eagleis required for the speciﬁcation of serotonin neurons ... · the lineage of insect serotonin...

463Development 125, 463-472 (1998)Printed in Great Britain © The Company of Biologists Limited 1998DEV8413

eagle is required for the specification of serotonin neurons and other

neuroblast 7-3 progeny in the Drosophila CNS

Martha J. Lundell 2,* and Jay Hirsh 1

1Department of Biology, University of Virginia, Charlottesville, VA 22903, USA2Division of Life Sciences, University of Texas at San Antonio, San Antonio, TX 78249, USA*Author for correspondence (e-mail: [email protected])

Accepted 13 November 1997: published on WWW 13 January 1998

During development of the Drosophila nerve cord,neuroblast 7-3 gives rise to a pair of mitotic sister serotoninneurons in each hemisegment. Here we show that the zincfinger gene eagle, which is expressed in neuroblast 7-3, isessential for specifying the fate of serotonin neurons. Wefind that loss-of-function eagle mutations produce anunusual differential phenotype with respect to the sisterserotonin cells and that eagle is necessary for themaintenance of engrailed and zfh-2 expression in theserotonin neurons. We present a model that uniquelyidentifies all progeny neurons in the neuroblast 7-3 lineage

based on the expression of specific molecular markers,position within the nerve cord and the effect of eagle loss-of-function mutations. Although serotonin is an importantneurotransmitter conserved throughout the animalkingdom, we show that hypomorphic alleles of eaglecanproduce viable adults that have a dramatic reduction in thenumber of serotonin-producing neurons.

Key words: eagle, engrailed, pdm1, zfh-2, Ddc, Serotonin,Drosophila, CNS, Neuroblast

SUMMARY

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INTRODUCTION

We are investigating the mechanism that specifies serotoneurons in the ventral nerve cord of the fruit fly, Drosophilamelanogaster. Serotonin is an evolutionarily conservneurotransmitter that has roles in locomotion and behavioboth invertebrates (Carew, 1996; O’Gara et al., 1991; Seget al., 1995) and vertebrates (Grillner et al., 1995; Kandeal., 1991). The general pattern of serotonin cells in segmeninvertebrates is rather well conserved in animals from lobsto insects, implying a conserved developmental pathway these neurons. These cells are derived from a relatively simlineage (Taghert and Goodman, 1984; Higashijima et al., 19Lundell et al., 1996; Dittrich et al., 1997) and are one of ona few neuronal types where the terminal differentiatphenotype is easy to assay. These characteristics makelineage an excellent model for examining moleculmechanisms of cell specification.

Neuronal diversity in the insect CNS arises through invaridivision of neuroblasts (NBs) and through environmental cufrom neighboring neuronal and nonneuronal cells (ChLaGraff and Doe, 1993; Huff et al., 1989). NBs are stem cethat undergo several asymmetric divisions producing a specnumber of ganglion mother cells (GMCs). Each GMC dividonce to form two neuronal or glial progeny. The Drosophilaventral nerve cord develops from stereotyped divisions ofNBs in each hemisegment, which form a two-dimension

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array in which each NB has a unique position (Goodman aDoe, 1993). Recently we have shown that the serotonneurons arise from NB 7-3 (Lundell et al., 1996). Within mossegments of the Drosophila ventral cord are two bilaterallysymmetric pairs of serotonin neurons (Fig. 1A).

The lineage of insect serotonin neurons was first examinin grasshopper by injecting NB 7-3 with the vital dye lucifeyellow (Taghert and Goodman, 1984). NB 7-3 in grasshoppproduces four GMCs, the last of which degenerates. The paiserotonin neurons are derived from division of the first GMCIn the first thoracic segment, there are three serotonin neurper hemisegment; this third cell originates from division of thsecond GMC. The fate of the other cells in this lineage aunknown. The axons of the serotonin neurons projeanteriorly and cross to the contralateral side via the postercommisure. Although the two sister serotonin cells armorphologically very similar they have slightly differentgrowth rates and distinguishable projections. Given thsimilarities between the grasshopper and Drosophila NB 7-3lineage, it is most likely that the serotonin neurons iDrosophilaare also mitotic sisters.

Since direct injection of neuroblasts in Drosophila is moredifficult, initial studies on the NB 7-3 lineage have been donusing molecular markers. NB 7-3 expresses several genincluding: engrailed(en), huckebein(hkb), seven-up (svp), pdm1(also known as nubbin) and eagle(eg) (Broadus et al., 1995;Dick et al., 1991; Higashijima et al., 1996; W. Chia, person

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communication). Most of these genes are expressed in mother NBs as well and, since NB 7-3 is one of the last neuroblto delaminate from the neuroepithelium, the overall CNS pattof expression for these genes at the time that the NB7-3 lineinitiates is very complex.eg, however, is expressed in only fouNBs, including NB 7-3 (Higashijima et al., 1996), making it relatively simple marker for the NB 7-3 lineage.

Higashijima et al. (1996) show, with eg-lacZenhancer trapsand eg RNA in situ, that NB 7-3 produces four neurons in abdominal region of the ventral cord. Three of the foneurons, the EW neurons, project anteriorly to the postecommisure, and one, the GW neuron, projects ipsilateral posteriorly. Recently these results have been confirmed witheg antibody and DiI-labeled NB 7-3 clones (Dittrich et a1997). A direct relationship between the EW neurons and serotonin neurons is demonstrated by Dittrich et al. (199which appeared while this manuscript was under revieMutant alleles ofegshow the correct number of eg-expressingNB 7-3 progeny but the neurons have abnormal projectio(Higashijima et al., 1996; Dittrich et al., 1997) . This suggethat eg has a role in neuronal specification rather than tlineage divisions of NB 7-3. The restriction of eg expressionto just four neuronal lineages produces an egmutant phenotypewhere the overall morphology of the CNS is well preserved

The name eagledescribes an adult phenotype of wings heout at right angles to the body (Fig. 1C), which was initialcharacterized in 1930 by Thomas Hunt Morgan. egencodes azinc finger protein with homology to the steroid receptfamily (Higashijima et al., 1996; Rothe et al., 1989). Besidits limited expression in the CNS, the only other reportexpression of egis transient expression in the embryonic gon(Rothe et al., 1989). Recently eg was isolated in a screen fosuppressors of a rough eye phenotype caused by overexpression of Ras1, suggesting that egmay be involved inthe Ras1 signal transduction pathway (Hay et al., 1997).

Our initial interest in egwas to use an egdriven lacZ reporterconstruct as a simple marker of the serotonin neurons latembryogenesis. These experiments turned out to be diffibut interesting because P-element insertions in eg produce anunusual differential effect in the two sister serotonin neurothat alters the expression pattern of several genes and disserotonin synthesis. These changes in cell fate enable us tofurther insight into the developmental pathway of serotonneurons and to present a model that uniquely identifiesprogeny in the NB 7-3 lineage.

MATERIALS AND METHODS

Fly strainsAll wild-type flies are Oregon R. The egT6 and eg289 alleles containlacZ P-element insertions 5′ of eg. egmz360contains a gal-4 P-elementinsertion 5′ of eg. eg225Aand eg18B are P-element excision of egmz360.eg18B is a deficiency that removes the first two exons and translational start of eg(Dittrich et al., 1997). All eg alleles weregenerously provided by J. Urban and G. Technau. egT6 was generatedby R. White and eg289 was generated by B. Genisch and G. Korge

ImmunohistochemistryDissected CNSs were fixed in 4% paraformaldehyde and incubawith primary and secondary antisera as described previously (Lunand Hirsh, 1994).

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Rabbit and rat Ddc antiserum (Beall and Hirsh, 1987) were used20×dilution. Rat serotonin monoclonal antibody (Accurate Chemicawas used at 100× dilution. Rabbit Tyrosine Hydroxylase antiserum(Pel Freeze) was used at 40× dilution. Rabbit anti-β-galactosidase(Cappel) and mouse anti-β-galactosidase (Promega) were used a1000× dilution. en immunoreactivity was detected with a mousinvected monoclonal antiserum 4D9 used at a 2× dilution (a gift fromC. Doe). Rabbit pdm1 antiserum was used at 1000×dilution (a giftfrom W. Chia). Rat zfh-2 antiserum was used at 100× dilution (a giftfrom A. Tomlinson). All FITC secondary antibodies (Jackson) and aL-RITC antibodies (Tago) were used at a 200× dilution. All antibodieswere preabsorbed against first-instar larval tissue.

5-hydroxytryptophan (5HTP) loadingDissected larval CNS were incubated in PBS with 0.1 mM 5hydroxytryptophan and 1.0 mM CaCl2 for 30 minutes at roomtemperature prior to fixation.

MicroscopyAll confocal projections were generated on a Molecular Dynamic2000 or a Biorad MRC 1024 confocal laser scanning microscope.Figs 1B and 4, the images were assembled from multiple projectioalong the anterior/posterior CNS axis using Photoshop (Adobe).the black and white images shown, the contrast has been invertedclarity.

RESULTS

eg-lacZ is expressed in the serotonin neuronsTo directly demonstrate the relationship between eg-expressingneurons and serotonin-synthesizing neurons, we used the aeg289, which contains a lac-Z P-element inserted 5′ of eg. egRNA is not detectable after 11 hours of developme(Higashijima et al., 1996), and serotonin synthesis is ndetectable until 16 hours (Lundell and Hirsh, 1994). In spite this temporal gap, the perdurance of β-galactosidase from thelacZ reporter allows one to assess which cells expressed egatan earlier time.

Serotonin neurons can be detected with either DOPdecarboxylase (Ddc) or serotonin immunoreactivity. Since Dcatalyzes the last step in the biosynthesis of both serotonin dopamine, a Ddc antibody will detect both cell types (Beaand Hirsh, 1987; Konrad and Marsh, 1987; Lundell and Hirs1994; Valles and White, 1986). In the wild-type larval nervcord, depicted in Fig. 1A, three types of Ddc-immunoreactivcells are distinguished in each segment: the paired venlateral serotonin cells (VL), a single midline dopamine cell (Mand two single dorsal lateral dopamine cells (DL). In the moposterior abdominal segment A8, the serotonin neurons single cells instead of pairs and in the most anterior thorasegment, T1, the serotonin neurons are triplets and there atriplet of medial dopamine neurons instead of a single cell.

Double labeling for eg-lacZ and Ddc demonstrates that eg-lacZ is expressed equally in both serotonin neurons. This resis shown in Fig. 1D, which shows the abdominal segmentsa first instar larval CNS heterozygous for eg289, labeled withnuclear localized β-galactosidase in red and Ddc in green. Thexpression of eg-lacZin the entire larval CNS is shown in Fig.1B. In addition to the serotonin cells, eg-lacZis also expressedin far lateral clusters in the abdominal region, in midline celand in a large number of cells in the thoracic ansubesophageal region. There is no expression of eg-lacZin the

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Fig. 1. Expression of eg-lacZin the serotoninneurons. (A) A third instar larval CNSimmunostained with a Ddc antibody. Opencircles are serotonin cells and closed circlesare dopamine cells. VL, ventral lateralserotonin neurons; M, medial dopamineneurons; DL, dorsal lateral neurons; T1-T3,thoracic segments; A1-A8, abdominalsegments. (B) A wild-type first instar larvalCNS heterozygous for the eg289 allele andlabeled for β-galactosidase. The black verticalarrowheads indicate the ventral lateral cellclusters that contain the serotonin neurons andthe yellow horizontal arrowheads indicatehemisegments that show a third eg-lacZ-immunoreactive cell closely associated withthe serotonin cells. (D) A highermagnification of the abdominal region of afirst instar larval CNS heterozygous for theeg289 allele. This demonstrates the colocalization of Ddc immunoreactivity (green) and β-galactosidase immunoreactivity (red) in the serotoninneurons. Similar to B, the yellow arrowheads indicate a third eg-lacZcell closely associated with the serotonin cells that does not express Ddc.For clarity, the dorsal lateral and midline dopamine cells are not included in Fig. 1D. There is no expression of eg-lacZin dopamine cells (datanot shown). (C) The perpendicular wing phenotype characteristic of all egalleles. Scale bars in all figures, 20 µm.

Fig. 2. Hemisegments in egmutants show single serotonin cellsinstead of the normal mitotic pair. CNS from five different egalleleswere stained for Ddc immunoreactivity. All panels show the nervecord from a third instar larvae except eg18Bwhich is from a stage 17embryo. eg18B is the null phenotype. The immunoreactive Ddc cells atthe posterior tip of eg18B (horizontal arrow) are cells only detectabletransiently during embryogenesis. Staining was done simultaneouslyand confocal parameters were identical during collection of data suchthat the relative intensities of staining for each CNS is comparable.VL, ventral lateral serotonin neurons; M, medial dopamine neurons;T1-T3, thoracic segments; A1-A8, abdominal segments.

brain lobes. A similar pattern of expression is seen with anotenhancer trap, egmz360 (data not shown).

The coexpression of egand serotonin in descendants of NB7-3 makes a direct connection between the serotonin cells the three previously identified EW neurons (Higashijima et a1996), all of which send their projections contralateralthrough the posterior commisure. In Fig. 1D mohemisegments show a third eg-expressing red cell in closeproximity to the serotonin neurons (arrowheads), which whypothesize is the third EW neuron based on its lateral posito the serotonin neurons. This third neuron can also be seeFig. 1B in the hemisegments marked with yellow arrowheaOccasionally three Ddc-immunoreactive cells can be detecin abdominal hemisegments (unpublished results) and,segment T1 and the most posterior subesophageal segmenserotonin cells normally occur as triplets, all of which seprojections through the posterior commissure. Apparently,early development, the three EW neurons show simicharacteristics in gene expression and growth patterns, ethough later in development only two of these cells wsynthesize serotonin in the abdominal segments.

eg is necessary for the differentiation of serotoninneuronsIn eg loss-of-function mutants, we observe an unususerotonin cell phenotype that varies in severity with differeegalleles. Fig. 2 shows five egalleles immunostained for Ddc;egmz360, egT6 and eg289 are all P-element insertions 5′ of the egcoding region and eg225Aand eg18B are excisions of the egmz360

allele (Dittrich et al., 1997). eg18B is a null allele (Dittrich etal., 1997) and it is a late embryonic/early larval lethal. All thimages are third instar larval CNS except eg18B, which is a lateembryonic CNS. Fig. 2 demonstrates that, although segments show Ddc immunoreactivity in the midline dopamicells, many hemisegments do not show Ddc immunoreactivin the serotonin cells and, strikingly, those hemisegments tdo show Ddc-immunoreactive serotonin cells often havesingle cell rather than the normal doublet.

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Table 1. The number of serotonin neurons is reduced in egloss-of-function alleles

No. ofDdc-positive cells/hemisegment

Genotype 0 1 2 x–±s.e.m. n

egwt − − 100% 2.00±0.00 52eg360 2% 26% 72% 1.70±0.07 54eg225 20% 54% 26% 1.06±0.09 54egT6 74% 19% 7% 0.33±0.08 54eg289 66% 30% 4% 0.37±0.08 54eg18B 73% 24% 3% 0.24±0.04 140

CNS from five egalleles were stained for Ddc immunoreactivity and thenumber of serotonin cells in individual hemisegments from the secondthoracic segment to the seventh abdominal segment were counted. Thesehemisegments normally have two serotonin cells. The results are presenta percentage of the number of hemisegments counted (n). All CNS werelarval stage except eg18B which were embryonic stage 17 CNS.

-of-function mutants synthesize serotonin but have other abnormal (A,D) or egT6CNS (B,C,E,F), were double labeled for Ddc (A-C) and

. The one-to-one cell correspondence demonstrates that the remainingynthesize serotonin. The solid arrow indicates a serotonin neuron with ahe open arrow indicates a serotonin neuron with an aberrant ipsilateralt incubated in exogenous 5-HTP and then stained for serotoninidline dopamine cells uptake the precursor and become immunoreactiveonin cells are still undetected. VL, ventral lateral serotonin neurons; M,, thoracic segments; A1-A8, abdominal segments.

The projections in Fig. 2 are arranged with increasiseverity of the serotonin phenotype. The egmz360allele showsan almost wild-type pattern of serotonin cells in the abdomiregion but only single serotonin cells in the thoracic segmeeg225Ashows a few abdominal segments with doublets anlarge number of segments with single cells, but no serotocells in thoracic segments. egT6 and eg289 show a severereduction in the number of serotonin cells in both the thoraand abdominal segments. Even in the null allele, eg18B, afew Ddc-immunoreactiveserotonin cells persist. Thissuggests that there areredundant mechanisms thatallow the formation ofserotonin cells in the absenceof Eg protein. Most often thisredundant function onlyallows for development of asingle serotonin cell eventhough eg is expressed inboth serotonin cells (Fig.1D). This allelic variation inthe serotonin cell phenotypeis summarized in Table 1,which quantifies the numberof hemisegments that showzero, one or two serotonincells for each allele. Anaverage value for each alleleis calculated to show thereproducibility of thephenotype. Note that thephenotypes of thehypomorphic alleles egT6

and eg289 are very similar tothe phenotype of the nullallele eg18B.

Confirmation that theremaining single cells in egmutants can synthesizeserotonin is shown by directexamination of serotoninimmunoreactivity in Fig.

Fig. 3. Serotonin neurons in eg lossproperties. A larval wild-type CNSserotonin (D-F) immunoreactivityserotonin cells in egmutants can snormal contralateral projection. Tprojection. The CNS in G was firsimmunoreactivity. In this assay, mfor serotonin but the absent serotmedial dopamine neurons; T1-T3

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3E,F. Although these cells can synthesize serotonin, they ohave abnormal axonal projections. The normal axonal pathwfor the serotonin neurons is to project anteriorly, cross to contralateral side via the posterior commisure and then proboth posteriorly and anteriorly (Taghert and Goodman, 198In the egmutants, we find a mixture of normal and abnormprojections: some of the remaining serotonin cells appearcross to the contralateral side Fig. 3E (closed arrow) but othare defective in that their projections remain ipsilateral Fig. (open arrow). Abnormal projections of NB 7-3 progeny in egmutants are also observed during early developme(Higashijima et al., 1996; Dittrich et al., 1997). The serotonneurons in the brain lobes are unaffected by loss of eg function(data not shown), as expected, since egexpression is restrictedto the nerve cord (Fig. 1B).

The similarity of the grasshopper and Drosophila NB 7-3lineages suggests that the serotonin cells of the ventral necord are mitotic sisters. Mutant alleles of eg show the correctnumber of NB 7-3 progeny early in development (Higashijimet al., 1996); therefore, the undetectable sister serotonin must adopt an alternative fate that no longer expresses norlevels of Ddc and serotonin. In an attempt to detect the seccell, we incubated eg mutant CNS in exogenous 5-hydroxytryptophan (5HTP), the metabolic precursor oserotonin that is decarboxylated by Ddc. Both serotonin adopamine neurons have the ability to uptake exogenous 5Hwhich can be readily detected by serotonin immunoreactiv

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in cells that contain even very low levels of Ddc (unpublishdata). Fig. 2G shows a larval egT6 CNS that was first incubatedin 5HTP and then immunostained for serotonin. The midlidopamine cells are now detectable, but the missing serotocells are still undetectable with this assay. If the undetectacells are present, they are defective in their uptake mechanand/or are totally deficient for Ddc, suggesting a dramaalteration in cell fate.

Adult eg mutants can survive with a reducednumber of serotonin neurons in the ventral cordAlthough most hypomorphic egT6 and eg289 flies die as pupae,15% survive to viable adults. Given the stochastic variationdevelopment of serotonin cells in the eg mutants (Fig. 2), wesuspected that those eg mutant flies that survive to adulthoodwould have a more normal complement of serotonimmunoreactive cells. Fig. 4 demonstrates that this is not case. This figure compares the adult ventral cords from wtype and egT6, immunostained with serotonin in green antyrosine hydroxylase in red to specifically detect dopamicells. After metamorphosis, the abdominal serotonin cells located in the posterior tip of the ventral cord. Although tnumber of dopamine neurons in the egT6 mutant is almostequivalent to wild type, the number of serotonin neuronsreduced sixfold to sevenfold, reflecting the phenotype sepreviously in the larval CNS (Figs 2, 3).

Serotonin has been implicated as a stimulatory modulatolocomotion in invertebrates (O’Gara et al., 1991; Segalat et 1995). Since the egmutants show a reduced number of neurosynthesizing serotonin, we observed both larvae and adult for locomotor and other behaviors. Both egT6 and eg289 larvae

Table 2. Changes in the expression of eg-lacZ(A) eg-lacZ

No. of eg-lacZpositive cells

Genotype 1 cell 2 cells 3 cells

eg289/+ 0.7% 30.0% 69.3%eg289/eg289 7.2% 10.3% 55.5%

(B) pdm1No. of cells coexpressing eg-lacZ/pdm1/hemisegment

Genotype 1 >1 0

eg289/+ 100% Ø Øeg289/eg289 89.0% 4.7% 7.8%

(C) zfh-2 No. of cellsexpressing only eg-lacZ/hemisegment

Genotype 1 >1 n

eg289/+ 98.9% 1.1% 90eg289/eg289 40.3% 59.7% 77

(D) enNo.of cells expressing only eg-lacZ/hemisegment

Genotype 0 1 >1

eg289/+ 97.7% 2.3% Øeg289/eg289 7.0% 32.1% 60.8%

eg289/+ and eg289/eg289 CNS were double stained for eg-lacZand either pdm1(seventh abdominal segment were counted for the number of cells that onlyen. In B-D only hemisegments with three or four detectable NB 7-3 cells whemisegments in experiments B-D. The numbers presented are a percent

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and flies appear to be normal for phototaxis and geotaxis bby simple inspection, the adults are much less active locomotion. Although flight is impossible due to the extendewings this feature is not responsible for the loss of locomotiosince the same phenotype is readily visible in flies with clippewings. We also find that eg mutants have difficulty holdingonto glass surfaces when climbing vertically or when rotateupside down but, have no obvious morphological defects in tlegs or tarsi. Whether these observed phenotypes are a diconsequence of reduced serotonin levels is uncertain sinceegis expressed in three other neuronal lineages. Given the mphysiological effects of serotonin that have been observedother organisms, we are surprised that Drosophilacan surviveas viable fertile adults with so few serotonin neurons.

The paired serotonin neurons of each hemisegmentare distinguishable and, in an eg loss-of-functionmutant, the remaining single cell has a phenotypesimilar to the more lateral serotonin cellAlthough eg is expressed in both serotonin cells, the eg loss-of-function mutants often affect the development of only onserotonin cell of each pair (Figs 2, 3). We questioned wheththe development of one specific cell in each pair is consistenaffected. In grasshopper, the two sister cells have slighdifferent growth patterns and projections (Taghert anGoodman, 1984), but these are impossible to discern Drosophila. We previously found that the two cells can bdistinguished from each other by differential expression of zfh-2 (Lundell and Hirsh, 1994). Here we show that the differentiexpression of zfh-2and of another antigen, pdm-1, can be usedto determine that the remaining single cell in eg mutants

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Fig. 4. Adult nerve cords from eg loss-of-function mutants also havea reduced number of serotonin neurons. Adult wild-type (A) and egT6

(B) nerve cords were double labeled for serotonin immunoreactivityin green and tyrosine hydroxylase immunoreactivity in red, which isspecific for dopamine-producing cells. Although the number ofdopamine cells is similar the number of serotonin cells is reducedsixfold. Staining was done simultaneously and confocal parameterswere identical during collection of data such that the relativeintensities of staining for each CNS is comparable.

Fig. 5. eg loss-of-function mutants have a more severe effect on thedifferentiation of the more medial serotonin cell. CNS from wildtype (A,B), egT6 (C,D) and eg18B (E,F) were double labeled for Ddcimmunoreactivity in red and either Zfh-2 (A,C,E) or Pdm1 (B,D,F)immunoreactivity in green. In the wild-type CNS, coexpression isobserved only in the lateral cell of each serotonin pair and in themidline dopamine neurons. A single dopamine neuron is shown atthe top of A and B. In egT6 and the null eg18B, the remainingserotonin cells coexpress zfh-2andpdm1 indicating they have aphenotype characteristic of the more lateral serotonin cell (indicatedby horizontal arrowheads in E and F). Most of the midline dopaminecells are included in the egT6 projections but have been excludedfrom the wild-type and eg18Bprojections for clarity of the serotonincells. The wild-type and egT6 images are from third instar larvae andthe eg18B images are embryonic, stage 17. VL, ventral lateralserotonin neurons; M, medial dopamine neurons.

expresses markers characteristic of the more lateral serotcell.

In a wild-type CNS, both zfh-2 and pdm1 are selectivelyexpressed in the more lateral serotonin cell but not in the mmedial cell (Fig. 5A,B). Both antigens are also expressedthe midline dopamine cells as shown by the single dopamcell at the top of Fig. 5A,B, and by the entire midline showin Fig. 5C,D. The remaining single serotonin cells in mutants consistently expresses both zfh-2and pdm-1 (Fig. 5C-F), characteristic of the more lateral serotonin cell. This resis seen in egT6 (Fig. 5C,D) where the larval CNS has a higlevel of Ddc expression, and also in the embryonic CNS of null allele, eg18B (Fig. 5E,F). Therefore, even though eg isnormally expressed in both serotonin cells, the absence oprotein has a more dramatic effect on the fate of the mmedial neuron.

eg loss-of-function mutations alter the specificationof serotonin cell fate without disrupting the lineagedivisions of NB 7-3To determine the fate of the medial serotonin cell, we examieg-lacZexpression late in embryonic development using teg289 allele. In a wild-type line heterozygous for eg289, two tothree NB7-3 descendants can be identified in eahemisegment with eg-lacZ (Figs 6A, 1B,D). These threeneurons are the EW neurons that generally lie in a rperpendicular to the midline and which project anteriorly to t

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posterior commisure (Higashijima et al., 1996). For thpurpose of discussion, we suggest naming these neurons EEW2 and EW3 from the midline out. Examination of a larvaCNS double labeled for eg-lacZand Ddcshows that EW1 andEW2 are the serotonin neurons and that EW3 is more variain its position and detectability (Fig. 1D).

In the mutant line homozygous for eg289, which dramaticallyreduces the number of serotonin cells detectable with Ddc (F2, Table 1), there are actually more cells expressing eg-lacZrather than less (compare Fig. 6A to B). This result


Fig. 6. Coexpression of eg-lacZ with Zfh-2, Pdm1 and En in wild-type and eg289 mutant embryos. (A,C,E,G) The ventral cord from eg289/+stage 17 embryos. (B,D,F,H) The ventral cord from eg289/eg289 stage 17 embryos. The CNS were double labeled for eg-lacZ immunoreactivityin red and either Pdm1 (C,D), Zfh-2 (E,F) or En (G,H) immunoreactivity in green. (H) Composite of two CNS with the green line indicating themidline for each. The three EW neurons are labeled 1, 2 and 3 from the midline out. Definitive identification of EW3 and GW neurons cannotbe achieved due to the movement of the cells during contraction of the ventral cord. (F) The closed arrowheads indicate hemisegments thatshow two medially located cells which only express eg-lacZ, suggesting the loss of zfh-2expression from the EW2 neuron. (H) The closedarrowheads indicate hemisegments that are likely to have no detectable serotonin cells and the open arrowheads indicates hemisegments that arelikely to have one detectable serotonin cell. Asterisks indicate hemisegments that show two medial cells that express only eg-lacZand twolateral cells that coexpress eg-lacZand either zfh-2or en. Suggesting that the GW neuron can express both zfh-2and en. The two en-expressingcells in G and H that are in close proximity to the serotonin cells, are presumably from a lineage distinct from NB 7-3.

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Fig. 7. A model comparing gene expression in the NB 7-3lineage of wild-type and eg289 mutant embryos. Theexpression of eg-lacZ, en, zfh-2and pdm1at stage 17 isdepicted in NB 7-3 progeny neurons for a wild-type(eg289/+) hemisegment with two detectable serotonin cells,an eg289/eg289 hemisegment with one detectable serotonincell and a eg289/eg289 hemisegment with no detectableserotonin cells. The open GW neuron in the wild-typehemisegment illustrates that the neuron is there but does notexpress eg-lacZ. The question mark signifies that theexpression of en, zfh-2and pdm1are unknown in thisneuron. The black lines indicate the axonal projectionswhich are often aberrant in egmutants. A, anteriorcommisure; P, posterior commisure; M, midline.

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summarized in Table 2A, which shows that 26% of themisegments in a eg289 homozygote have four or moreeg-lacZ-positive cells, versus none in the eg289 /+ heterozygote.This result confirms the work of Higashijima et al. (199showing that eg mutations affect cell fates rather than thnumber of NB 7-3 progeny. Clearly the cells that are ndetectable in egmutants with Ddc or serotonin are present btheir specification as serotonin neurons is dramatically alter

The linear arrangement of the eg-lacZ cells in wild-typeCNS (Figs 1D, 6A) suggests that they are the three Eneurons. We presume that the fourth cell that appears in theghomozygote is the GW neuron, which projects ipsilateral aposteriorly. In a wild-type CNS, the GW neuron must nexpress as much eg-lacZas the EW neurons or the egpromoterturns off earlier. Consistent with this latter proposal, egtranscripts in all four NB 7-3 progeny are seen in a wild-tystage 13 embryo, several hours earlier than the stage 17 shown in Fig. 6A (Higashijima et al., 1996; Dittrich et al1997). This suggests a negative autoregulatory function oegthat is uniquely active in the GW neuron, a function that coube responsible for its unique properties relative to the Eneurons.

eg loss-of-function mutations alter the expressionpattern of zfh-2 and engrailed in the serotoninneuronsTo investigate how egspecifies cell fates in the NB 7-3 lineagewe addressed whether eg289 shows any specific changes in genexpression. In particular, we examined whether the differenexpression of pdm1 and zfh-2is maintained and whether theris any effect on the expression of engrailed (en). We haveshown previously that enis required for development of theserotonin neurons (Lundell et al., 1996). Fig. 6 shows coexpression of these three genes with eg-lacZin wild-typeeg289/+ and mutant eg289/eg289embryos. The combinatorial useof these four markers allows us to uniquely identify all progein the NB 7-3 lineage.

Unique characteristics of EW1, EW2 and EW3 ademonstrated by the immunostained wild-type CNS in F6C,E,G. In the eg-lacZ/Pdm1 double-labeled wild-type CN(Fig. 6C), the only eg-lacZcell to express pdm1in the NB 7-3 lineage is a single cell that we infer from Fig. 5B to be tmore lateral serotonin cell (EW2). In the eg-lacZ/Zfh-2 doublabeled wild-type CNS (Fig. 6E), all eg-lacZ cells of the NB7-3 lineage show zfh-2expression except the most medial cewhich we infer from Fig.5A must be the medial serotonin c(EW1). These results are surprising since pdm1, which isexpressed in NB 7-3 (W. Chia personal communication)restricted to just one progeny neuron late in embryogenesiszfh-2, which is detectable only after NB formation (Lai et a1991), must be independently expressed in the progeny of mthan one NB 7-3 GMC. In the eg-lacZ/en double-labeled witype CNS, all eg-lacZcells of the NB 7-3 lineage show enexpression (Fig. 6G). In the ventral lateral region of a stagenerve cord, there is cluster of five en-expressing cells. We haveshown previously (Lundell et al., 1996) that the two momedial cells in the encluster are the serotonin cells, EW1 anEW2. In Fig. 6G, all hemisegments show coexpression ofenand eg-lacZ in the serotonin neurons but, in somhemisegments, the presence of three coexpressing indicates that EW3 can also expresses en. Therefore the three

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EW neurons can be uniquely identified by their position aexpression of pdm1, zfh-2 anden (Fig. 6C,E,G and summarizedin Fig. 7). EW1, the medial serotonin neuron, does not exprpdm1or zfh-2but does express en; EW2, the lateral serotoninneuron, expresses all three gene products; and EW3, whicsometimes detectable with the Ddc antibody (data not showexpresses zfh-2and enbut not pdm1.

Having established the wild-type pattern of expression, wnext asked how the expression of pdm1, zfh-2and enchangein the mutant allele eg289. In summary, we find that expressionof zfh-2and en is dependent on eg function but expression ofpdm1is independent of egfunction. The results for each of thethree genes are discussed below.

Loss of eg function appears to have no affect on thexpression of pdm1(Fig. 6D; Table 2B). Although there is anincrease in the number of eg-lacZ-expressing cells in eg289,expression of pdm1is still restricted to EW2 (Fig. 6D). Table2 shows that 89% of the hemisegments in eg289 retain a singlecoexpressing cell. Since 89% of the hemisegments still shpdm1expression but 66% of the hemisegments develop wno detectable serotonin cells (Table 1), then clearly tserotonin cell phenotype in eg mutants is not directly relatedto the expression of pdm1.

Loss ofeg function affects the expression of zfh-2 in EW2(Fig. 6F; Table 2C). Comparison of the eg-lacZ/Zfh-2 imagfor wild type (Fig. 6E) and eg289 (Fig. 6F) shows that theproportion of cells per hemisegment that express eg-lacZbutnot Zfh-2 increases in eg289. In wild-type nerve cords, almostall (99%) hemisegments with three eg-lacZcells show two cellsthat also express zfh-2 (EW2 and EW3), and one cell thatexpresses only eg-lacZ(EW1) (Fig. 6E; Table 2C). In eg289

only 40% of hemisegments show this pattern; instead remaining hemisegments show two or more cells that expronly eg-lacZ. This increase in cells that only express eg-lacZcould be explained either by the appearance of the GW neuwhich does not express zfh-2or by the loss of zfh-2 expressionfrom EW2 or EW3. We favor the later hypothesis because cells expressing only eg-lacZconsistently occur as mediallylocated doublets (yellow arrowheads, Fig. 6F), suggesting texpression of zfh-2is absent in EW2 (Fig. 6F). In addition,some eg289 hemisegments where four eg-lacZ neurons areclearly detectable (asterisk, Fig. 6F), show two medial cethat lack zfh-2expression and two lateral cells that coexprezfh-2, suggesting that the GW neuron can express zfh-2ineg289.

Loss of eg function affects the expression of en in bothserotonin neurons (Fig. 6H; Table 2D). Comparing the elacZ/En images of wild type (Fig. 6G) with eg289 (Fig. 6H) themost obvious difference is the appearance of one or two cin eg289 that express eg-lacZ but not en, per hemisegmentThese cells tend to be the cells of the encluster closest to themidline, EW1 and EW2. Table 2D indicates that, in eg289, 7%of the hemisegments show only coexpressing eg-lacZ/encellslike wild type, 32% show one cell that lacks enexpression and61% show two or more cells that lack en expression. Thisdistribution is similar to the distribution of detectable serotoncells in eg289 (Table 1); 4% show two cells, 30% show one ceand 66% show no cells. Therefore we propose thhemisegments with two cells that only express eg-lacZ(closedarrowheads in Fig. 6H) would likely correspond themisegments with no detectable serotonin cells a


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hemisegments containing one cell that only expresses eg-lacZ(open arrowheads in Fig. 6H) would likely correspond hemisegments, where only the single lateral serotonin c(EW2) is detectable. In some eg289 hemisegments, where foureg-lacZneurons are clearly observed (asterisk, Fig. 6H), thare two medial cells that lack en expression and two lateralcells that coexpress en, suggesting that the GW and EWneurons express en in eg289.

DISCUSSION

Our previous work demonstrated that the serotonin neuronsdescendants of the NB 7-3 lineage (Lundell et al., 1996). Tmanuscript assigns a unique identity to each of the NB progeny neurons based on the expression of specific molecmarkers, position within the nerve cord and the effects of egloss-of-function mutations. We show that egmutations alter thespecification of cell fates in this lineage. Our results asummarized with a model presented in Fig. 7.

During mid-embryogenesis, stages 11-13, the thoracic aabdominal segments of the ventral cord show four neurons are derived from NB 7-3 (Higashijima et al., 1996; Dittrich al., 1997). The four NB 7-3 progeny comprise: (a) the thrEW neurons that initially lie in a lateral row, project anteriorto the posterior commisure and cross to the contralateral sand (b) the GW neuron, which is initially located slightlposterior to the EW neurons and projects posteriorly on ipsilateral side (Higashijima et al., 1996). Here we demonstrthat the two EW neurons closest to the midline, EW1 and EWbecome serotonin-producing neurons (Fig. 1D). By analogygrasshopper (Taghert and Goodman, 1984), we assume EW1 and EW2 are mitotic sisters of the first ganglion mothcell, GMC 1. Also by analogy to grasshopper, the EW3 neuris most likely a progeny cell of GMC 2. The GW neuron either a mitotic sister of EW3 or represents a single progeof GMC 3.

During late embryogenesis, stage 17, only three eg-lacZ-expressing descendants of NB 7-3 are detectable in a wild-tCNS (Figs 1D, 6A, 7); two are the serotonin cells and the thiEW3, we identify by its lateral position. Additionally, this thirdcell infrequently expresses Ddc and projects to the anteriorcommisure, similar to the EW1 and EW2 neurons (data shown). The combinatorial use of three molecular markepdm1, zfh-2and en has allowed us to uniquely identify all threeEW neurons. In a wild-type nerve cord EW1 expresses oen, EW2 expresses all three markers and EW3 expresseenand zfh-2 (Figs 6C,E,G, 7). The GW neuron is no longedetectable with eg-lacZduring late embryogenesis so we dnot know the expression of these genes in a wild-type Gneuron.

Our experiments with eg mutants confirm and extend theconclusions of Higashijima et al. (1996) showing that losseg function does not alter the number of NB 7-3 progeny, brather alters the specification of cell identity in this lineagAlthough eg mutations reduce the number of cells detectabwith DDC and serotonin (Figs 2, 3) all progeny of this lineaare detectable with eg-lacZ (Figs 6B, 7). Therefore, thespecification of serotonin cell fate is altered in eg mutants. Inthe absence of Eg protein, the mitotic sister serotonin neurbecome quite distinct. EW1 is much more sensitive to redu

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Eg, resulting in single serotonin cells in many hemisegme(Figs 2, 3). In contrast, serotonin synthesis in EW2 can completely independent of eg function since we observe thescells as pdm1- and zfh-2-expressing serotonin cells even innull allele eg18B (Fig. 5E,F).

The simplest explanation for the difference between EWand EW2 is that EW2 selectively contains a redundamechanism that allows continued synthesis of serotonin in absence of Eg protein. This redundant mechanism is not 10efficient since not all segments in an eg mutant CNS containserotonin cells. What specific factors are present in EW2, not in EW1, that might account for this redundancy? We hashown that pdm1and zfh-2are differentially expressed in EW2(Fig. 5A,B). Both proteins are potential transcription factorPdm-1 is a POU-protein (Billin et al., 1991; Dick et al., 199Lloyd and Sakonju, 1991) required for appropriadevelopment of the first ganglion mother cell in the RPlineage (Bhat et al., 1995; Yeo et al., 1995) and Zfh-2 ishomeodomain zinc-finger protein (Fortini and Rubin, 1990which binds to a serotonin cell-specific regulatory elementthe Ddc promoter (Lundell and Hirsh, 1992). Our results shthat zfh-2, but not pdm1, expression is affected in eg mutants(Figs 6, 7; Table 2). Therefore zfh-2may be a potential factorfor this redundant pathway that establishes eg-independentserotonin synthesis. The model in Fig. 7 presents the possibthat loss of zfh-2expression in EW2 is correlated with the losof serotonin synthesis in these cells. Definitive proof of thhypothesis will require direct examination of zfh-2mutants.

In this manuscript, we demonstrate that, in an eg loss-of-function mutant, the loss of Ddc expression is alwaysaccompanied by a loss of en expression, but can occuindependently in the two serotonin cells: in a hemisegmwhere both cells fail to express Ddc, neither cells show enexpression, in a hemisegment where only EW2 continuesexpress Ddc, EW2 shows enexpression but EW1 does no(Figs 6H, 7). Previously we have shown that enis required fordevelopment of the serotonin cells, with both serotonin ceaffected equally in an en loss-of-function mutant (Lundell etal., 1996). Recently it has been shown that in the sameenmutant eg expression is absent in NB 7-3 (Matsuzaki anSaigo, 1996). Therefore enand eg show different relationshipsduring the development of this lineage: at the neuroblast staenis required to maintain egexpression, whereas after divisioof GMC1, egis required to maintain enexpression in each cell.Continued expression of en and Ddc does not require egexpression, since eg expression ends at stage 13 (Higashijimet al., 1996).

The results presented in this manuscript are establishinhierarchy of genetic interactions that leads to the specificaof serotonin cell fate. Other genes that have been shownaffect development of the NB 7-3 lineage include wingless,hedgehog, patched, gooseberryand huckebein (Higashijima etal., 1996; Lundell et al., 1996; Matsuzaki and Saigo, 199Patel et al., 1989), but their specific contribution to tdifferentiation of serotonin neurons is unknown. Thexpression of egin only a small subset of neurons results CNS where the overall organization is well preserved in egmutants. This is not only beneficial for the study of cespecification but leads to the recovery of hypomorphic aduOur interesting finding that adult flies can survive with lolevels of serotonin, albeit with reduced locomotor activity, be

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for further analysis into the function of the ventral corserotonin neurons. Our identification of unique properties feach cell in the NB 7-3 lineage provides new tools for furthinvestigation into both the function and mechanisms that leto the specification of serotonin neurons during neurogene

The work was supported by NIH grant GM 27318 to JH. We thaJ. Urban and G. Technau for the eagle stocks, C. Doe, W. Chia A. Tomlinson for antisera, S. Britt and A. Cassill for laboratory spaand reagents at UTSA, and Claire Cronmiller and the members ofHirsh laboratory for helpful discussions and comments on tmanuscript.

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eagleis required for the speciﬁcation of serotonin neurons ... · the lineage of insect serotonin...

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