruli, aberrant profuse dendrite arborisation throughout the antennal lobe, as well as mistargeting to other parts of the CNS. ey-FLP-based MARCM analysis of olfactory sensory neurons reveals an additional requirement for Jing in axonal targeting; mutational inactivation of Jing causes specific mistargeting of some olfactory sensory neuron axons to the DA1 glomerulus, reduction of targeting to other glomeruli, as well as aberrant stalling of axons in the antennal lobe. Taken together, these findings indicate that Jing acts as a key transcriptional control element in wiring of the circuitry in the developing olfactory sensory system in Drosophila. & 2013 Published by Elsevier Inc.

Introduction important role in these processes is played by sets of transcription factors which act in sensory neurons or in their interneuron The establishment of peripheral and central sensory circuitry targets as cell autonomous regulators of the spatio-temporal involves the directed outgrowth, pathfinding and correct targeting expression of downstream effectors such as cell surface molecules of neural processes from sensory cells to the neural processes of and signalling systems that control neuronal identity and process their appropriate interneuronal targets in order to form appro- guidance. Some of these transcriptional regulators have been priate synaptic contacts. The cellular and molecular mechanisms characterized in detail in the development of the olfactory system that control this process have been investigated in several organ- in Drosophila (Komiyama and Luo, 2006; Brochtrup and Hummel, isms, and the developing olfactory system has proven to be an 2011). excellent model for this type of analysis. In olfactory system In Drosophila, the olfactory sensory neurons (OSNs) are located development, a complex suite of molecular mechanisms has been in the hair-like sensillar organs of the antennae and maxillary shown to orchestrate neuronal specification, axonal pathfinding, palps. OSNs derive from sensory organ precursors in a fixed dendrite outgrowth and target recognition in both vertebrate and lineage-specific manner and subsequently project their axons to invertebrate model systems (Jefferis and Hummel, 2006; Luo and the antennal lobe of the brain. There the OSN axons segregate into Flanagan, 2007; Rodrigues and Hummel, 2008; Sakano, 2010). An distinct neuropile aggregates called glomeruli and synapse with the dendrites of projection neurons (PNs) and local interneurons (LNs), which represent the first order (antennal lobe) interneurons

n in the olfactory system. Each OSN expresses a characteristic Corresponding author. Fax: +91 80 363 6662. E-mail address: [email protected] (K. VijayRaghavan). odorant receptor (as well as a more ubiquitously expressed 1 Deceased November 10, 2010. co-receptor), and OSNs that express the same odorant receptor

0012-1606/$ - see front matter & 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.ydbio.2013.06.023

Please cite this article as: Nair, Indu.S., et al., The zinc finger transcription factor Jing is required for dendrite/axonal targeting in Drosophila antennal lobe development. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.06.023i 2 Indu.S. Nair et al. / Developmental Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ make synaptic connections in the same antennal lobe glomerulus embryonic-lethal deletion lines covering the right arm of the (Vosshall and Stocker, 2007). Establishment of these specific second chromosome and embryonic lethality of non-balanced connections between OSNs and PNs/LNs during postembryonic progeny from the crosses was used as the readout for checking development is a multistep process and depends on positional non-complementation (data not shown).This allowed us to loca- information, cell autonomous differentiation programs, initial lize the mutation in the 42A14-42C7 region (corresponding to the formation of a coarse map of projections into and within the smallest deletion line Df(2R) BSC326 (Bloom 24351) that did not developing antennal lobe, refinement of these projections and complement the embryonic lethality of the mutation). A total of 41 establishment of appropriate targeting in a process involving genes are uncovered by this deficiency line (ref. Flybase). Lethality- considerable cell–cell interactions, and finally formation of specific based complementation analyses was carried out using lethal synapses (Brochtrup and Hummel, 2011). mutations in 15 out of the 41 genes for which lethal mutant lines In recent years, some insight into the transcriptional control of (all balanced over CyO) were available (Table S1). olfactory interneuron wiring specificity in the antennal lobe has been achieved (see Komiyama and Luo, 2006; Rodrigues and Hummel, 2008; Brochtrup and Hummel, 2011). Thus, the two MARCM experiments POU domain transcription factors Acj6 and Drifter, the LIM- homeodomain transcription factors Islet and Lim1, the LIM- For wild-type or jing mutant MARCM clones in PNs, FRT42B cofactor Chip, the homeodomain transcription factors Cut and UAS-GFP; hs-FLP or FRT42B jingn UAS-GFP; hs-FLP females, respec- Ems, and the zinc finger transcription factors Squeeze and Lola are tively, were crossed to GH146-Gal4 UAS-mCD8GFP/FM7a; FRT42B all required for dendrite targeting of antennal lobe interneurons tubulin-Gal80/CyO males. (Komiyama et al., 2003; Komiyama and Luo, 2007; Spletter et al., For wild-type or jing mutant MARCM clones in LNs, FRT42B 2007). Less is known about the cell intrinsic transcriptional UAS-GFP; hs-FLP or FRT42B jingn UAS-GFP; hs-FLP females, respec- mechanisms that underlie the specific identity and target recogni- tively, were crossed to LN2-Gal4/FM7a; hs-FLP FRT42B tubulin- tion features of developing OSNs. Currently, only Ems, Acj6 and Gal80/CyO males. Pdm3 are known to be cell autonomously required for correct For jing mutant UAS-Jing rescue MARCM in PNs, FRT42B jingn targeting of subsets of OSNs in the antennal lobe (Komiyama et al., UAS-GFP; UAS-Jing females were crossed to GH146-Gal4 UAS- 2004; Tichy et al., 2008; Sen et al., 2010). mCD8GFP/FM7a; hs-FLP, FRT42B tubulin-Gal80/CyO males. Embryos In this report, we identify the zinc finger transcription factor Jing from these MARCM crosses were collected on standard medium as a cell autonomously acting transcriptional regulator that is over a 4-h time window and cultured at 25 1C for 21–25 h before required for dendrite targeting of PNs and LNs as well as for axonal giving a one-hour heat shock in a water bath maintained at 371. targeting of OSNs during the development of olfactory circuitry in After heatshock, the flies were raised at 25 1C until eclosion. the antennal lobe. Immunocytochemical analysis shows that Jing is For wild-type or jing mutant MARCM experiments in adult OSNs, widely expressed in the neural cells of the CNS during postem- FRT42B UAS-GFP; ey-FLP or FRT42B jingn UAS-GFP; ey-FLP females, bryonic development. MARCM-based clonal mutant analysis of PN respectively, were crossed to males of elav-Gal4 UAS-mCD8GFP; and LN lineages reveals a requirement for Jing in dendrite targeting FRT42B tubulin-Gal80/CyO –GFP (for pan-neural OSN visualisation) in the antennal lobe. Jing loss-of-function results in loss of innerva- and Or-Gal4/FM7a; FRT42B tubulin-Gal80/CyO or FRT42B tubulin-Gal80/ tion in specific glomeruli, ectopic innervation of inappropriate CyO; Or-Gal4/MKRSb (for individual OSN class-specific visualisation). glomeruli, aberrant profuse dendritic arborisation throughout the For jing mutant UAS-Jing rescue MARCM in OSNs, elav-Gal4UAS- antennal lobe as well as mistargeting to other parts of the CNS.ey- mCD8GFP; FRT42B tubulin-Gal80; UAS-Jing males were crossed to FLP-based MARCM mutant analysis of OSNs reveals an additional FRT42B jing22F3;ey-FLPfemales. The crosses were raised throughout requirement for Jing in axonal targeting in the antennal lobe. at 25 1C until the eclosion of the progeny. Mutational inactivation of Jing causes specific mistargeting of OSN axons to the pheromone-responsive glomerulus DA1, reduction of targeting to other glomeruli, as well as aberrant stalling of axons in Antibody generation the antennal lobe. Taken together, these findings indicate that Jing acts cell autonomously as a key transcriptional control element in Three antigenic peptides of the Jing protein were synthesised postsynaptic dendrite and presynaptic axonal wiring of the devel- AMSKPTTPQRHGNPC(amino acids 47–61), CRKHPRKPPKVTPPA oping olfactory sensory system in Drosophila. (amino acids 1460–1474) and CLPKREPESETDTLK (amino acids 851–865) and conjugated to KLH and used subsequently to generate anti-Jing goat antisera from which the Jing-specific Materials and methods antibodies were obtained by peptide-affinity purification (Gen- Script USA Inc.). Antibody specificity was confirmed by examining Fly strains and genetics embryos homozygous for a deficiency in jing (Df(2R)BSC326) and after ectopic expression of Jing (engrailed-Gal4/UASJing) in the Most of the fly stocks used in this report, including the wing discs (data not shown). deficiency set for the 2R chromosome as well as mutants for the subsequent complementation analyses were obtained from the Bloomington Stock Centre (IN, USA). FRT42B jing22F3 and UAS-Jing Immunolabeling transgenic fly stocks were kindly provided by Culi et al. (2006). All stocks were grown on cornmeal media at 25 1C. For staging, white Brains were dissected and immunostained as described earlier (Wu prepupae (0 h after puparium formation; APF) were collected on a and Luo, 2006). Primary antibodies used were: Goat anti-Jing (1:200, moist filter paper and aged under humid conditions at 25 1C. GenScript USA Inc), Rabbit anti-GFP (1:10,000; Molecular Probes, Invitrogen, Delhi, India), mouse anti-Bruchpilot (mAbnc82, 1:20; Genetic mapping DSHB, Iowa, USA), Rat anti-Elav (7E8A10, 1:10; DSHB, Iowa, USA). Alexa-488, Alexa-568 and Alexa-647 coupled secondary anti- In order to map the embryonic-lethal mutation of interest on bodies generated in goat/donkey (Molecular Probes) were used at the second chromosome, the mutant line was crossed to 67 1:400 dilutions.

Montell, 2001; Culi et al., 2006), and obtained comparable mistargeting phenotypes (data shown only in Fig. S4). Taken together, these ﬁndings implicate the zinc ﬁnger transcription factor encod- ing jing gene in the development of correct targeting of PN and LN dendrites and OSN axons in the antennal lobe.

Jing is widely expressed in neural cells during postembryonic brain development

The zinc finger transcription factor Jing has been shown previously to play important roles in embryonic central nervous system (CNS) development, in tracheal development as well as in ovarian border cell migration (Liu and Montell, 2001; Sedaghat et al., 2002; Sedaghat and Sonnenfeld, 2002). During embryogen- esis, Jing is expressed in the neural cells of the CNS from stage 9 onward and appears to be required for the development of most major axon pathways (commissural and longitudinal), both in the embryonic ventral nerve cord and brain (Sedaghat et al., 2002; Fig. 1. Genetic mapping of an embryonic-lethal mutation identifies the jing gene. Sedaghat and Sonnenfeld, 2002). To identify the mutated gene, a deficiency mapping study was carried out using To determine if Jing is also expressed in neural cells of the brain deficiency kit covering the right arm of the second chromosome. This analysis initially localized the mutation in the 42A14-42C7 region based on failure of Df(2R) during postembryonic development, we carried out an immuno- BSC326 to complement the embryonic lethality. This was followed by lethality- cytochemical analysis of Jing expression in the CNS of pupal stages, based complementation studies using the mutations in all of the genes contained viz., 18 h APF and 22 h APF. In both these stages, Jing immunor- in this region for which lethal mutant lines were available, which revealed jing eactivity was seen widely in the neural cells of the brain. All of the (highlighted in green) as the only gene which did not complement the mutation (see Table S1). postmitotic neurons in the brain hemispheres, co-labeled with the neuron-specific marker ELAV, co-expressed the Jing transcription factor (Fig. 2A–C). Given this widespread expression of Jing in neurons of the brain during postembryonic development, it Microscopy and image processing seemed likely that the developing antennal lobe interneurons also express Jing. To confirm this, we focused on the developing PNs Immunostained whole mount brains were mounted and that express the enhancer trap line Gal4-GH146, which labels imaged on an Olympus Fluoview (FV1000) confocal microscope. subsets of postmitotic PNs in three antennal lobe neuroblast Optical sections were acquired at 1 μm intervals with a picture size lineages viz., the anterodorsal (adNB), lateral (lNB), and ventral of 512 512 pixels or 1024 1024 pixels. Images were digitally (vNB) lineages, once these PNs begin to extend their neurites processed using Adobe Photoshop CS3. (Stocker et al., 1997; Jefferis et al., 2001). All of the neurons labeled by Gal4-GH146 were immunopositive for Jing in postembryonic brain development (Fig. 2D–F). This widespread expression of Jing Results in postmitotic neurons was also seen in the larval and adult brain, (Fig. S1). Genetic mapping of an embryonic-lethal mutation implicates the jing gene in olfactory neuron targeting Jing is required in olfactory interneurons for correct dendrite In a MARCM-based analysis of embryonic lethal mutations targeting in the developing antennal lobe located on the second chromosome, we recovered a mutant line with striking targeting defects in the adult olfactory system that Since jing loss-of-function results in embryonic lethality, we are characterized by mistargeting of PN and LN dendrites and OSN analysed the role of jing in the postembryonic development of axons in the glomeruli of the antennal lobe (for a detailed antennal lobe interneurons using MARCM-based clonal methods phenotype analysis, see below). To identify the mutated gene, in the adNB, lNB, and vNB lineages (Lee and Luo, 2001). For this, which was localized to chromosome 2R, we carried out a defi- Gal4-GH146 was used to drive a UAS-mCD8::GFP reporter in ciency mapping study focused on this chromosomal arm. This experiments in which MARCM clones were induced at 0–4 h ALH analysis initially localized the mutation in the 42A14-42C7 region. (after larval hatching) and recovered in the adult. Gal4-GH146 Subsequent lethality-based complementation studies using lethal driven labeling of wild-type control clones manifested the mutations corresponding to all of the genes contained in this expected highly specific pattern of dendrite innervation in identi- region for which lethal mutations were available, revealed jing as fied glomerular subsets by PNs whose cell bodies were located the only gene that did not complement the mutation (see the either at the anterior-dorsal, lateral, or the ventral margin of the “Materials and methods” section; Fig. 1). Two of the previously antennal lobe neuropile (e.g. Jefferis et al., 2001). In contrast, characterized jing mutant alleles jing01094 and jingk03404 were labeled jing loss-of-function mutant clones showed pronounced tested and both failed to complement the mutation in contrast dendrite targeting defects, which differed for the PNs of the adNB to the other genes in this region that were tested (Table S1). This lineage versus the PNs of the lNB and vNB lineages. implies that the observed targeting effects are due to a mutation in Mutant GH146-Gal4-positive PNs in the adNB lineage mani- the jing gene. To confirm that the observed mutant phenotypes fested two major mistargeting phenotypes. First, mutant PNs were indeed due to jing loss-of-function, we repeated our failed to innervate specific glomeruli that were innervated by the MARCM-based analysis using the previously characterized jing22F3 corresponding wild-type interneurons. Thus, mutant PNs failed to allele, which represents an EMS-induced, embryonic lethal muta- innervate the VA1lm glomerulus in all of the clones examined (10 tion that encodes a truncated, non-functional protein (see Liu and out of 10 antennal lobes analysed) versus 100% innervation (13 out

Fig. 2. Jing is widely expressed in neural cells during postembryonic brain development, (A–C) A single Z-section of 18 h APF brain immunolabeled with anti-Elav (green) (A) and anti-Jing (red) (B). Jing is expressed in all the postmitotic neurons that also express Elav (C). Region inside the white outline in A–C have been shown separately in the insets to show the co-expression of Elav and Jing clearly. (D–F) A single Z-section of 22 h APF central brain immunolabeled with anti-Jing (D). Single Z-sections of 22 h APF central brain with GH146-Gal4 driven UAS-mCD8GFP immunolabeled with anti-GFP (green) and anti-Jing (red) (E and F). The cell bodies of PNs of the anterodorsal lineage (indicated by yellow arrows in E) as well as that of the lateral lineage (indicated by yellow arrowheads in F) show Jing expression. Regions inside the white outline have been zoomed in the insets to show Jing expression in the adPNs and lPNs clearly. Vertical white line indicates the brain midline. AL-Antennal lobe; VNC-Ventral Nerve Cord. Scale bar: 30 lm. of 13 antennal lobes analysed) observed for wild-type clones fasciculated into aberrant bundle-like structures within the (Fig. 3A–H). Similar innervation defects were found in the VA3 antennal lobe. and DL1 glomeruli, both in neuroblast clones (n¼10/10; Fig. 3I–X) Since the lNB lineage comprises LNs as well as PNs, we also and in single cell clones (n¼13/13); see Fig. S2). Second, mutant analysed the role of jing in LN dendrite targeting in MARCM clones PNs ectopically innervated inappropriate glomeruli. For example, using the LN2-Gal4 driver which specifically labels a subset of the mutant PNs ectopically innervated the DL2 glomerulus in all of the LNs in the lNB lineage (Lai Sen-Lin et al., 2008; Das et al. 2008). clones examined (n¼10/10) compared to little or no innervation The wild-type LN2-Gal4 positive LNs targeted their dendrites to by wild-type adPN clones (n¼13/13; white asterisks in Fig. 3Q–X). the glomeruli of the ipsilateral antennal lobe which were uni- In addition, mutant PNs also misprojected to regions in between formly innervated (Fig. 5A and B; n¼2). In contrast, the mutant identified glomeruli resulting in an overall diffuse innervation LN2-Gal4 positive LNs, while also innervating the ipsilateral pattern in parts of the lobe (n¼10/10) (e.g. Fig. 3E–G). The antennal lobe, manifested two types of mistargeting phenotypes. remaining landmark glomeruli innervated by the wildtype neu- They projected neurites across the midline to the contralateral rons of the adNb lineage were properly innervated by the mutant antennal lobe and sent processes out of the ipsilateral antennal neurons as well (Table S2). Also, the axons of mutant adPNs lobe into the neuropile of the subesophageal ganglion (Fig. 5C–H; seemed to target correctly from the antennal lobe to the higher n¼6). centers viz., mushroom body calyx and lateral horn like the The misprojections observed in mutant PNs could be rescued wildtye adPNs. by GH146-Gal4 driven transgenic expression of wild-type Jing Mutant GH146-Gal4-positive PNs in the lNB as well as in the protein in jing loss-of-function mutant MARCM clones (Table S2 vNB lineage manifested a different set of mistargeting phenotypes. and Fig. S3). The typical pattern of dendrite innervation in specific Wild-type PNs of the lNB (n¼10) and vNB (n¼12) lineage, glomeruli manifested by wild-type PNs in labeled neuroblast innervated sets of discrete, individual glomeruli (Fig. 4A–D, I–L). clones and in labeled single cell clones was restored in the mutant In contrast, mutant PNs in these lineages never targeted individual clones upon transgenic expression of wild-type Jing. Given that glomeruli, rather they formed profuse branching throughout the the GH146-Gal4 driver is only active in postmitotic neurons, these antennal lobe (Fig. 4 E–H, M–P; n¼6 for lNB and n¼8 for vNB). rescue experiments imply that the post-embryonic requirement Moreover, they manifested misprojections out of the antennal lobe for Jing in dendrite targeting of antennal lobe interneurons is towards the subesophageal ganglion, and their dendrites often postmitotic in nature.

Fig. 3. Jing is required for the correct dendrite targeting of adPNs in the antennal lobe. GAL4-GH146 UAS-mCD8::GFP MARCM clones immunolabeled with anti-GFP (green) and neuropile marker anti Bruchpilot (nc82; red). Stacked confocal sections of single adult antennal lobes with dorsal to top and medial to left. The VA1l/m, VA3 and DL1 glomeruli have been outlined by white dots and indicated by white arrows and DL2 glomerulus by pink dots and white asterisk. Scale bar: 30 lm.(A–D, I–L and Q–T) Wild- type MARCM clones of the adNB lineages show proper innervation of the VA1l/m, VA3 and DL1 glomeruli. (E–H, M–P and U–X) jing mutant MARCM clones of the adNB lineage show loss of dendrite innervation of VA1l/m, VA3 and DL1 glomeruli. (U–X) Ectopic innervation of inappropriate glomerulus DL2 by jing mutant adNB clones in contrast to little or no innervation of DL2 by wild-type clones. 6 Indu.S. Nair et al. / Developmental Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

Fig. 4. jing mutant GH146-Gal4-positive PNs in the lNB and the vNB lineages show dendrite mistargeting out of the antennal lobe. (A–D and I–L) Wild type PNs of the lNB and vNB lineages show characteristic innervation patterns in the antennal lobe. (E–H and M–P) In contrast, jing mutant PNs in these lineages formed abnormally profuse branching throughout the antennal lobe and misprojected dendrite processes out of the antennal lobe towards the subesophageal ganglion (white arrows). Stacked confocal sections of single adult antennal lobes with dorsal to top and medial to left. The antennal lobe boundary has been outlined with blue dots. Anti-GFP (green); anti-Bruchpilot (nc82, red). Scale bar: 30 lm.

Taken together, these mutant analyses show that Jing is Jing is required in olfactory sensory neurons for correct axonal required for correct dendrite targeting of PNs in the adNB, lNB targeting in the developing antennal lobe and vNB lineages as well as of LNs in the lNB lineage during postembryonic development. Moreover, given the clonal nature of Given the requirement of Jing in dendrite targeting of antennal these MARCM analyses, this requirement is likely to be cell lobe interneurons, and the dual role of other transcription factors autonomous. (e.g. Acj6, Ems) in both dendrite targeting of central PNs/LNs and

Fig. 5. jing mutant LNs labeled by LN2-Gal4 show dendrite crossovers to the contralateral lobe and mistargeting out of the antennal lobe. (A and B) The wild-type LNs labeled by LN2-Gal4 driven UAS-mCD8GFP show dendrite branching restricted exclusively to the ipsilateral lobe. (C–H) In contrast, the jing mutant LNs, while also innervating the ipsilateral antennal lobe, send dendrite processes across the midline to the contralateral antennal lobe (pink arrows in C, D, G and H) as well as out of the ipsilateral antennal lobe into the neuropile of the subesophageal ganglion (white arrows in C–H). The GFP channel in A, C and E, G has been shown separately in grayscale in B, D, F and H, respectively. The antennal lobe boundary has been outlined with blue dots. Anti-GFP (green); anti-Bruchpilot (nc82, red). Scale bar: 30 lm.

axonal targeting of peripheral OSNs, we further investigated if Jing methods based on ey-FLP MARCM. In these experiments, FLP might also be implicated in axonal targeting of OSNs in the recombinase is driven by the eyeless promoter to induce recombi- antennal lobe. To investigate this, we used mutational mosaic nation in the antenna/maxillary palp such that 30–50% of the OSNs

Fig. 6. Jing is necessary for correct targeting of the olfactory sensory neuron axons in the antennal lobe. Wild-type and jing mutant OSN clones generated using ey-FLP based MARCM and visualized using pan-neural elav-Gal4 driven UAS-mCD8 GFP show strikingly different patterns of axonal targeting in the adult antennal lobes. (A–D) The wild- type OSN axons branched throughout the antennal lobe and innervated all of the glomeruli. (E–H) In contrast, mutant OSN axons showed drastic increase in the innervation of the DA1 glomerulus compared to the other antennal lobe glomeruli in the antennal lobe. Some axon branches misprojected in the antennal lobe without apparent targeting to any glomeruli (arrowheads in F and H). White arrows point to the DA1 glomerulus. Anti-GFP (green); anti-Bruchpilot (nc82, red). Scale bar: 30 lm.

are labeled and made homozygous mutant while the interneurons specifically innervated the VA1d glomerulus (n¼8 ALs); in the of the CNS remain unaffected (Newsome et al., 2000; Hummel mutant, these axons always mistargeted to the DA1 glomerulus et al., 2003). As drivers in these ey-FLP MARCM experiments, (n¼22 ALs) (Fig. 7A and B). In the wild-type, the axons labeled by either the pan-neuronal elav-Gal4 driver or the Or83b-Gal4 driver OR33b-Gal4 innervated the DM3 and DM5 glomeruli(n¼20 ALs); (data not shown), which is expressed in the majority of the OSNs, in the mutant, these axons invariably mistargeted to the DA1 were used. glomerulus(n¼21 ALs) (Fig. 7C and D). In two other OR-Gal4 lines, In wild-type control experiments, the ey-FLP MARCM labeled aberrant axonal targeting was observed occasionally. Thus, the axonal processes of OSNs branched throughout the antennal lobe axons labeled by OR47a-Gal4, which targeted the DM3 glomerulus and formed labeled arborizations in all of the glomeruli (Fig. 6A–D; in the wild-type (n¼12 ALs), mistargeted to the DA1 glomerulus in n¼18 ALs). As expected for the ey-FLP MARCM method, there is 10% of the cases (n¼2/20 ALs) (Fig. 7E and F). Similarly, the axons some variability in the spatial distribution of labeled arbors in labeled by OR33c-Gal4, which targeted the VC1 glomerulus in the different preparations. However, in all cases the intensity of label wild-type (n¼20 ALs), mistargeted to the DA1 glomerulus in in the OSN axon arbors was comparable throughout the antennal approximately 25% of the mutant cases (n¼6/24 ALs)) (Fig. 7G lobe glomeruli. and H). In the remaining two ORGal4 lines, mistargeting of mutant In contrast, strikingly different OSN axonal targeting patterns axons was not observed. The axons labeled by OR67d-Gal4 and by were observed in mutant ey-FLP MARCM experiments (Fig. 6E–H; GR21a-Gal4 innervated the same, appropriate glomeruli in the n¼28 ALs). Three phenotypic features of these mutant targeting wild-type (n¼6 each) and in the mutant (n¼12 and 14 ALs, patterns are noteworthy. First, in the majority of cases observed, respectively) (Fig. 7I–L). the innervation of the DA1 glomerulus was markedly stronger Taken together, these findings demonstrate that Jing is required than that of any other antennal lobe glomerulus. (The wild-type during OSN development in the antennal disc for correct axonal DA1 glomerulus normally receives sensory input from pheromone targeting in the antennal lobe glomeruli. Moreover, they imply detecting OSNs; e.g. Couto et al., 2005; Fishilevich and Vosshall, that this requirement is specific for some, but not all OSN 2005; Datta et al., 2008). Second, labeled arborizations were subclasses. markedly reduced in the other antennal lobe glomeruli, notably in comparison to the intense labeling of the DA1 glomerulus. Third, some axon branches misprojected in the antennal lobe Discussion without apparent targeting to any glomeruli (arrowheads in Fig. 6 F and H). In contrast, no obvious change in the number or overall In this report, we identify the zinc finger transcription factor arrangement of the antennal lobe glomeruli was observed in these Jing as a key transcriptional control element required for dendrite experiments, with the exception of the DA1 glomerulus which and axonal wiring of the developing olfactory sensory system in appeared increased in size when targeted by exceptionally numer- Drosophila. We show that Jing is cell autonomously required for ous mutant OSN axons (e.g. Fig. 6E). These mutant phenotypic appropriate dendrite targeting of PNs and LNs as well as for correct features of OSN targeting could be partially rescued by incorpor- axonal targeting of OSNs in the development of the antennal lobe ating a UAS-Jing (wild-type) transgene into the ey-FLP MARCM circuitry. These findings place Jing among a small number of other experiment (Fig. S4). transcription factors that have been shown to be required both for To assay the effects of jing loss-of-function on axonal targeting the development of the peripheral OSNs and for the development of individual OSN classes as defined by the expression of specific of their central targets, the PNs and LNs, during olfactory circuit OR genes, we combined ey-FLP MARCM-based mutational inacti- formation. Thus, the POU domain transcription factor Acj6, the vation of jing with six different OR-Gal4 driver lines (see homeodomain transcription factor Ems and, as reported here, the Fishilevich and Vosshall, 2005). Aberrant axonal targeting patterns zinc-finger transcription factor Jing are all required in OSNs as well in the antennal lobe were consistently observed for two of the six as in PN/LN lineages. Moreover, all these transcription factors have OR-Gal4 lines. In the wild-type, the axons labeled by OR88a-Gal4 dual roles in peripheral and central olfactory system development

Fig. 7. jing loss-of-function causes specific axonal mistargeting of some, not all, OSN subclasses to DA1 glomerulus. Wild-type and jing mutant OSN clones generated using ey-FLP based MARCM and visualized using six different OR-Gal4 lines driving UAS-mCD8 GFP.(A–D) Or 88a and OR33b OSN axons invariably show specific mistargeting to DA1 (BandD) instead of their wild-type target glomeruli, viz., VA1d (OR88a) and DM3 and DM5 (for OR33b) in the antennal lobe (A and C). (E–H) OR47a and OR33c wild-type OSN axons target the glomeruli DM3 and VC1 respectively (E and G) upon jing loss-of-function, the mutant axons of these two OSN classes show occasional mistargeting of a few axons to DA1 (F and H). White arrowhead in F show a few axon branches misprojecting in the antennal lobe without apparent targeting to any glomeruli. (I–L) OR67d and GR21a OSN jing mutant axons (J and L) innervated the same glomeruli, viz., DA1 and VA6 (OR67d)andV(GR21a) as their wild-type counterparts (I and K), thus indicating that jing is not required for proper axonal targeting of these OSN classes. White arrows point to the DA1 glomerulus. White asterisks indicate the wild-type targets of the respective OSN classes. Anti-GFP (green); anti-Bruchpilot (nc82, red). Scale bar: 30 lm. 10 Indu.S. Nair et al. / Developmental Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ in that they are required for appropriate axonal/dendrite targeting might operate in the formation of olfactory circuitry in insects and of presynaptic/postsynaptic elements (Komiyama et al., 2003, mammals during development. One example for this is repre- 2004; Das et al., 2008; Lichtneckert et al., 2008; Sen et al., 2010; sented by the Ems/Emx family of homeodomain transcription this report). factors. Thus, the ems transcription factor is required for develop- We speculate that these, and other as yet unidentified tran- ment of peripheral and central olfactory neurons in Drosophila, scription factors, could represent a transcription factor code that and the vertebrate homologs of ems, the Emx1/2 genes, are acts in a cell autonomous manner in specifying and interconnect- similarly required for the development of peripheral and central ing the presynaptic sensory neurons to their postsynaptic inter- olfactory neurons in mammals (Bishop et al., 2003; Shinozaki neuronal targets in olfactory system development. A number of et al., 2004; Das et al., 2008; Lichtneckert et al., 2008; Sen et al., other transcription factors that have been shown to be required for 2010). Given the role of Jing in peripheral and central olfactory proper wiring of the olfactory circuit viz., POU domain transcrip- system development in Drosophila, it will now be important to tion factor Drifter, the LIM-homeodomain transcription factors analyse the role of the homologous AEBP2 gene in mammalian Islet and Lim1, the LIM-cofactor Chip, the homeodomain transcrip- olfactory system development. tion factors Cut and Ems, and the zinc finger transcription factors Squeeze and Lola (Komiyama et al., 2003; Komiyama and Luo, 2007; Spletter et al., 2007) could also be components of the Acknowledgements different transcription factor codes or downstream transcriptional programs required for specification and wiring of different subsets We dedicate this paper to the memory of our co-author, of olfactory neurons. Such a transcriptional program could lead to Veronica Rodrigues. This work was supported by core funding the expression of comparable sets of cell surface recognition from TIFR and the Department of Biotechnololgy, Government of molecules (cell adhesion molecules and ligand/receptors) in the India; the Swiss NSF and the Indo-Swiss Joint Research Program. developing pre- and postsynaptic neurons and, hence, facilitate We are grateful to Denise Montell, Joaquim Culi and Tzumin Lee; the formation of appropriate synaptic interconnectivity (Jefferis the Bloomington Drosophila Stock Centre, DSHB for fly stocks and and Hummel, 2006). Cell surface molecules such as Dscam, Robo, reagents. We thank Greeshma Ray for technical help with the N-Cadherin, Semaphorin, Plexin A and leucine-rich repeat trans- deficiency mapping crosses. We are thankful to Ajeet Pratap Singh membrane protein Capricious have been shown to be required for and Sonia Sen for comments on the manuscript. We thank the dendrite/axonal targeting in the antennal lobe (Hummel et al., Department of Science and Technology, Government of India for 2003; Hummel and Zipursky, 2004; Jhaveri et al., 2004; Zhu and the Olympus FV1000 confocal microscopes in the Central Imaging Luo, 2004; Zhu et al., 2006; Komiyama et al., 2007; Lattemann and Flow Cytometry Facility (CIFF) at NCBS. et al., 2007; Sweeney et al., 2007; Hong et al., 2009). An investigation of changes in the expression of these identified cell surface molecules following mutational alteration of the postu- Appendix A. Supporting information lated Jing, Ems, Acj6 transcription factor code in OSNs and PNs/LNs should be both feasible and instructive. Supplementary data associated with this article can be found in Alternatively, given its ubiquitous expression in developing the online version at http://dx.doi.org/10.1016/j.ydbio.2013.06.023. neural cells, the Jing transcription factor may not be an integral part of a putative transcription factor code for neuronal specificity and targeting, but rather be required for maintaining the stable References expression of the more specific transcription factors that consti- tute the postulated code. Evidence for this type of a more general Bishop, K.M., Garel, S., Nakagawa, Y., Rubenstein, J.L., O'Leary, D.D., 2003. Emx1 and role for Jing in the determination of cell fate during development Emx2 cooperate to regulate cortical size, lamination, neuronal differentiation, fi comes from experiments which indicate that Jing together with development of cortical efferents, and thalamocortical path nding. J. Comp. 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