dual role for hox genes and hox co-factors in conferring leg … · tubgal80/frt82b, frt82b...

RESEARCH ARTICLE 2027

Development 140, 2027-2038 (2013) doi:10.1242/dev.090902© 2013. Published by The Company of Biologists Ltd

INTRODUCTIONAnimal locomotion requires coordinated muscle contractions thatare controlled by motoneurons. Motoneurons receive commands inthe CNS and execute these commands by controlling musclecontractions in the periphery. Most animals capable of movementadopt one of two types of locomotion. Undulatory movements,exhibited by Drosophila larvae and Caenorhabditis elegans, requirecoordinated body wall muscle contractions along the dorsoventral(DV) and anteroposterior (AP) axes. By contrast, walking byanimals such as mice and adult Drosophila requires well-coordinated muscle contractions in each leg. Accordingly, walkingdepends on the accurate specification of and connections betweenthe motoneurons, sensory neurons and interneurons that make upthese circuits (Dasen and Jessell, 2009; Arber, 2012).

How the exquisitely stereotyped morphologies exhibited byneurons are established during development is an importantunsolved problem in biology. In insects such as Drosophila, neuronsare derived from neural stem cells, called neuroblasts (NBs). EachNB is located in a stereotyped AP and mediolateral position in theventral nerve cord (VNC) and gives rise to a unique set of progeny(Schmid et al., 1999; Truman et al., 2004). A series of transcriptionfactors expressed at sequential times during the life of each NBprovides temporal information that is integrated with spatialinformation to generate neuronal identities (Pearson and Doe,2004). NBs divide in one of two waves of activity, the first duringembryogenesis (with 30 NBs per thoracic hemisegment) and thesecond during larval stages (with ~25 NBs per thoracichemisegment) (Truman and Bate, 1988; Truman et al., 2004).During the embryonic wave of neurogenesis, ~17 NBs generate ~40MNs, which target 30 larval body wall muscles in each abdominal

hemisegment (Schmid et al., 1999). During the larval wave ofneurogenesis, nine NBs give rise to ~50 MNs, which target 14muscles in each adult leg (Baek and Mann, 2009).

In both vertebrates and invertebrates, the Hox family oftranscription factors has emerged as a group of key players innervous system development. Previous work on non-leg MNDrosophila lineages demonstrated that, depending on the context,Hox genes can regulate both apoptosis and the timing of cell cycleexit of NBs, and apoptosis in post-mitotic neurons. For example, inthe abdomen the Hox gene abdominal A (abd-A) induces NBapoptosis in a subset of lineages that continue to produce progenyin thoracic segments (Bello et al., 2003; Cenci and Gould, 2005;Maurange et al., 2008). Abdominal Hox genes also have the abilityto induce NB cell cycle exit in some abdominal lineages (Karlssonet al., 2010). Finally, the Hox gene Abdominal-B (Abd-B) can blockor promote apoptosis of neurons, depending on the context (Miguel-Aliaga and Thor, 2004; Suska et al., 2011). In each of these cases,Hox genes execute functions that diversify the nervous system alongthe AP axis, analogous to their more classically defined functions inother tissues.

In vertebrates, Hox genes have been shown to be importantregulators of MN identity in the spinal cord (Dasen et al., 2003;Dasen et al., 2005; Jung et al., 2010). In the mouse, Hox6 and Hox10paralogs are needed to define the lateral motor columns (LMCs)present at brachial and lumbar levels, respectively, whereas Hox9paralogs suppress the LMC fate at thoracic levels. Notably, the Hoxaccessory factor encoded by Foxp1 gates Hox functions in a leveldependent manner, depending on the column (Dasen et al., 2008).Within the brachial LMC, cross-regulatory interactions between 11of the 39 mouse Hox genes establish the identities of MN pools,providing them with their muscle-specific targeting properties inthe limb (Dasen et al., 2005). The specification of pool identities byHox genes is apparently distinct from their role in AP patterning,because these cross-regulatory interactions are restricted to limb-forming regions at brachial and lumbar levels of the rostral-caudalaxis.

In addition to walking, each pair of legs in Drosophila executessegment-specific behaviors. For example, the second thoracic (T2)legs are important for flight takeoff, whereas the T1 and T3 legs are

Departments of 1Biological Sciences and 2Biochemistry and Molecular Biophysics,Columbia University, 701 W. 168th Street, New York, NY 10032, USA.

*Current address: Howard Hughes Medical Institute, Smilow Neuroscience Program,Department of Physiology and Neuroscience, New York University School ofMedicine, New York, NY 10016, USA‡Author for correspondence ([email protected])

Accepted 4 March 2013

SUMMARYAdult Drosophila walk using six multi-jointed legs, each controlled by ~50 leg motoneurons (MNs). Although MNs have stereotypedmorphologies, little is known about how they are specified. Here, we describe the function of Hox genes and homothorax (hth),which encodes a Hox co-factor, in Drosophila leg MN development. Removing either Hox or Hth function from a single neuroblast(NB) lineage results in MN apoptosis. A single Hox gene, Antennapedia (Antp), is primarily responsible for MN survival in all threethoracic segments. When cell death is blocked, partially penetrant axon branching errors are observed in Hox mutant MNs. Whensingle MNs are mutant, errors in both dendritic and axon arborizations are observed. Our data also suggest that Antp levels in post-mitotic MNs are important for specifying their identities. Thus, in addition to being essential for survival, Hox and hth are requiredto specify accurate MN morphologies in a level-dependent manner.

KEY WORDS: Drosophila melanogaster, Hox genes, Motoneurons, Cell death and survival, Neuroblasts

Dual role for Hox genes and Hox co-factors in conferring legmotoneuron survival and identity in DrosophilaMyungin Baek1,*, Jonathan Enriquez2 and Richard S. Mann2,‡

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used for grooming the head and abdomen, respectively (Szebenyi,1969; Kaplan and Trout, 1974; Dawkins and Dawkins, 1976;Trimarchi and Schneiderman, 1993). These observations raise thequestion of whether Hox genes play a role in diversifying MNidentities or circuitries between thoracic segments. In the workdescribed here, we provide evidence that during the development ofleg MNs Hox genes execute both AP patterning and segment-independent functions. Newly born leg MNs initially express thesame Hox gene, Antennapedia (Antp), in all three thoracic segments.Genetic analysis demonstrates that Hox genes and the geneencoding the Hox co-factor homothorax (hth) are required for post-mitotic MN survival in all three segments. By blocking cell death inthese mutants and by examining individual mutant MNs, we showthat these genes are not required to specify a leg MN identity or fortheir specific lineage identity. However, these experiments reveal arequirement for Hox genes to specify both axon and dendritemorphologies of these MNs. Interestingly, we also find that withina single NB lineage Antp is present at different levels that correlatewith MN birthdate, and that these levels are important forinstructing axon targeting. Thus, Hox genes are needed for both MNsurvival and for specifying their morphologies in a level-dependentmanner in all three thoracic segments. Later in development, as legMNs differentiate during metamorphosis, their Hox expression codechanges in a segment-specific manner. The unique Hox codeexhibited by pupal stage leg MNs suggests an additional late role forHox genes in diversifying their properties in the different thoracicsegments, perhaps to allow them to execute segment-specificfunctions.

MATERIALS AND METHODSFly stocksUnless otherwise noted, fly stocks were obtained from the BloomingtonDrosophila Stock Center.

yw hs-flp; ; FRT82Byw hs-flp; ; FRT82B tubG80yw hs-flp FRT19Aw hs-flp tubG80 FRT19AScrC1 AntpNS+RC3 UbxMX12 (Struhl, 1982)yw hs-flp; FRT 82B AntpNS+RC3/MKRSyw hs-flp tub-Gal4 UAS-GFPFRT82B Ubx1/TM6BFRT82B hthP2/MKRS (Sun et al., 1995)w hs-flp FRT19A tubGal80vGlut-Gal4 (also called OK371-Gal4) (Mahr and Aberle, 2006)UAS-CD8GFP (on II)UAS-p35 (on II) (from Laura Johnston, Columbia University Medical

Center, NY, USA)vGlut-lexA (C. S. Mendes and R.S.M., unpublished)LexO-rab3YFP (C. S. Mendes and R.S.M., unpublished)UAS-6myc-Antp (M. A. Crickmore and R.S.M., unpublished)UAS-AntpRNAi (Bloomington Drosophila Stock Center, stock number

27675)

ImmunohistochemistryPrimary antibodies were: mouse anti-Abd-B [1:5; Developmental StudiesHybridoma Bank (DSHB)], mouse anti-Ubx (1:20; DSHB), mouse anti-Antp (8C11; DSHB), mouse anti-prospero (DSHB), mouse anti-d-CSP2(1:200; DSHB), rat anti-Elav (1:50; DSHB), mouse anti-Repo (DSHB),rabbit anti-Exd (Mann and Abu-Shaar, 1996), guinea pig anti-Hth (GP52)(Ryoo and Mann, 1999), rat anti-Abd-A (from B. Gebelein, CincinnatiChildren’s Hospital Medical Center, OH, USA), rabbit anti-Scr (CLU395,1:500) (Joshi et al., 2010), rabbit anti-Pb (e9, 1:100) (Cribbs et al., 1992),guinea pig anti-Dfd (1:200; from W. Mcginnis, University California, SanDiego, CA, USA), guinea pig anti-Lab (1:50,000; from B. Olstein,Columbia University Medical Center, NY, USA), guinea pig anti-Deadpan

(1:1000, from J. Skeath, Washington University in St Louis, MO, USA),guinea pig anti-Dll (Estella et al., 2008) and rabbit anti-myc (MolecularProbes). Secondary antibodies were: AlexaFluor488, AlexaFluor555 andAlexaFluor647 conjugates (Molecular Probes).

CNS dissections and immunostaining were carried out as described(Baek and Mann, 2009). Samples were incubated with primary antibodiesfor two days and secondary antibodies for one day at 4°C. PBST (PBS with0.1% Triton X-100, 0.5% BSA and 5% goat serum) was used for theincubation and washing steps. Adult legs attached to bodies were fixedovernight at 4°C and washed three times in PBST for 15 minutes at roomtemperature. CNS and legs were dissected and mounted onto glass slidesusing Vectashield mounting medium (Vector Labs).

Mosaic analysis with a repressible cell marker (MARCM) analysisTo generate MARCM clones, embryos were collected for 12 hours andincubated for 24 hours at 25°C. First instar larvae were transferred to vialsand given heat shocks at appropriate time points. Entire lineage clones weregenerated from heat-shocks at 24-36 hours after egg laying (AEL). Strainsused are listed below.

Standard MARCM clonesyw hs-flp; vGlut-Gal4 UAS-CD8GFP; FRT82B tubGal80/FRT82B,FRT82B ScrC1AntpNs+RC3UbxMX12, FRT82B Antp NS+RC3, FRT82B Ubx1 orFRT82B hthP2.

yw hs-flp/yw hs-flp tub-Gal4 UAS-GFP; vGlut-Gal4 UAS-CD8GFP/+;FRT82B tubGal80/FRT82B, FRT82B ScrC1AntpNs+RC3 UbxMX12, FRT82BAntp NS+RC3, FRT82B Ubx1 or FRT82B hthP2.

Cell death inhibitionyw hs-flp/yw hs-flp tub-Gal4 UAS-GFP; vGlut-Gal4 UAS-CD8GFP/UAS-p35; FRT82B tubGal80/FRT82B, FRT82B ScrC1AntpNs+RC3UbxMX12 orFRT82B hthP2.

Compensatory axonal targetingyw hs-flp; vGlut-Gal4 UAS-CD8GFP/vGlut-LexA lexO-rab3YFP; FRT82BtubGal80/FRT82B, FRT82B ScrC1AntpNs+RC3UbxMX12 or FRT82B hthP2.

Ectopic Antp expression in MARCM clonesyw hs-flp FRT19A/w hs-flp tubGal80 FRT19A; +/vGlut-Gal4 UAS-CD8GFP; +/+ or UAS-6xmycAntp.

Knockdown of Antp levelsvGlut-Gal4, UAS-rab3YFP/+; UAS-AntpRNAi/+.

Microscopy and image analysisConfocal images were taken as described (Baek and Mann, 2009) using thesame confocal settings for each antibody. z-stack maximum merged imageswere generated and, if necessary, non-leg motoneuron clones were removedusing ImageJ.

RESULTSHox gene expression patterns in leg MNsIn Drosophila, there are eight Hox genes: labial (lab),proboscipedia (pb), Deformed (Dfd), Sex combs reduced (Scr),Antp, Ultrabithorax (Ubx), abd-A and Abd-B (McGinnis andKrumlauf, 1992). We examined the expression pattern of theproteins encoded by all eight Hox genes as well as hth andextradenticle (exd), which encode Hox co-factors. As in othertissues (Mann and Morata, 2000), nuclear Exd correlated with thepresence of Hth, so only the pattern of Hth is reported here. Around50 leg MNs are born from nine post-embryonic NB lineages in eachthoracic hemi-segment (Baek and Mann, 2009). In the present study,we focused on LinA (also called Lin 15) (Truman et al., 2004)because it generates the largest number of leg MNs and can bereadily identified by its position in the VNC (Fig. 1A-C). LinA givesrise to ~28 adult leg MNs (Baek and Mann, 2009; Truman et al.,2010). The timing and birth order for LinA MNs are stereotyped:LinA MNs are generated from ~50-90 hours after egg laying (AEL)

RESEARCH ARTICLE Development 140 (9)

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(Baek and Mann, 2009). By late third instar (~120 hours AEL),LinA MN cell bodies reside medially in the VNC and send axonsthat exit the VNC towards the leg imaginal discs through the legnerve (Fig. 1A-C) (Truman et al., 2004). At the pupal stage, LinAleg MN cell bodies move to their final positions, and dendritic andaxonal arborizations begin to elaborate (Fig. 1A-C). In addition, wefound that LinA also gives rise to ~22 glial cells that end upsurrounding each thoracic neuropil in the adult VNC (Fig. 1D; datanot shown).

We found that three Hox proteins, Pb, Antp and Ubx, and bothHox co-factors are expressed in the thoracic segments of the VNC,where leg MN cell bodies are located, at late larval and mid-pupalstages (supplementary material Fig. S1; Fig. S3A). Because thereare no specific markers for the leg MNs, we used mosaic analysiswith a repressible cell marker (MARCM) (Lee and Luo, 1999),combined with the stereotyped position of LinA within the VNC toidentify this lineage unambiguously. At the third larval stage, allLinA MNs in all three thoracic segments expressed Antp(Fig. 2B,C,E).

In the third larval VNC, most NBs, including the LinA NB, arepositioned along the ventral side of the VNC and divide to produce

progeny in the dorsal direction (Fig. 2A). Consequently, earlier-bornneurons are located more dorsally and later-born neurons are locatedmore ventrally, closer to the NB (Maurange et al., 2008).Interestingly, within single LinA MARCM clones, Antp wasdetected more strongly in ventrally located cells compared withdorsally located cells (Fig. 2B,E; supplementary material Fig. S2).Besides Antp, Ubx was the only other Hox protein observed in LinAMNs at the third instar stage: about three dorsally positioned LinAMNs, and only in the T3 segment, expressed Ubx at this stage(Fig. 2D,E). Pb, which is required for the development of the adultproboscis, was not expressed in LinA in the third instar VNC (datanot shown).

The patterns of Hox expression changed by the mid-pupal stage(3-4 days post-pupariation), at a time when both axon targeting anddendritic arbors are elaborating. At this time, Antp was expressedstrongly in all LinA MNs in the T2 segment but was undetectablein LinA MNs in T1 and T3 (Fig. 2G,J). By contrast, Ubx wasobserved in all LinA MNs of the T3 segment but not in T1 or T2(Fig. 2H-J). As in the larval stage, no other Hox proteins weredetected in LinA MNs at the mid-pupal stage. Hth and Exd werepresent in all LinA MNs at both larval and pupal stages

2029RESEARCH ARTICLEHox function in Drosophila MNs

Fig. 1. LinA MNs during development. (A-C) LinA MARCM clones in T2, labeled withvGlut-Gal4 >CD8GFP. (A) Cell bodies anddendrites of LinA MNs at late L3, mid-pupa andadult stages. Leg neuropil region is shadedpink. (B) LinA MARCM clones stained for d-CSP2 (red, labels the neuropil) and Dll (blue,labels the leg imaginal discs). VNCs areoutlined with blue dotted lines. Regions shownin C are marked with white boxes. The pupalstage leg is outlined in pink. (C) LinA MN axonsin the periphery. Shown is the GFP channelfrom the regions boxed in B. Late L3 legimaginal disc, pupa stage leg and adult leg areoutlined in pink. The VNC at the L3 stage isoutlined with blue dotted lines. (D) LinAMARCM clones stained with anti-Repo (red) tomark glia. The left-most image shows a low-magnification view of the VNC (as depicted inthe schematic); the region boxed is shown athigher magnification in the images to theright. About 22 LinA-derived glia are visualized;these surround the neuropil in the adult (datanot shown).

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(supplementary material Fig. S3A). In the adult, these segment-specific Hox expression patterns were maintained (supplementarymaterial Fig. S3C-F). In summary, Hox expression in LinA isdynamic: in late third instar VNCs, all LinA MNs express Antpregardless of the thoracic segment (Fig. 2E). At this stage, the levelsof Antp correlate with the MN birthdate within LinA: older MNshave lower levels of Antp than do younger MNs. Approximately72 hours later, during metamorphosis, LinA MNs in T1 express noHox genes, those in T2 express Antp and those in T3 express Ubx(Fig. 2J).

Hox genes and hth are required for MN survivalTo determine the function of Hox genes and hth in LinA, we usedthe MARCM method to genetically remove their activities from this

lineage. To ensure that we were examining the loss of all thoracicHox function, most experiments used the triple-mutant chromosomeScr Antp Ubx, although, as described below, our key findings wereconfirmed with single Hox mutant chromosomes. Depending on theexperiment, MARCM clones were labeled with mCD8GFP drivenby either vGlut-Gal4, which is a post-mitotic MN driver, or bycombining tub-Gal4, a ubiquitous driver, with vGlut-Gal4. Theubiquitous Gal4 driver was added to ensure that we were monitoringall LinA progeny, not just those that expressed vGlut-Gal4.

In either Scr Antp Ubx or hth mutant clones, the number of LinA-derived neurons (Elav+) was dramatically reduced when examined atthe late third instar larval stage (~11 compared with 35 neurons inwild type) or in the adult [about four (hth) or nine (Scr Antp Ubx)compared with 25 MNs in wild type] (Fig. 3A,C,E,G; supplementary


Fig. 2. Hox expression patterns. (A) Schematic of LinA in the larval VNC. The LinA NB (light blue) is positioned ventrally in the VNC and its progeny(green) extend dorsally. Consequently, earlier born MNs are located more dorsally and later born MNs are located more ventrally. Planes 1 to 5 refer tothe z-stack slices shown in B-D. (F) Schematic of LinA in a mid pupa (3-4 day APF) VNC. LinA MN cell bodies (green) are located ventrally. Planes 1 to 5represent the z-stack slices in G-I. (B-D,G-I) LinA MARCM clones in late L3 (~120 hour AEL; B-D) and at the mid-pupa stage (G-I) showing five z-stackslices from ventral (1) to dorsal (5). Magnified areas are indicated with the yellow arrows of clones in the low-magnification images on the left andregions examined are depicted in the schematics. (B) LinA clones in T1 (top row), T2 (middle row) and T3 (bottom row) stained for Antp (red). LinAclones in T2 and T3 were generated in a single sample. (C) Antp−/− LinA clone in T3 segment during late larval stages stained for Antp (red); the absenceof staining in the clone confirms the specificity of the signal seen in B. (D) LinA clones in T1 and T3 (in a single sample) stained for Ubx (red) and Scr(blue). Approximately three cells in the LinA clone in T3 express Ubx. (G) Antp (red) is expressed strongly in all T2 LinA MNs, but not in T1 or in T3. Antplevels do not correlate with their dorsal-ventral position at this stage. (H) LinA clones in T1 and T3 stained for Ubx (red) and Scr (blue). Ubx is expressedin all LinA MNs in T3. Most MNs express high levels of Ubx. Scr is not expressed in LinA MNs. (I) Ubx−/− LinA clone (green) in T3 segment stained for Ubx;the lack of staining confirms the specificity of the signal seen in H. (E) Summary of Hox expression patterns in LinA MN progeny in late L3. (J) Summaryof Hox expression patterns at the mid-pupa stage. In T2, but not T1 or T3, Antp is expressed in all LinA MNs, mostly at high levels. In T3, Ubx is expressedin all LinA MNs. Hth is expressed in all LinA MNs in all three thoracic segments in both larval and pupal stages but its levels vary (supplementary materialFig. S3A).

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material Fig. S4A). These phenotypes were observed in all threethoracic segments. Because these experiments included the ubiquitousdriver tub-Gal4, they argue against the possibility that MNs are beingtransformed to non-MN cell types in Hox or hth mutants.

Antp single mutant LinA clones also resulted in a reduced numberof MNs in T1 and T2 but, curiously, not in T3 (in the adult, aboutnine LinA MNs were observed in T1 and T2 segments and ~25neurons in T3) (supplementary material Fig. S4A). Ubx mutantLinA clones also did not show a reduction in MN number in T3: inthe adult, ~25 MNs were observed in Ubx mutant LinA clones in allthree segments (supplementary material Fig. S4A). Wehypothesized that the difference in the phenotypes observed in T3for Scr Antp Ubx clones (about nine MNs) and the singly mutantAntp or Ubx clones (~25 MNs) might be due to de-repression of

Antp in Ubx mutant clones in this segment and a functionalredundancy of these two genes for promoting survival. Consistentwith this notion, when examined at the pupal stage, we observedAntp protein in Ubx mutant T3 LinA clones (supplementarymaterial Fig. S4B).

Neuronal apoptosis in Hox and hth mutant LinAclonesThe reduced number of MNs observed in Hox or hth mutant LinAclones could be caused by several mechanisms, which are notmutually exclusive: for example, early NB cell cycle exit, prematureNB cell death, or post-mitotic MN cell death. To distinguishbetween these mechanisms, we first checked whether the LinA NBundergoes premature cell death. At the early third instar larva stage,


Fig. 3. Hox and hth are required to generate the correct number of LinA progeny. (A-F) LinA MARCM clones labeled with tub-Gal4; vGlut-Gal4>CD8GFP (green, outlined with light blue dotted lines), stained for Elav (red, marks mature neurons) and Pros (blue, marks young neurons). Magnifiedareas are indicated by boxes and yellow arrows and the position of clones in the VNC are shown by the red boxes in the schematics on the left. (A) WT,(B) WT +p35, (C) Scr−/−Antp−/−Ubx−/−, (D) Scr−/−Antp−/−Ubx−/− +p35, (E) hthP2, (F) hthP2 +p35. (G,H) Quantification of neurons (G; Elav+) and glia (H;identified by position and the lack of Elav staining; see Fig. 1D) in individual LinA clones at late L3 (~120 hour AEL). Each point represents the cellnumber for a single MARCM clone. Ectopic p35 expression rescued the number of neurons (G) but not the number of glia (H). Compared with wild-typeLinA clones, about ten additional neurons were generated when p35 was expressed, suggesting that apoptosis normally eliminates these cells (Trumanet al., 2010). Elav– (non-glia) cell numbers were not significantly changed in mutant LinA clones [median values: WT (8); WT +p35 (3); Scr−/−Antp−/−Ubx−/−

(4); Scr−/−Antp−/−Ubx−/− +p35 (8); hthP2 (4); hthP2 +p35 (2)]. Black bars represent median values. (See also supplementary material Fig. S4A.)

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NBs, labeled by Deadpan (Dpn) (Lee et al., 2006), were observedin both wild-type and Hox mutant LinA clones (supplementarymaterial Fig. S4C-F). At this time point, the LinA NB wasassociated with a similar number (~18) of neuronal progeny (asassessed by Elav+ staining) in both wild-type and mutant LinAclones (supplementary material Fig. S4C-F). These data argueagainst the idea that either premature LinA NB cell death or earlycell cycle exit account for the lower number of LinA MNs. Todetermine whether apoptosis is responsible for the reduced numberof MNs, we blocked cell death by expressing the caspase inhibitorp35 (Hay et al., 1995). When examined at the late third instar stage,the expression of p35 in Hox or hth mutant LinA clones(simultaneously using tub-Gal4 and vGlut-Gal4) rescued thereduced number of MNs (Fig. 3D,F,G). In addition, wild-type ormutant LinA clones expressing p35 generated a greater number ofneurons than did wild-type LinA clones not expressing p35 (from~35 to 45 cells; Fig. 3B,G). These findings are consistent withprevious observations showing that a subset of LinA progenynormally undergoes apoptosis (Truman et al., 2010). An alternativeexplanation is that the LinA NB, which normally disappears at themid-third instar stage (Truman et al., 2004), is kept alive by p35.When expression of p35 was limited to post-mitotic MNs (by usingonly vGlut-Gal4) we observed only a partial rescue (data notshown), suggesting that both mechanisms might be operational.However, we cannot rule out the possibility that p35 levels are tooslow to accumulate in time to efficiently rescue MN survival, in partowing to perdurance of Gal80 in these MARCM clones.

In the MARCM experiments using tub-Gal4, which labels bothMNs and glia, we found that the wild-type LinA NB also gives riseto ~22 glial cells (as assessed by anti-Repo staining) that end up

surrounding the leg neuropil (Fig. 1D; data not shown). As withMNs, the number of glia was strongly reduced in Hox or hth mutantLinA clones (Fig. 3H). Surprisingly, however, although ectopic p35expression in mutant LinA clones rescued the number of MNs, thenumber of glia was not rescued (Fig. 3H), arguing that apoptosis isnot the cause of the reduced number of glia in Hox or hth mutantLinA clones. With the exception of very low levels of Antp, we alsodid not detect expression of Hox genes, hth or exd in LinA-derivedglia (supplementary material Fig. S3B). Taken together, these datasuggest that Hox and Hox co-factors might be required in the LinANB to specify the glia cell fate.

Defects in axon targeting of Hox and hth mutantleg MNsTo determine whether, in addition to survival, Hox genes and hth arerequired for MN identities, we examined both axon targeting anddendritic arbors of mutant leg MN clones. Wild-type LinA MNsmainly target the femur and tibia and have a complex dendriticmorphology in the neuropil (Fig. 4A) (Baek and Mann, 2009;Brierley et al., 2009; Brown and Truman, 2009). In either Hox (ScrAntp Ubx) or hth mutant LinA clones, the surviving MN axonstargeted the same region of the leg as did wild-type LinA clones.Compared with wild-type clones, fewer branches were observed,consistent with the fewer number of cells (Fig. 4A shows examplesin T2; see supplementary material Fig. S5A,B for examples in T1and T3). The distal region of the tibia was most highly affected;other axon branching defects appeared to be randomly distributedwithin the LinA-targeted region of the leg (summarized insupplementary material Fig. S5D). Although axon branching errorswere not observed in Ubx mutant LinA clones, Antp mutant LinA


Fig. 4. Axon targeting and dendritic defects in Hox andhth mutant LinA MNs. All panels show axons in adult T2 legsand dendrites in the VNC of LinA MARCM clones. In the VNC,the leg neuropil and midline are marked by blue dottedcircles and red dotted lines, respectively. See supplementarymaterial Fig. S5A,B for examples in T1 and T3, andsupplementary material Fig. S5D for a summary. (A) LinA MNsprojecting to T2 legs. Axon targeting defects are marked withred lines. Wild-type and Hox mutant LinA clones have thindendrites that cross the midline (blue arrows); these aremissing in hth LinA clones (red arrow). (B) LinA MNsexpressing p35 projecting to T2 legs. Ectopic expression ofp35 partially rescues the axon targeting phenotypes for bothHox and hth mutants. hth mutant LinA clones lack dendritescrossing the midline (red arrow).

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clones displayed targeting defects in all three segments, includingT3, despite no affect on MN cell number in this segment(supplementary material Fig. S5C). These results argue that Hoxgenes, and Antp in particular, play a crucial role in specifying MNmorphologies in addition to maintaining their survival. Althoughthe dendritic phenotype is more difficult to assess owing to the largenumber of MNs in a LinA clone, some defects could be observed.Most clearly, we found that a small number of midline-crossingdendrites were absent in hth mutant LinA clones, in all threethoracic segments (Fig. 4A; supplementary material Fig. S5A,B).This phenotype was not observed in Hox mutant clones (Fig. 4A;supplementary material Fig. S5A,B).

To explore further the idea that Hox genes are required forconferring MN identities, we examined the phenotypes of Hoxmutant LinA-derived MNs in which survival was rescued by theexpression of p35. p35 expression in otherwise wild-type LinAclones generated MNs with axon-targeting properties very similarto those of wild-type LinA clones (Fig. 4B). Hox mutant axons thatwere rescued by p35 also targeted the same region along the

proximodistal axis of the leg as did wild-type LinA MNs (Fig. 4Bfor examples in T2; supplementary material Fig. S5A,B forexamples in T1 and T3). However, most samples exhibited sometargeting defects (summarized in supplementary material Fig. S5D).These errors were not limited to one region of the LinA-targetedleg and typically consisted of an absence of terminal arborizations.In the neuropil, hth; p35+ clones exhibited a similar midline-crossing defect as seen in hth clones not rescued by p35 (Fig. 4B;supplementary material Fig. S5A,B). Together, these datademonstrate that in the absence of Hox or hth inputs LinA MNs arenot appropriately specified, even when death is blocked. However,they also show that in this lineage Hox genes and hth are notrequired for forming leg MNs and for specifying a LinA identity.

Removing Hox functions from individual MNsresults in axon- and dendritic-patterning errorsThe experiments described above examined entire LinA clones,each labeling >25 MNs. Consequently, these experiments do nothave cellular resolution. To examine the role of Hox genes and hth


Fig. 5. Aberrant axon branching and dendriticmorphologies in individual Hox or hth mutantMNs. Examples of single-cell MARCM clones. Foreach clone, both axon and dendrites are shown.The neuropil and midline are marked by bluedotted circles and red dotted lines, respectively.(A) Removing Hox and hth functions results indistinct phenotypes. Dendritic and axon defectsare indicated by blue arrowheads and red lines,respectively. Coxa- and femur-targeting MNs arefrom LinB; the tibia-targeting MN is from LinA.(B,C) Examples of mutant single MNs in whichthere is a mismatch between axon and dendritemorphologies. The left-hand images show wild-type MNs, each outlined in red or blue. The right-hand images show Hox (B) or hth (C) mutant MNsthat share either the axon or dendritemorphologies with one of the two wild-type MNsas indicated. (See also supplementary materialTable S1.)

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in individual LinA MNs, we changed the MARCM protocol togenerate individual MNs that are mutant for these genes (Baek andMann, 2009). Altogether, we were able to recover about two-thirdsof the MNs generated by LinA, suggesting that most LinA MNs cansurvive when the functions of these genes are removed at this lastcell division. Moreover, more than half of these mutant MNsshowed an aberrant morphology, although with variable penetrance(supplementary material Table S1). For many Hox or hth mutantMNs, dendritic or axonal arborization defects were observed,although the requirement for these factors often differed (Fig. 5;supplementary material Table S1). For example, a coxa-targetedMN had ectopic dendrites when mutant for hth, but not Hox(Fig. 5A, top row), whereas a tibia-targeted MN had ectopicdendrites when mutant for Hox, but not hth (Fig. 5A, bottom row).In both of these examples, axon branching appeared normal. Bycontrast, a femur-targeted MN had fewer axon branches when eitherHox or hth functions were removed, but ectopic dendrites were onlyobserved when the MN was mutant for hth (Fig. 5A, middle row).From these data it appears that individual MNs often have a uniquerequirement for hth and Hox functions.

In most cases, individual MNs mutant for hth or Hox exhibiteddefective, as opposed to transformed, dendritic morphologies andaxon branches. However, in two examples (out of 125 total singleMN clones), we observed MNs that appeared as though they werehybrids of two different MNs. In these cases, the mutant MNs haddendritic and axon morphologies that are usually observed in differentwild-type MNs, suggesting that the identities of these MNs werepartially transformed (Fig. 5B,C). For example, Fig. 5B shows a Hoxmutant MN that targets the distal femur but has a dendriticmorphology normally found in a MN that targets the proximal tibia.Fig. 5C shows an hth mutant MN that targets the distal femur but hasa dendritic morphology normally found in a MN that targets theproximal tibia. Although they are rare, these examples suggest thatseparable genetic programs control dendritic and axonalmorphologies, and that Hox and hth often execute distinct functionsat the single-cell level.

Different levels of Antp are instructive for MNmorphologiesOne of the intriguing observations described above is that Antplevels vary among LinA progeny such that younger MNs express

higher levels than older MNs (Fig. 2B; supplementary material Fig.S2). In our previous analysis of this lineage (Baek and Mann, 2009),we found that older MNs from LinA had a tendency to target theproximal femur and proximal tibia, whereas younger MNs targetedthe distal regions of these same segments. To test whether Antplevels play a role in this targeting bias, we generated LinA MARCMclones in which Antp was ectopically expressed via the post-mitoticdriver vGlut-Gal4. This manipulation did not affect MN survival orfinal cell number (data not shown). However, we found that therewas a clear reduction of axon branches in the proximal femur andincreased branching in the distal femur (Fig. 6; supplementarymaterial Fig. S6). Weaker effects in the same direction wereobserved in the tibia. In a separate experiment, we knocked downAntp levels in all post-mitotic MNs by driving Antp-RNAi usingvGlut-Gal4 and observed a distal-to-proximal shift in axon targeting(supplementary material Fig. S7). Together, these observationssuggest that high levels of Antp, normally present in younger LinAprogeny, promote the identity of MNs that target the distal regionsof the femur and tibia. Conversely, MNs born earlier in the lineagetarget the proximal regions of these segments, in part owing to lowerlevels of Antp.

No compensatory targeting from MNs from otherlineagesIn all of the experiments described above, manipulating the activityof Hox or hth did not affect the MN or LinA identity of progenycells. For example, cells derived from Hox mutant LinA and rescuedby p35 expression remained MNs and targeted the same region ofthe leg as did wild-type LinA MNs. These observations suggest thatthe identities of progeny MNs are hardwired according to the NBlineage from which they are derived. As an additional test of thisidea, we asked whether MNs derived from other lineages might beable to take the place of MNs that die in the absence of Hox or hth.To answer this question, we generated positively labeled Hox or hthmutant NB clones in a background in which all MNs were labeledby the expression of rab3-YFP (Zhang et al., 2007). In thisbackground, we generated Hox and hth mutant LinA and LinBMARCM clones, labeled by mCD8GFP driven by vGlut-Gal4(Fig. 7A). Axons and neuromuscular junctions (NMJs) in mutantMN clones were compared with those in wild-type MNs. For bothLinA and LinB, legs innervated by either Hox or hth mutant clones


Fig. 6. The levels of Antp are instructive for MNtargeting. (A) Antp levels are highest in late-bornLinA progeny and lowest in early-born LinAprogeny, and early-born MNs target proximalregions of the leg segments, whereas late-born MNstarget distal regions (Baek and Mann, 2009). (B) Examples in T2 (left) and T3 (right) of LinA MNsexpressing either GFP (top row) or Antp (bottomrow) via the vGlut-Gal4 driver. Pink and blue boxeshighlight the proximal femur (PF) and distal femur(DF), which show a decrease and increase inbranching, respectively. Defective midline-crossingdendrites are marked by light-blue arrowheads. (Seealso supplementary material Fig. S6.)

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had regions that remained unlabeled by both rab3-YFP and GFP(Fig. 7B,C). We conclude that, despite the lower number of MNsproduced by these mutant lineages, there was no compensatorytargeting by wild-type MNs from wild-type non-LinA lineages.Thus, even when many muscle targets are available, leg MNsapparently maintain their lineage-derived identity.

DISCUSSIONIn addition to their well-documented role in specifying differencesalong the AP axis during animal development (McGinnis andKrumlauf, 1992), Hox genes also set up differences along the APaxis within the nervous system. In Drosophila, some NB lineagesproduce more or less progeny depending on where they are alongthe AP axis and, therefore, which Hox gene they express (Bello etal., 2003; Maurange et al., 2008). In the vertebrate nervous system,sub-regions of the hindbrain, known as rhombomeres, obtain theiridentities as a result of which Hox genes are expressed (McGinnisand Krumlauf, 1992). And, more recently, Hoxc9 has been shown

to play a crucial role in specifying thoracic as opposed to limb-innervating motoneurons in the mouse spinal cord (Jung et al.,2010). In the work described here, we present evidence for a Hoxfunction that is independent of AP patterning: the survival of post-mitotic MNs in the Drosophila thorax. Interestingly, in all threethoracic segments this function appears to be carried out primarilyby Antp, although in T3 either Antp or Ubx can apparently executethis function. Moreover, for MN survival, similar (though notidentical) phenotypes were observed for Hox and hth mutant LinAclones, suggesting that for this function, Hox and hth might functiontogether to regulate the same target genes, as they do in othercircumstances. Because our experiments depend on clonal analysis,our results also demonstrate that Hox genes are requiredautonomously within a single NB lineage for keeping motoneuronsalive. Although this could represent a cell-autonomous functionwithin individual MNs, we cannot exclude the possibility that thereduced number of LinA-derived glia in mutant LinA clonescontributes to the poor survival of MNs.


Fig. 7. No compensatory targeting inlegs with Hox or hth mutant LinAclones. (A) Experimental design. In thisexperiment, Hox or hth mutant (M*) LinA MARCM clones (labeled by vGlut-Gal4; UAS-CD8GFP) were generated in abackground in which all MNs werelabeled by vGlut-LexA; LexO-rab3YFP.(B,C) Examples of Hox mutant clones(B) and hth clones (C) in LinA and LinB.For comparison, the wild-type (WT)contralateral legs from the sameanimals are shown on the right.Targeting defects are indicated by redlines and are still observed when allMNs are visualized (YFP+GFP).

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In vertebrates, MN survival is typically dependent upon thereception of and retrograde signaling by neurotrophic factorsderived from peripheral tissues (Zweifel et al., 2005; Kanning et al.,2010). By contrast, in Drosophila and other insects, MN survival isindependent of the target muscle (Goodman and Bate, 1981;Whitington et al., 1982; Nässel et al., 1986; deLapeyrière andHenderson, 1997). Nevertheless, a family of neurotrophins thatmediate neuronal survival has been identified in Drosophila thatacts through Toll-like receptors (Zhu et al., 2008). Based on ourresults, we speculate that Hox and hth are required for the receptionor production of these neurotrophins in LinA.

Although the number of MNs that die in these mutantbackgrounds was consistent from sample to sample, the neuronsthat were affected were apparently randomly distributed within thelineage. One possible explanation for this observation is that withoutHox or hth inputs the identities of surviving MNs becomerandomized, resulting in random targeting. However, our single-cellclone analysis argues against this idea, as MN identities remainedlargely intact despite exhibiting aberrant morphologies.Alternatively, we favor the idea that removing Hox or hth results inpartial failure in the reception of survival factors, resulting instochastic and partially penetrant cell death among all the MNswithin the lineage.

MN identities in the absence of Hox inputIn addition to promoting MN survival, our data also reveal arequirement for Hox gene functions in the specification ofmotoneuron morphologies. When entire LinA lineages were mutantfor either Hox or hth, and MN survival rescued by p35, LinA MNstargeted the same positions along the proximodistal (PD) axis as didwild-type LinA MNs; axons targeting to ectopic positions along theleg were not observed. These observations suggest that Hox or hthinputs are not required for this level of MN specification; the cellsremain MNs and their ‘LinA identity’ remains intact. However,within the correctly targeted region, mutant LinA MNs exhibitedfrequent axon-targeting defects. These defects were not limited to aspecific subregion of the LinA-targeted domain, suggesting that nospecific subset of LinA MNs has an absolute requirement for thesefunctions.

The maintenance of a LinA identity by Hox or hth mutantlineages is reminiscent of the observation in vertebrates that thetargeting of limb-level MNs appears superficially normal in theFoxP1 mutant (Dasen et al., 2008; Rousso et al., 2008). In this case,targeting defects were only revealed when individual MN poolswere labeled by backfilling from specific muscles. Our single-cellmutant analysis argues against a similar scrambling of identitywithin LinA occurring in Hox or hth mutant MNs. Out of 87individual Hox mutant LinA neurons that were analyzed (distributedacross all three thoracic segments), 55 had a non-wild-typemorphology. However, despite clear defects, these MNs did notappear to have a transformed identity. Instead, most mutant MNscontinued to approach the same muscle target and either exhibitedan aberrant dendritic (44/87) or axon branching (18/87)morphology. Moreover, for many LinA MNs that showed aberrantphenotypes when mutant, the same MNs had wild-typemorphologies in other samples (supplementary material Table S1).One explanation to account for these partially penetrant phenotypesis that they result from the perdurance of the Hox or hth geneproducts following clone induction. It is also feasible that removingonly a single component from potentially large transcription factorcomplexes has only a limited effect because of multiple, partiallyredundant inputs. Answering this question will ultimately require

the identification and characterization of target genes that aredirectly regulated by Hox and hth in developing MNs.

The differences between the role of Hox genes in Drosophilacompared with their role in specifying MN identities in vertebratesmight be a consequence of the additional requirement forestablishing motoneuron pools in vertebrates, which, for forelimbMNs, depends on at least 11 different Hox gene products (Dasen etal., 2005). By contrast, the relatively lower number of motoneuronsinnervating the muscles of the Drosophila leg might not requiresuch a complex combinatorial transcription factor code. Wespeculate that the expansion of Hox gene inputs governing MNdevelopment in vertebrates, itself a consequence of a large increasein Hox gene number, might have enabled the evolution of morecomplex limbs and musculature.

Different requirements for Hox and Hox co-factorinputsAs noted above, we found that both Hox and hth inputs are similarlyrequired for MN survival, suggesting that these factors mightfunction as co-regulators of the same target genes, as has beenobserved for other Hox functions (Rieckhof et al., 1997; Mann andAffolter, 1998). However, other phenotypes were different whenHox or hth functions were removed (Figs 4, 5). For example, for afemur-targeting MN we found that Hox inputs were required foraccurate axonal targeting, whereas hth was required to block theaberrant growth of dendrites towards the midline in the CNS(Fig. 5). By contrast, for a tibia MN, we found that Hox functionswere required to block aberrant dendrite growth, whereas hthappeared to play no role in either dendrite or axon morphology(Fig. 5). In a third example, hth was required for a wild-typedendritic pattern of a coxa-targeting MN, whereas Hox functionsapparently played no role in this MN (Fig. 5). Taken together, thesedata support the idea that independent genetic programs controldendritic patterning and axonal patterning. In addition, for theseaspects of MN morphology it appears that Hox proteins do not useHth (and probably also not Exd) as a co-factor. Instead, these datasuggest that Hox and hth are regulating different sets of target genesthat independently affect dendritic or axonal targeting, depending onthe MN. Previous work suggests that, for specifying dendritemorphologies, one pathway that may be targeted is the Robo/Slitpathway (Brierley et al., 2009).

Antp levels as a timing mechanismThe progeny of individual NBs in Drosophila differ in part owingto their birthdates within their respective lineages. For several NBlineages, this temporal information is controlled by a series ofsequentially expressed transcription factors (Pearson and Doe,2004). Here, we provide evidence that another mechanism todiversify cell fates within a lineage is by varying the levels of theHox transcription factor Antp. The experiments supporting thisconclusion were clearest when we ectopically expressed Antp inpost-mitotic progeny using the vGlut-Gal4 driver. In theseexperiments, we observed a clear shift from MNs targeting theproximal femur, which are born early in the lineage, to distallytargeting MNs, which are born later in the lineage. Conversely,knockdown of Antp levels in post-mitotic MNs by RNAi resultedin a distal-to-proximal shift in axon targeting. In contrast to theseknockdown experiments, when we eliminate Hox gene expressionusing null alleles we found that both distally and proximallytargeting MNs were affected. Based on these findings, we concludethat low levels of Antp results in a phenotype that is distinct fromthat observed in the absence of Antp. The data suggest that low Antp


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levels (normally present in early-born MNs) instruct MNs to targetdistal muscles, whereas MNs with no Antp show neither a proximalnor a distal bias in their axon targeting.

Although to our knowledge this mechanism has not beenpreviously observed for a Hox protein, the levels of the BTB/zincfinger transcription factor Chinmo are instructive in NB lineages inthe Drosophila mushroom body (Zhu et al., 2006). In this case,Chinmo levels are controlled translationally, by sequences in the5�UTR of its mRNA. Although such a mechanism may account forvarying Antp levels in LinA, it is also possible that Antp iscontinuously expressed in the LinA NB, but not in its progeny,resulting in its gradual decline in post-mitotic neurons. Level-dependent functions have also been observed for the Hox accessoryprotein FoxP1 in vertebrate MNs and for Hth in Drosophilaembryonic lineages (Dasen et al., 2008; Karlsson et al., 2010).However, these two cases are distinct from the Chinmo and Antpexamples, which influence cell fates within individual NB lineages.By contrast, in the case of FoxP1, its levels are important forestablishing differences between MN columns: FoxP1 levels arehigh in limb-level LMCs, but low in intervening MNs that projectto thoracic body wall muscles. In the case of Hth, an abrupt increasein levels during embryogenesis is important for accurate generegulation in the progeny of NB 5-6 in the Drosophila thorax.Nevertheless, the findings presented here reinforce the idea thattranscription factor levels play an important role in their function,and in conferring temporal information to NB lineages.

AcknowledgementsWe thank G. Struhl, J. Skeath, C. Doe, L. Johnston, B. Ohlstein, W. McGinnis,B. Gebelein, B. McCabe, C. Mendes, Hybridoma Bank, BloomingtonDrosophila Stock Center for materials; and B. Noro, W. Grueber and J. Dasenfor comments on the manuscript.

FundingM.B was partially supported by a Gatsby studentship (The Gatsby Initiative inBrain Circuitry). The project was funded by grants from Project A.L.S [awardedto R.S.M.]; and the National Institutes of Health [grant number R01NS070644to R.S.M.]. Deposited in PMC for release after 12 months.

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.090902/-/DC1

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