Neurosecretory Identity Conferred bythe apterous Gene: Lateral Horn

Leucokinin Neurons in Drosophila

PILAR HERRERO,* MARTA MAGARINOS, LAURA TORROJA,

AND INMACULADA CANAL

Departamento de Biologıa, Fisiologıa Animal, Universidad Autonoma de Madrid,28049 Madrid, Spain

ABSTRACTThe LIM-HD protein Apterous has been shown to regulate expression of the FMRFamide

neuropeptide in Drosophila neurons (Benveniste et al. [1998] Development 125:4757–4765).To test whether Apterous has a broader role in controlling neurosecretory identity, weanalyzed the expression of several neuropeptides in apterous (ap) mutants. We show thatApterous is necessary for expression of the Leucokinin neuropeptide in a pair of brainneurons located in the lateral horn region of the protocerebrum (LHLK neurons). ap nullmutants are depleted of Leucokinin in these cells, whereas hypomorphic mutants showreduced Leucokinin expression. Other Leucokinin-containing neurons are not affected bymutations in ap gene. Co-expression of apterous and Leucokinin is observed exclusively in theLHLK neurons, from larval stages to adulthood. Rescue assays performed in null ap mutants,by expressing Apterous protein under apGAL4 and elavGAL4 drivers, demonstrate therecovery of Leucokinin in the LHLK neurons. These results reinforce the emerging role of theLIM-HD proteins in determining neuronal identity. They also clarify the neuroendocrinephenotype of apterous mutants. J. Comp. Neurol. 457:123–132, 2003. © 2003 Wiley-Liss, Inc.

Indexing terms: LIM homeodomain; neuropeptide regulation; lateral protocerebrum

The LIM homeodomain (LIM-HD) genes encode a familyof transcription factors that participate in a wide varietyof developmental processes (Dawid et al., 1998). All theknown LIM-HD genes have expression domains in thenervous system, and functional studies indicate thatmany LIM-HD genes can confer specific neural identitiesand regulate axonal pathway formation (Dawid et al.,1998; Hobert and Westphal, 2000).

The Drosophila apterous (ap) gene is a member of theLIM-HD family. It is involved in diverse developmentalprocesses such as wing and muscle development, axonguidance, and neurosecretory identity (Hobert and West-phal, 2000). In addition, ap mutants are deficient in juve-nile hormone, which has been associated with their steril-ity and low female sexual receptivity (Altaratz et al.,1991). So far, apterous contribution to neuropeptide choicehas been restricted to the direct regulation of the FMRF-amide gene exclusively in the Tv-cells (Benveniste et al.,1998). It is not known whether Apterous also affects neu-ropeptide selection in other neuronal cell types. However,the infertile and uncoordinated phenotype of ap mutantadults suggests a broader involvement of Apterous in de-termining neurosecretory identity. To address this issue,

we assayed several neuropeptide antisera in the search forabsent neuropeptide expression in ap mutants. We findthat one brain cell type is depleted in Leucokinin in apnull mutants.

Leucokinins (LKs) were first isolated from the cock-roach Leucophaea maderae (Holman et al., 1986a,b). Theyconstitute a neuropeptide family of amidated octapeptidesthat induce contractions in the hindgut muscle of someinsects and stimulate secretion from the malpighian tu-bules in all insect species studied so far (Chen et al., 1994,Nassel et al., 1992, Veenstra et al., 1997). Besides these

Grant sponsor: Spanish Ministry of Science and Technology; Grant num-bers: DGES PB96-0081 and MCYT BFI 2000-0304; Grant sponsor: Comu-nidad Autonoma de Madrid; Grant number: CAM 07B/0013/1998.

*Correspondence to: Pilar Herrero, Departamento de Biologıa, FisiologıaAnimal, Universidad Autonoma de Madrid, 28049 Madrid, Spain.E-mail: pilar.herrero@uam.es

Received 29 March 2002; Revised 30 July 2002; Accepted 20 September2002

DOI 10.1002/cne.10555Published online the week of January 20, 2003 in Wiley InterScience

(www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 457:123–132 (2003)

© 2003 WILEY-LISS, INC.

functions, additional roles for LK in cardioregulation andrespiration have been proposed, based on the projectionsthat the abdominal ganglial LK-containing neurons sendto the heart, the abdominal spiracles, and the neurohemalregions of the abdominal wall (Cantera and Nassel, 1992).Nothing is known about its central neurotransmitting/neuromodulatory role in brain interneurons.

In this study, we have identified one pair of neurons inthe Drosophila brain that express LK and are regulatedby the LIM homeoprotein Apterous. We describe and an-alyze the development of the brain LK neurons from lateembryogenesis to adulthood. We show that LK-containingDrosophila brain cells form extensive arborizations in the

protocerebrum, particularly in areas surrounding the pe-duncle of the mushroom bodies. Our findings contribute tothe understanding of the role of LIM-HD proteins in es-tablishing neuronal identity in a highly cell-type-specificfashion. It also further defines the apterous pleiotropicneuroendocrine phenotype.

MATERIALS AND METHODS

Drosophila melanogaster strains

Wild-type Oregon flies served as a control strain in allexperiments. We used apUGO35 (Rincon-Limas et al.,

Fig. 1. Tachykinin and pigment dispersing factor (PDF) expres-sion in wild-type and apterous third-instar larval CNS. A,B: LarvalCNS whole-mount showing the complete pattern of Tachykinin ex-pression in wild-type (A) and in apUGO mutant (B). A strong signal isseen in five pairs of small brain cells and two pairs of large brain cellsin both larvae. C,D: Larval CNS whole-mount showing the complete

pattern of PDF expression in wild-type (C) and apUGO mutant (D).Two pairs of brain cells that project into the anterior medial region,two subesophageal neurons, and a small cluster of cells in the poste-rior end of the abdominal ganglion can be seen in both CNSs. Scalebar � 100 �m in D (applies to A–D).

124 P. HERRERO ET AL.

2000), apP44 (Bourgouin et al., 1992), and ap-GAL4MD544

(named apGAL4), apGAL4 insertion in the ap locus (Callejaet al., 1996), as ap null alleles. The hypomorphic ap allelesincluded the spontaneous mutants ap4and ap56f (Ringo etal., 1991), and aprk568, an enhancer detector line thatexpresses lacZ in the nuclei of ap expressing cells (Cohenet al., 1992).

For rescue experiments, we used the following GAL4lines: c155 (hereafter referred to as elavGAL4), an en-hancer trap line in the elav locus that is expressed inall postmitotic neurons (Lin and Goodman, 1994); ap-GAL4MD544, expressed in the ap-expressing domains(Calleja et al., 1996); and VNC-GAL4, expressed in asmall subset of ap-expressing neurons (Rincon-Limas etal., 1999). The UAS-ap line encoding the wild-typeApterous protein has been described in Rincon-Limaset al. (1999). The UAS:super bright GREEN FLUORES-CENT PROTEIN (sbGFP) line was used to label theGal4-expressing neurons in colocalization experiments.

The c503GAL4 line (kindly sent by V. Budnick) wasused as a marker for mushroom bodies.

Immunocytochemistry

The only Leucokinin neuropeptide known in Drosophilamelanogaster, Drosophila leucokinin (DLK), was isolatedwith an antibody prepared against the Leucophaea mad-erae Leucokinin I (DPAFNSWGamide). This antibodyshows broad specificity for the LK family of neuropeptides(Nassel and Lundquist, 1991; Terhzaz et al.,1999). Never-theless, because the known Drosophila DLK (NSVVL-GKKQRFHSWGamide) has greater homology with leu-cokinin IV from Leucophaea (DASFHSWGamide), weused the anti-LK IV for this study. The similarities be-tween the patterns of staining obtained with the twoanti-LK antibodies (see Results) indicate that the anti-LKIV used in this study recognizes the real DLK.

For whole-mount immunocytochemistry, Drosophilacentral nervous systems (CNSs) of larvae (first, second,and third instar) and adults were fixed for 30–60 minutesin 4% paraformaldehyde in PBT (PBS containing 0.3%Triton X-100 , pH 7.4) at room temperature. After several30-minute washes and a methanol immersion at 4°C for15 minutes, CNSs were blocked in 3% normal goat serum

Fig. 2. Leucokinin expression in wild-type third-instar larvalCNS. A: CNS whole-mount showing the complete pattern of LK ex-pression. B: Detail of a lateral horn Leucokinin (LHLK) cell in ananterodorsal position in the brain lobe. The image is a reconstructionmontage from different focal planes of the same brain. C,D: Relativeposition of an LHLK neuron (red) with respect to the mushroom body(MB; green) in a lateral (C) and frontal (D) view. Arrowheads point to

the LHLK neuronal soma. Note the LK varicosities surrounding theMB peduncle (p) and MB calyx (ca). MB are visualized as the result ofc503Gal4;UASGFP expression. Kc, Kenyon cell; se, subesophagealneuron; ab, abdominal neuron. In A and B, anterior is on the left, andin C and D, anterior is at the top. Scale bars � 100 �m in A; 25 �min B–D.

125APTEROUS REGULATES LK EXPRESSION IN FLY BRAIN NEURONS

(NGS) for 1 hour and incubated overnight at 4°C in pri-mary antibody diluted in PBT (pH 7.4). The primary an-tibodies used in this study were as follows: rabbit anti-LKIV (Chen et al., 1994) at 1:1,000; rabbit anti-Tachykinin,raised against Drosophila Tachykinin (Siviter et al.,2000), at 1:6,000; rabbit anti-FMRFamide (BioTrend, Ger-many) at 1:2,000; and rat anti-PDF, raised against Dro-sophila PDF (Park et al., 2000), at 1:500. Followingwashes in PBT, secondary antibody incubations were per-formed at room temperature for 1.5 hours. Secondary an-tibodies were conjugated with fluorescein isothiocyanate(FITC), Texas-Red (TxR) (Jackson ImmunoResearch,West Grove, PA), or horseradish peroxidase (HRP) and

used at 1:250. CNSs incubated with HRP-conjugated sec-ondary antibody were developed with diaminobenzidineDAB (Sigma, St. Louis, MO), dehydrated, and mounted inDEPEX (Merck, West Point, PA). Tissues prepared forfluorescence microscopy were cleared and mounted inglycerol-propylgallate.

For double fluorescent labeling, whole-mount CNSswere first processed using one primary and TxR-labeledsecondary antibodies. After several washes in PBT, la-beled CNSs were incubated with the second primary andFITC-labeled secondary antibodies.

Microscopy and imaging

For confocal imaging, we used a Bio-Rad (Hercules, CA)Confocal Radiance 2000 Microscope and Lasersharp2000 v.4 software. The Confocal Assistant program wasused for Z-series projection and to merge images fromdouble-labeled CNSs.

For optical microscopy we used a Leica Wild MPS52microscope. Photos were taken with an adapted Leicacamera, developed, and scanned for digitalization. Figureswere generated using Adobe Photoshop v. 4.

RESULTS

Apterous function in regulation of FMRFamide genetranscription (Benveniste et al., 1998) might reflect a

Fig. 3. Leucokinin expression in wild-type adult CNS. A: Brainwhole-mount showing one pair of lateral horn leucokinin (LHLK) cellsand one pair of subesophageal LK neurons (se). B: Ventral gangliashowing the abdominal LK neurons (ab). Dorsal axonal varicositiesfrom these neurons can be observed. C,D: Higher magnification im-ages and schematic drawings showing the LHLK neuron and itsprojections, from a ventral (C) and anterior view (D). Two main

collateral branches can be observed in the two views. In C, arboriza-tions are seen in the medium protocerebrum surrounding the pedun-cle (p) of the MB, which is presented in a transverse optical section.Images are reconstructions made from different focal planes of thesame brain; drawings were made with a camera lucida. In A and C,anterior is at the top, in B, anterior is on the left, and in D, arrowlabeled d indicates dorsal. Scale bars � 100 �m in A,B; 25 �m in C,D.

TABLE 1. Leucokinin Immunoreactivity in LHLK Neuronsin Wild-Type and ap Mutants1

Adult Larvae Total %

Wild type 90/92 44/44 98.5apUGO35/apUGO35 0/40 0/50 0apP44/apP44 0/20 0/18 0apGAL4/apGAL4 0/24 0/12 0aprk568/aprk568 14/20 6/14 58.8ap56f/ap56f 35/36 20/20 98.2ap4/ap4 16/38 8/12 48

1The scores represent the number of lateral horn leucokinin (LHLK) neurons withanti-LK immunoreactivity relative to the total number of expected LHLK cells (two perbrain).

126 P. HERRERO ET AL.

broader role for this protein in the control of neuropeptideexpression. To address this possibility, we analyzed theexpression of the neuropeptides PDF, Tachykinin, andLeucokinin (LK) in apterous mutants. Mutations in theapterous gene did not alter the pattern of anti-PDF (Parket al., 2000) and anti-Tachykinin (Siviter et al., 2000)

immunoreactivity (Fig. 1). However, one pair of LK-expressing neurons seemed to be absent in apterous nullmutants. As LK expression in Drosophila has only beenstudied in larval stages (Cantera and Nassel, 1992), wewill first describe the pattern of expression of LK-immunoreactive cells in the adult CNS.

Expression pattern of the leucokininpeptide in wild-type drosophila

melanogaster

Positive LK immunoreactivity was observed in the CNSof D. melanogaster larvae and adults, mainly distributedin the protocerebrum and ventral ganglion. In larval CNS,the pattern of LK immunoreactivity (Fig. 2) resembledthat previously detected with an antibody against anotherLK neuropeptide (LK-I; Cantera and Nassel, 1992). Theventral ganglion showed one pair of prominent LK-immunoreactive neurons in seven abdominal neuromeres(Fig. 2A) that send their processes to the lateral abdomi-nal nerves (Cantera and Nassel., 1992). Two pairs of sube-sophageal neurons and one pair of large anterodorsal neu-rons in the brain lobes completed the pattern of larvalLK-immunopositive neurons (Fig. 2A).

In adults, the ventral ganglion contained eight to tenpairs of LK-immunoreactive cells, located dorsolaterallyin the abdominal neuromeres (Fig. 3B). The position ofthese cells indicates that they correspond to the larvalabdominal neurons. In the adult brain, only two pairs ofLK-expressing neurons could be detected. One pair corre-sponds to the subesophageal neurons (Fig.3A), which pro-jected upward from their ventromedial position to thetritocerebrum midline. The second pair of brain LK neu-rons was comprised of two large anterodorsal neurons inthe protocerebrum (Fig. 3A). The cell body of these neu-rons was located in the lateral horn area, one on each sideof the brain. We have named these cells the lateral hornleucokinin (LHLK) neurons.

The LHLK neurons had a highly characteristic patternof projection (Fig. 3C,D). Their thin processes ran ven-trally, before bifurcating into two main collateralbranches that projected toward the medial part of theprotocerebrum. The collateral branches reached the ven-tral or dorsal side of the medial protocerebrum, wherethey formed a network of large LK-immunoreactive vari-cosities. These varicosities appeared to surround the pe-duncles of the mushroom bodies (Fig. 3C). Some othersmaller secondary branches could be observed, mainly offthe ventral collateral branch. The varicosities did not ex-tend further away from the protocerebrum.

In larvae, the position and branching pattern of theLK-immunoreactive neuron located in each brain lobe in-dicated that these are the LHLK neurons, which persistinto adult stages (Fig. 2A,B). To confirm this, we followedthe development of these cells throughout larval life. TheLK-immunoreactive larval neurons appeared between 10and 12 hours after hatching. The soma of each larvalLHLK cell was clearly seen in the dorsal anterior area ofthe brain lobe, where they began to project toward aventromedial location. This gross position remained un-changed from first to third instar. However, bifurcation ofthe main neurite into two collaterals could be appreciatedduring late larval stages. Using an enhancer trap line tolabel the larval mushroom bodies (c503GAL4,UAS:sbGFP),the two collaterals from the larval LHLK neurons were

Fig. 4. Leucokinin expression in apterous mutants. A,B: LK ex-pression in apUGO35 larval CNS (A) and adult brain (B). Note thatLHLK cells are not visible in either larva (compare with Fig. 2A) oradult (compare with Fig. 3A). C: LK expression in LHLK neurons inap4 hypomorphic mutant brain. Thin arrows point to LHLK cells.Notice the different intensity of LK immunoreactivity of the twoneurons. The insets show higher magnification images of the rightLHLK neuron (c1), with its varicosities clearly visible, and the leftLHLK neuron (c2), with few detectable varicosities (white arrow).Scale bars � 100 �m in A–C; 50 �m in inset.

127APTEROUS REGULATES LK EXPRESSION IN FLY BRAIN NEURONS

seen in close proximity to the peduncles and calyces, in aposition reminiscent of the adult projections (Fig. 2C,D).

To gain insight into the possible functions of the LHLKneurons, we performed co-localization experiments withanti-LK and antibodies against other neuropeptidesknown to label neurons in similar positions. We triedantibodies against the neuropeptides FMRFamide, PDF,and Tachykinin, but none of them labeled the LHKL neu-rons (not shown).

Apart from the aforementioned LK neurons, no otherneurons showed LK staining in the CNS of larvae andadults. We did not detect anti-LK immunoreactive cells inthe ring gland, gut, or malpighian tubules.

apterous is necessary for LK expressionin LHLK neurons

To investigate the role of apterous gene in the specifi-cation of neurosecretory identity, we examined the pat-

Fig. 5. Coexpression of apterous and leucokinin in the CNS.A,B: CNS from apGAL4/CyO;UAS:sbGFP larvae (A) and adult (B)showing expression of apterous (green; revealed by GFP expression)and LK (red). Final images are projections of confocal images. Insetsare higher magnification images of the boxed areas, which include thethree types of LK neurons: lateral horn leucokinin (LHLK), sube-

sophageal (se), and abdominal (ab) neurons. Note that LHLK cells arethe only neurons that show colocalization of LK (cytoplasmic) and ap(nuclear) in both larva (A) and adult (B). No colocalization was de-tected in subesophageal (se) or abdominal (ab) neurons. Anterior atthe top. Scale bars � 100 �m for nervous system larva and brain ofA,B; 50 �m for insets of A,B.

128 P. HERRERO ET AL.

tern of LK immunoreactivity in several homozygous apnull mutants (Cohen et al., 1992). These included twotranscriptionally null alleles, apP44 and apUGO35, and oneprotein null allele, apGAL4 (O’Keefe et al., 1998). In allthree cases, the LHLK neurons were not visible (Table 1),whereas the subesophageal and abdominal neurons werepresent in normal numbers and had a normal appearance(Fig. 4). The absence of anti-LK staining in the LHLKneurons was total in both larval (Fig. 4A) and adult (Fig.4B) ap-null CNSs.

To test whether LK expression in LHLK neurons wassensitive to the amount of Apterous protein, we analyzedthe expression of LK in the hypomorphic ap alleles ap4,aprk568, and ap56f (Table 1). Both aprk568 and ap4 showeda significant reduction in the expression of LK in LHLKcells, although normal levels of LK were present in 58.8and 48.0% of the cells, respectively (Fig. 4C). Often, neu-ronal soma with very weak anti-LK staining was observedin the position of the LHLK neuron (Fig. 4C). It is ofparticular note that many ap hypomorphic brains con-tained only one LHLK cell with positive LK immunoreac-tivity. LK expression in ap56f mutant CNSs was indistin-guishable from that in wild type (Table 1).

LHLK neurons express apterous

To understand the mechanism by which Apterous reg-ulates LK expression, we first examined whether apterousand LK are co-expressed. We used two enhancer trap linesin the ap gene, apGAL4 and aprk568. Flies carrying apGal4and the UAS-sbGFP reporter transgene were double-labeled with anti-GFP and anti-LK. aprk568, which ex-presses �-galactosidase in the nuclei of all ap-expressingcells, was double-labeled with anti-�-gal and anti-LK. Pre-vious studies have confirmed that both apGAL4 and aprk568

are expressed in the same neurons as the ap gene (Cohenet al., 1992; Benveniste et al., 1998).

In larval and adult brains, ap was expressed in the opticlobes and in numerous neurons of the central brain (Fig.5). However, only the LHLK neurons showed co-expression of ap an LK (Fig. 5). This co-expression wasconstant throughout all developmental stages. None of theother LK-immunoreactive cells, i.e., the subesophagealand abdominal neurons, expressed apterous (Fig. 5).

ap in the LHLK neurons rescuesLK expression

LK expression is affected in ap mutants only in thosecells that co-express ap and LK, i.e., the LHLK neurons.This suggests that ap might directly regulate LK expres-sion. To corroborate this, we studied LK expression in fliesin which Apterous protein is expressed in different neu-ronal domains, in an otherwise ap-null background. To dothis, we used the GAL4/UAS system (Brand and Perri-mon, 1993), with various neuronal Gal4 lines driving apexpression from a UAS-ap transgene. The results of theserescue experiments are summarized in Table 2.

Flies apGAL4/apUGO35; UAS-ap showed LHLK stainingin 92% of the brains analyzed (Fig. 6C). In many cases,only one of the two LHLK neurons was detected (Fig. 6D),so that 79.1% of the LHLK cells showed positive LK im-munoreactivity. This genetic combination is able to pro-vide almost complete rescue of other apGAL4/apUGO35 mu-tant phenotypes (Rincon-Limas et al., 2000).

Similar results were obtained when elavGAL4, a gener-alized neuron-specific driver, was used to express ap in an

apUGO35/apUGO35 mutant fly (Table 2). In this combina-tion, 80% of the LHLK cells showed LK staining (Fig. 6B),and 90% of the brains contained at least one LHLK cellwith LK immunoreactivity. However, we did not observeany ectopic neuronal LK immunoreactivity when ap� wasexpressed with this driver, in an either homozygous or het-erozygous apUGO35 background, even though elavGAL4drives expression of ap in all neurons, including those thatdo not normally express ap.

A Gal4 line that is expressed in subsets of neurons thatdo not include the LHLK neurons (data not shown), suchas VNCGAL4 (Rincon-Limas et al., 1999), did not rescueLK expression in LHLK neurons (Fig. 6A, Table 2). There-fore, this result suggests that ap must be expressed in theLHLK neurons in order to activate LK expression.

DISCUSSION

In this study, we demonstrate that Apterous is neces-sary for the expression of LK in only one type of neuron,the LHLK brain cells. Other neuropeptides, i.e., PDF andTachykinin, and all other LK-expressing cells, are notaffected in ap mutants. This situation is comparable to thealteration of FMRFamide in ap mutants, which are af-fected only in some of the FMRFamide-expressing cells(Benveniste et al., 1998). Overall, these results indicatethat the role of ap in controlling neurosecretory identity isnot general, rather, ap regulates the expression of certainneuropeptides only in subsets of neuropeptide-expressingcells.

Other LIM-HD proteins have been shown to specifyneurosecretory properties in Drosophila, C. elegans, andmammals (Thor and Thomas, 1997; Girardin et al., 1998;Hobert et al., 1999). Similar to ap, lim-6 regulates�-aminobutyric acid (GABA) synthesis in a subset ofGABAergic neurons in C. elegans (Hobert et al., 1999), andDrosophila islet is necessary for expression of serotoninand dopamine only in those neurons that co-express isletand the neurotransmitter (Thor and Thomas, 1997). Re-cent work on ttx-3, the ap ortholog in C. elegans, hasshown that specific combinations of transcription factorsin a hierarchical and parallel way are necessary to deter-mine the differentiation outcome of a given neuron (Altun-Gultekin et al., 2001). A similar mechanism for Apterousaction would explain the lack of ectopic LK expressionwhen ap is ectopically expressed throughout the CNS.Thus, the emerging picture identifies LIM-HD proteins askey players in a transcription factor code that establishesthe terminal differentiation program in neurons.

TABLE 2. Summary of LHLK-Rescued Neurons in Adult Brains1

No. of fliesstudied

LHLK rescues2 % of LHLKrescued

neurons3�� � �

apGAL4/apUGO35 12 0 0 12 0 (0/24)apGAL4/apUGO35; UASap 36 24 9 3 79.1 (57/72)apUGO35;elavGAL4/UASap 10 7 2 1 80 (16/20)apUGO35;VNC-GAL4/UASap 10 0 0 10 0 (0/10)

1Lateral horn leucokinin (LHLK) cells with positive anti-LK immunoreactivity wereconsidered to be LHLK-rescued neurons.2Numbers of brains with: ��, complete rescue (two LHLK cells); �, one cell rescued; �,no rescue.3Numbers in parentheses refer to number of rescued LHLK neurons with respect toexpected number of total LHLK cells (two per brain).

129APTEROUS REGULATES LK EXPRESSION IN FLY BRAIN NEURONS

Regulation of the leucokiningene by apterous

The large number of Apterous-expressing cells in thelateral horn precluded the identification of the LHLK neu-ron based merely on its morphology and position. Thus,with the tools available, we cannot test whether the LHLKcells are present in ap mutants. However, our rescueexperiments with the neuronal elavGAL4 driver showthat LK expression can be restored when Apterous isproduced in postmitotic neurons, implying that Apter-ous is not necessary for the birth of LHLK neurons.

Moreover, the reduced LK content in hypomorphic apmutants suggests that Apterous controls LK transcrip-tion in the LHLK cells, rather than the development ofthese cells. This is also true for Apterous action onFMRFamide expression: the Tv neurons are morpholog-ically normal in ap mutants, although with reducedFMRFamide content (Benveniste et al., 1998). OtherLIM-HD proteins also regulate neurosecretory identityand other specific neuronal properties but are not es-sential for the emergence and maintenance of neurons(for review, see Hobert and Westphal, 2000).

Fig. 6. Rescue of LK expression in apterous mutant. A: apUGO35;VNC-GAL4/UASap adult brain, showing lack of LK immunostainingin LHLK neurons (arrows point to the position of LHLK neurons).Higher magnification images of the brain areas containing left andright LHLK neurons are seen in a1 and a2. B–D: LK expression inLHLK neurons in apUGO35; elavGAL4/UASap (B) and apGAL4/apUGO35;UAS-ap (C,D) adult brains. Arrows point to the LHLK cells. Insets

show a higher magnification image of LHLK neurons. Both elavGAL4(B) and apGAL4 (C) are able to provide full rescue of LK expression inboth LHLK neurons, with soma and varicosities clearly visible (b1, b2,c1, c2). In some cases (D), LK expression is rescued in only one of thetwo LHLK cells (see Table 2 and text). Notice the difference in inten-sity of LK immunoreactivity between left (d1) and right (d2) neurons.Scale bars � 100 �m in A–D; 50 �m for a–d.

130 P. HERRERO ET AL.

Apterous may regulate LK expression by a direct or anindirect mechanism. We have shown that LK expression isonly affected in those cells that express ap. Moreover, ourrescue experiments suggest that Apterous must bepresent in the LHLK cells to ensure proper LK synthesis.Thus, Apterous must regulate LK transcription, either bydirect binding to the LK promoter, or through activation ofanother, as yet unidentified, transcription factor in theLHLK neurons. The results of Benveniste et al. (1998)indicate that Apterous regulates FMRFamide transcrip-tion in embryonic stages through direct binding to theFMRFamide promoter, in cooperation with other tran-scription factors. In this regard, we have found 16 putativeApterous consensus binding motifs in the 7 kb upstream ofthe LK transcription unit (Terhzaz et al., 1999). The lesssevere phenotype in postembryonic FMRFamide expres-sion found in ap mutants might reflect the existence ofalternative, Ap-independent mechanisms that regulateFMRFamide transcription in the Tv cells later in develop-ment (Benveniste et al., 1998). However, reduction in LKexpression in LHLK cells was total in ap null mutants inthird-instar larvae and adults, suggesting that Apterousis required throughout development to maintain LK syn-thesis in these cells.

Suggested functions of LHLK cells

The expression of LK in Drosophila melanogaster third-instar larvae has been described before (Nassel and Lun-quist, 1991). Using an anti-LK I antibody, these authorsfound more LK-immunoreactive cells than we did in ourstudy. Although there is just one LK precursor gene inDrosophila (Terhzaz et al.,1999), this precursor may un-dergo post-translational modifications that give rise todifferent peptides. This phenomenon is common to manyneuropeptides, such as Drosophila FMRFamide (Schnei-der and Taghert, 1990) and LK from Aedes aegypti (Veen-stra et al., 1997). Therefore, the anti-LK IV-immunoreactive cells that we observe may express one ofthese LK forms and thus represent a subset of the LK-containing cells that are detected with the broad-specificity anti-LK I antibody.

Leucokinins are found in neural and non-neural tissuesin insects and have been shown to be involved in diuresis(Hayes et al., 1989). The diuretic action of LK is accom-plished by the abdominal LK cells. These cells secrete theneuropeptide into the neurohemal organs, from where it isreleased into the hemolymph and diffuses to the mal-pighian tubules (Chen et al., 1994). As in other insects,DLK stimulates fluid secretion from the malpighian tu-bules in Drosophila melanogaster (Terhzaz et al., 1999).DLK induces an intracellular increase of Ca2� levels inthe Drosophila stellate cells, which, in turn, raises thechloride conductance through the apical membrane chan-nels (O’Donnell et al., 1998).

ap mutant flies have a short life span, dying within afew days of eclosion. Genetic mosaic analysis suggeststhat this lethality is due to a defect in the function ofmalpighian tubules (Wilson, 1981). There is no apterousexpression in the tubules (Cohen et al., 1992), and so thisdefect probably implies a neuromodulatory failure. Al-though we cannot rule out the possibility, the LHLK cellsdo not seem to be the cause of the suggested ap malfunc-tion of the malpighian tubules because, in ap mutants, theabdominal and subesophageal LK neurons that project to

the lateral abdominal nerves are not affected. Some otherdiuretic hormones may be involved in this ap defect.

ap mutant flies show reduced levels of juvenile hormone(JH), which leads to female sterility as a result of reducedvitellogenesis (Altaratz et al., 1991). Regulation of JHlevels must depend on neuromodulatory signals from theApterous-expressing cells of the brain (Cohen et al., 1992),because Apterous protein is not found in the corpus alla-tum. Although it would be very tempting to link Leucoki-nin failure in the brain cells to the reduced JH levels of apmutants, we did not find any LHLK projections to the ringgland.

Finally, the projections of the LHLK neurons to thepedunculus of the mushroom bodies might suggest a rolein neuromodulation of associative learning and memory,as has been shown for the Amnesiac neuropeptidergiccells (Wadell et al., 2000). Although some aberrant sexualbehaviors of ap mutants have been associated with JHdeficiency (Ringo et al., 1991, 1992), they may also berelated to failures in olfactory memory. In fact, recentstudies point to the lateral protocerebrum region, wherethe LHLK neurons are located, as being the major pri-mary target for olfactory information in the memory cir-cuits of the insect brain (Davis, 2001; Jefferis et al., 2001).Further analysis of the role of the LHLK will be requiredto answer these questions, and apterous mutants may bean essential tool when LK mutants are not available.

ACKNOWLEDGMENTS

We thank Dr. Veenstra, Dr. Nassel, and Dr. Park forantibodies; J. Benito, B. Dorado, I. Molina, and E. Turie-gano for technical suggestions; Ma A. Munoz, C. Sanchez,and T.Villalba for assistance with confocal microscopy;and Dr. P. Mason for English assistance.

LITERATURE CITED

Altaratz M, Applebaum SW, Richard DS, Gilbert LI, Segal D. 1991. Reg-ulation of juvenile hormone synthesis in wild-type and apterous mutantDrosophila. Mol Cell Endocrinol 8:205–216.

Altun-Gultekin Z, Andachi Y, Tsalik EL, Pilgrim D, Kohara Y. 2001. Aregulatory cascade of three hoemobox genes, ceh-10, ttx-3 and ceh-23,controls cell fate specification of a defined interneuron class in C.elegans. Development 128:1951–1969.

Benveniste RJ, Thor S, Thomas JB, Taghert PH. 1998. Cell type-specificregulation of the Drosophila FMRF-NH2 neuropeptide gene by Apter-ous, a LIM homeodomain transcription factor. Development 125:4757–4765.

Bourgouin C, Lundgren SE, Thomas JB. 1992. apterous is a DrosophilaLIM domain gene required for the development of a subset of embry-onic muscles. Neuron 9:549–561.

Brand A, Perrimon N. 1993. Targeted gene expression as a means ofaltering cell fates and generating dominant phenotypes. Development118:401–415.

Calleja M, Moreno E, Pelaz S, Morata G. 1996. Visualization of geneexpression in living adult Drosophila. Science 274:252–255.

Cantera R, Nassel DR. 1992. Segmental peptidergic innervation of abdom-inal targets in larval and adult dipteran insects revealed with anti-serum against leucokinin I. Cell Tissue Res 269:459–471.

Chen Y, Veenstra JA, Davis NT, Hagedorn HH. 1994. A comparative studyof leucokinin-immunoreactive neurons in insects. Cell Tissue Res 276:69–83.

Cohen B, McGuffin ME, Pfeifle C, Segal D, Cohen SM. 1992. apterous, agene required for imaginal disc development in Drosophila, encodes amember of the LIM family of developmental regulatory proteins. GenesDev 6:715–729.

Davis RL. 2001. Mushroom bodies, Ca2� oscillations, and the memory geneamnesiac. Neuron 30:653–656.

131APTEROUS REGULATES LK EXPRESSION IN FLY BRAIN NEURONS

Dawid IB, Breen JJ, Toyama R. 1998. LIM-domains: multiple roles asadapters and functional modifiers in protein interactions. TrendsGenet 14:156–162.

Girardin SE, Benjannet S, Barale JC, Chretien M, Seidah NG. 1998. TheLIM homeobox protein mLIM3/Lhx3 induces expression of the prolac-tin gene by a Pit-1/GHF-1-independent pathway in corticotroph AtT20cells. FEBS Lett 431:333–338

Hayes TK, Pannabecker TL, Hinckley DJ, Holman GM, Nachman RJ,Petzel DH, Beyenbach KW. 1989. Leucokinins, a new family of iontransport stimulators and inhibitors in insect malpighian tubules. LifeSci 44:1259–1266.

Hobert O, Westphal H. 2000. Function of LIM homeobox genes. TrendsGenet 16:75–83.

Hobert O, Tessmar K, Ruvkun G. 1999. The Caenorhabditis elegans lim-6LIM homeobox gene regulates neurite outgrowth and function of par-ticular GABAergic neurons. Development 126:1547–1562.

Holman GM, Cook BJ, Nachman RJ. 1986a. Isolation, primary structureand synthesis of two neuropeptides from Leucophaea maderae: mem-bers of a new family of cephalomyotropins. Comp Biochem Physiol84C:271–276.

Holman GM, Cook BJ, Nachman RJ. 1986b. Isolation, primary structureand synthesis of two additional neuropeptides from Leucophaea mad-erae: members of a new family of cephalomyotropins. Comp BiochemPhysiol 84C:205–211.

Jefferis GS, Marin EC, Stocker RF, Luo L. 2001. Target neuron prespeci-fication in the olfactory map of Drosophila. Nature 414:204–208.

Lin DM, Goodman CS. 1994. Ectopic and increased expression of FasciclinII alters motorneuron growth cone guidance. Neuron 13:507–523.

Nassel DR, Lundquist CT. 1991. Insect tachikinin-like peptide: distribu-tion of leucokinin immunoreactive neurons in the cockroach and blow-fly brains. Neurosci Lett 130:225–228.

Nassel DR, Cantera R, Johard HAD, Lundquist CT, Muren E, Shiga S.1992. Organization of peptidergic pathways in insects. In: Singh RN,editor. Nervous systems. Principles of design and function. New York:Wiley Eastern. p 189–212.

O’Donnell MJ, Rheault MR, Davies SA, Rosay P, Harvey BJ, Maddrell HP,Kaiser K, Dow JAT. 1998. Hormonally controlled chloride movementacross Drosophila tubules is via ion channels in stellate cells. Am JPhysiol 43:R1039–R1049.

O’Keefe DD, Thor S, Thomas JB. 1998. Function and specificity of LIM

domains in Drosophila nervous system and wing development. Devel-opment 125:3915–3923.

Park JH, Helfrich-Forster C, Lee G, Liu L, Rosbash M, Hall JC. 2000.Differential regulation of circadian pacemaker output by separate clockgenes in Drosophila. Proc Natl Acad Sci USA 97:3608–3613.

Rincon-Limas DE, Lu CH, Canal I, Botas J. 2000. The level of DLDB/CHIPcontrols the activity of the LIM homeodomain protein Apterous: evi-dence for a functional tetramer complex in vivo. EMBO J 19:2602–2614.

Rincon-Limas DE, Lu CH, Canal I, Calleja M, Rodrıguez-Esteban C,Izpisua-Belmonte C, Botas J. 1999. Conservation of the expression andfunction of apterous orthologs in Drosophila and mammals. Proc NatlAcad Sci USA 96:2165–2170.

Ringo J, Werczberger R, Altaratz M, Segal D. 1991. Female receptivity isdefective in juvenile hormone deficient mutants of the apterous gene ofDrosophila melanogaster. Behav Genet 21:453–469

Ringo J, Werczberger R, Segal D. 1992. Male sexual signaling is defectivein mutants of the apterous gene of Drosophila melanogaster. BehavGenet 22:469–487.

Schneider LE, Taghert PH. 1990. Organization and expression of thePhe-Met-Arg-Phe-NH2 neuropeptide gene of Drosophila. J Biol Chem265:6890–6895.

Siviter RJ, Coast GM, Winther AME, Nachman RJ, Taylor CAM, ShirrasAD, Coates D, Isaac RE, Nassel DR. 2000. Expression and functionalcharacterization of a Drosophila neuropeptide precursor with homologyto mammalian preprotachykinin A. J Biol Chem 275:23273–23280.

Terhzaz S, O’Connell FC, Pollock VP, Kean L, Davies SA, Veenstra JA,Dow JAT. 1999. Isolation and characterization of a leucokinin-likepeptide of Drosophila melanogaster. J Exp Biol 202:3667–3676.

Thor S, Thomas JB. 1997. The Drosophila islet gene governs axon path-finding and neurotransmitter identity. Neuron 18:397–409.

Veenstra JA, Pattillo JM, Petzel DH. 1997. A single cDNA encodes all threeAedes leucokinins, which stimulate both fluid secretion by the mal-pighian tubules and hindgut contractions. J Biol Chem 272:10402–10407.

Wadell S, Armstrong JD, Kitamoto T, Kaiser K, Quinn WG. 2000. Theamnesiac gene product is expressed in two neurons in the Drosophilabrain that are critical for memory. Cell 103:805–813.

Wilson TG. 1991. A mosaic analysis of the apterous mutation in Drosophilamelanogaster. Dev Biol 85:434–445.

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