2923Development 121, 2923-2936 (1995)Printed in Great Britain © The Company of Biologists Limited 1995

Origin and specification of type II sensory neurons in Drosophila

Rachel Brewster and Rolf Bodmer*

Department of Biology, University of Michigan, 830 N University, Ann Arbor, MI 48109-1048, USA

*Author for correspondence: E-mail rolf@umich.edu

The peripheral nervous system (PNS) of Drosophila is apreferred model for studying the genetic basis of neuro-genesis because its simple and stereotyped pattern makesit ideal for mutant analysis. Type I sensory organs, theexternal (bristle-type) sensory organs (es) and the internal(stretch-receptive) chordotonal organs (ch), have been pos-tulated to derive from individual ectodermal precursorcells that undergo a stereotyped pattern of cell division.Little is known about the origin and specification of type IIsensory neurons, the multiple dendritic (md) neurons.

Using the flp/FRT recombinase system from yeast, we havedetermined that a subset of md neurons derives from esorgan lineages, another subset derives from ch organlineages and a third subset is unrelated to sensory organs.We also provide evidence that the genes, numb and cut, areboth required for the proper differentiation of md neurons.

Key words: Drosophila, peripheral nervous system, neurogenesis,sensory neuron, numb, cut

SUMMARY

INTRODUCTION

The peripheral nervous system (PNS) of Drosophila embryoshas provided many insights into the genetic mechanisms of howneural precursor cells are determined and assume a particulardevelopmental pathway of differentiation (for reviews seeCampuzano and Modellel, 1992; Ghysen and Dambly-Chaudiere, 1993; Jan and Jan, 1993). The model that hasemerged suggests that a given area of the early ectoderm wheresensory organs will develop becomes competent for producingneural precursors due to the action of ‘proneural’ genes, such asatonal and genes of the achaete-scute-Complex (AS-C) (e.g.Cabrera et al., 1987; Dambly-Chaudiere and Ghysen, 1987;Romani et al., 1989; Ruiz-Gomez and Ghysen, 1993; Jarman etal., 1993). Some of these genes are expressed in a limitednumber of ectodermal cells endowing each of these cells withthe potential to become a neural precursor. Another set of genes,the ‘neurogenic’ genes, such as Notch and Delta, are thenrequired to limit the number of cells that will become neural pre-cursors to one per cluster (Hartenstein and Campos-Ortega,1986; Goriely et al., 1991; Bodmer et al., 1993; for review onneurogenic genes see Artavanis-Tsakonas and Simpson, 1991;Campos-Ortega, 1993). Once a neural precursor has beensingled out, selector-type genes, such as cut and poxneuro(poxn), are required to initiate the correct developmentalprogram of a particular type of sensory organ (Bodmer et al.,1987; Blochlinger et al., 1991; Dambly-Chaudiere et al., 1992).Other genes are then responsible for specifying cellular identi-ties of sublineages of neural precursor cells. The gene numb, forexample, is required for the correct specification of second orderprecursor cells (Uemura et al., 1989; Rhyu et al., 1994; for othergenes affecting PNS development, see Salzberg et al., 1994).

Type I sensory neurons in Drosophila innervate the sensoryorgans to which they are related by lineage (Bodmer et al.,1989; Hartenstein and Posakony, 1989). Each of these sensoryorgans is thought to derive from a single ectodermal precursor(SOP) which gives rise to one or several monodendriticneurons and several support cells. Type I sensory organs havebeen classified into two major classes: mechano- or chemosen-sory organs that have external sensory structures in the cuticlesuch as bristles, campaniform and basiconical sensilla (esorgans) and chordotonal organs that are internally locatedstretch receptors (ch organs). In addition, the larval PNS alsocontains numerous type II neurons that have multiple dendrites(md neurons, Ghysen et al., 1986; Bodmer and Jan, 1987). Incontrast to es and ch neurons, md neurons (with one exception)do not seem to be associated with support cells. Md neuronsare thought to function as stretch or touch receptors. Threedifferent subclasses of md neurons have been distinguishedbased on their morphology (Bodmer and Jan, 1987): md-daneurons are the most abundant subclass and have extensivesubepidermal dendritic arborisations, md-bd neurons havebipolar dendrites and md-td neurons extend their dendritesalong tracheal branches. The origin and lineage relations ofthese cells with other PNS cells has not been established.Moreover, the genetic basis for md neuron differentiation ispoorly understood.

Mutations in some genes involved in sensory organ (esand/or ch) development have also been shown to affect theformation of md neurons. It is not clear, however, if mdneurons are affected independently of sensory organs or as aconsequence of alterations in sensory organ development. chand es organs are absent in atonal and AS-C mutants, respec-tively. Many md neurons are also missing in these mutants

2924 R. Brewster and R. Bodmer

(Dambly-Chaudiere and Ghysen, 1987; Jarman et al., 1993). Itis possible that md neurons have their own atonal- or AS-C-dependent precursor cells or, alternatively, md neurons mayderive from es or ch organ lineages. In the latter case, a failureto recruit es and ch SOPs will automatically result in the lossof md neurons.

In numb mutants, a normal number of es or ch SOPs areformed, but the second order precursor cells give rise mainlyto support cells instead of neurons and their glial-like siblingcells. Many md neurons are also missing in numb mutants(Uemura et al., 1989; Rhyu et al., 1994). The neural selectorgene, cut, is required for specifying es organ identity: in cutmutant embryos, the number of sensory organs and md neuronsis unchanged but es precursor cells develop as ch organs(Bodmer et al., 1987). In addition to the es organs, about twothirds of the md neurons also express cut (Blochlinger et al.,1990), many of them are in close physical association with esorgans. Interestingly, in deficiencies of AS-C all Cut-positivemd neurons are absent, whereas, in atonal mutants, a comple-mentary subset of (Cut-negative) md neurons is missing(Dambly-Chaudiere and Ghysen, 1987; Jarman et al., 1993; R.B. unpublished). In order to better understand the role of these(and other) genes in the specification of md neurons, it isnecessary to know their origins and lineages.

Observation of cell division patterns of sensory organs inother insects had suggested that the cells within an es or chsensory organ derive from a common precursor that dividesnear the organ’s final location (e.g., Wigglesworth, 1953;Lawrence, 1966; Jagers-Rohr, 1968; for review see Bate,1978). Due to the relatively small size of Drosophila cells, ithas not been possible to confirm these lineages by simple visu-alization of SOP divisions. BrdU labelling of Drosophilaembryos provided further insights concerning the divisionpattern of the putative SOPs (Bodmer et al., 1989). Exposureto BrdU at progressively later times resulted in fewer and fewerlabelled cells within the postulated sensory organ lineages,since many precursor cells had already completed their last S-phase (i.e. last possibility for BrdU incorporation) at the timeof exposure. A likely pattern of SOP division was inferred fromthe patterns of BrdU-labelled cells (Bodmer et al., 1989). Mdneurons were shown to be generated around the same time astype I SOPs, and it was speculated that they derive from typeII ectodermal precursors that divide close to their final position.The main drawback of BrdU incorporation studies with respectto SOP lineages stems from the inability to mark individualSOPs. Since all dividing PNS cells get labeled, the interpreta-tion of sensory organ lineages heavily relied on the underlyingassumption that the cells within a sensory organ lineage derivefrom a common precursor.

In order to reassess the lineages of type I sensory organs anddetermine the lineages of type II (md) neurons in the PNS ofDrosophila, we have used the flp/FRT site-specific recombi-nation system of yeast (Golic and Lindquist, 1989; Struhl andBasler, 1993). Using this method, we show that each type Isensory organ derives indeed from a single ectodermalprecursor cell. In addition, we have determined that many ofthe md neurons are part of the type I sensory organ lineages.Our results indicate that one subset of the md neurons is relatedto es organs, a second subset is related to ch organs and a thirdset does not appear to be related to either es or ch organs. Con-sistent with these proposed lineage relationships, we find that,

in numb mutants, the sensory organ related md neurons areusually absent or transformed into support cells. We also showthat, in cut mutant embryos, the identity of AS-C-dependent mdneurons (many of which are related to es organs) has changedto what seems to be characteristic of a subset of atonal-dependent md neurons. This suggests that AS-C and cut are notonly required for es organ formation, but also for specifying asubset of md neurons.

MATERIALS AND METHODS

Fly stocksThe fly stocks used to generate lacZ-positive clones were: hsp 70-flp(flipase construct inserted on the X chromosome, Struhl and Basler,1993) and Act-Draf-nuclacZ (construct inserted on the third chromo-some; Struhl and Basler, 1993). The Act-Draf-lacZ stock has a con-stitutive actin promoter separated from a lacZ reporter gene by asegment of DNA that contains a transcriptional stop codon and isflanked by two FRT repeats. The presence of Draf in this construct isincidental. The transformant flies were provided by G. Struhl.

In order to determine whether md neurons are affected in numbmutants, numbn7/CyO males (Uemura et al., 1989) were crossed tofemales from the E7-3-36 P-element enhancer trap line (second chro-mosome insertion), which marks all md neurons (Bier et al., 1989).Since the numb mutation and the E7-2-36 P-element insertion are bothon the second chromosome, recombinants from this cross wereselected (numb, E7-2-36/CyO).

Transformations of md neurons in cut mutants were assessed usingthe enhancer trap line, E7-3-49 (third chromosome insertion), whichmarks a subset of md neurons (Bier et al., 1989). This line was crossedinto a cut mutant background of the genotype: yw,ctdb7/FM7c. ctdb7

is a small deletion in the cut gene of ~1 kb that is homozygous lethaland considered a null mutation (Blochlinger et al., 1988).

Generation of lacZ-expressing clones in the embryolacZ-expressing clones were generated using the yeast flp/FRT site-specific recombination method according to Struhl and Basler (1993).Homozygous hsp 70-flp virgin females were crossed to Act-Draf-nuclacZ males. F1 embryos, hemizygous for hsp70-flp and heterozy-gous for Act-Draf-nuclacZ, were collected on grape plates with a dabof yeast and submitted to various levels of heat shock (28°, 30°, 32°or 34°C) for 30-40 minutes, at one of the following developmentalstages: 2-4 hours, 4-6 hours or 6-8 hours of development after egglaying. Heat shocks were administered either in a water bath or in anincubator in which water had been prewarmed to the desired temper-ature. The embryos were then allowed to develop at 18°C until theyreached 14-16 hours of development. They were then fixed andstained with antibodies. In order to avoid scoring the same clonestwice, the coordinates of each PNS clone were taken. Embryossubmitted to a mild heat-shock treatment (30°C) had less than 20clones and on average less than one clone per embryo that includedPNS cells embryos heat-shocked at 32°C had on average two or threePNS-containing clones. These two treatments were used in the vastmajority of the collected data. An excess of 5000 embryos was scored.

To test the efficacy of the flipase-mediated recombination, embryoswere heat shocked at 37°C for 30-40 minutes or given two consecu-tive 20-30 minute heat shocks at 37°C with an interval of 30 minutesat room temperature. With this regime, embryos expressed lacZ invirtually all cells. Another control consisted of raising the embryos at18°C until they reached 14-16 hours of development without heatshock. These embryos had very few lacZ-expressing clones (data notshown). Embryos bearing only the Act-Draf-nuclacZ construct werealso tested for lacZ expression (after heat shock for 30 minutes at32°C). These embryos did not express the reporter gene.

2925PNS lineages in Drosophila

Immunocytochemical staininghsp70-flp;Act-Draf-nuclacZ embryos or mutant embryos (for cut andnumb) marked with enhancer trap lines E7-2-36 or E7-3-49 weredouble labeled with a rabbit antibody against β-galactosidase (β-gal,Cappel, 1:2000) to monitor the lacZ-positive clones or md neurons,and one of the following antibodies: monoclonal antibody 22C10(1:100; all PNS neurons marked), monoclonal antibody 21A6 (1:10,labels scolopales and dendritic caps; Zipursky et al., 1984), anti-Prospero antibody (1:4, stains all thecogen and scolopale cells; Spanaand Doe, personal communication) or the antibody RK2 (1:500, stainsglial cells and ligament cells; Campbell et al., 1994). Embryos werethen incubated with the appropriate HRP-coupled secondary antibody(Biorad). The lacZ construct that we have used confers nuclear local-ization of this β-gal reporter gene product. Since the intensity of theanti-β-gal staining tended to mask that of 22C10, the two antibodieswere used sequentially. The staining procedure is essentially the sameas described in Bodmer and Jan (1987). In some cases, the DAB(Sigma) color reaction for one antibody was carried out in thepresence of 8% nickel chloride, which results in a black product thatcan then be distinguished from the brown DAB product. numbhomozygous mutants were recognized using the following criteria:absence of most peripheral neurons (determined with 22C10 anti-bodies) or absence and disorganization of md neurons (determined

Fig. 1. The embryonic PNS of Drosophila. (A) Diagram ofall the PNS cells in an abdominal hemisegment of a wild-type embryo. In blue: chordotonal (ch) organs, in yellow:external sensory (es) organs, in red: multiple dendritic neurons (md). NBodmer and Jan (1987) and Bodmer et al. (1989). Abbreviations: da, neinnervating neuron (drop shape); bd, bipolar dendrite neuron (triangle stormogen cell; l, ligament cell; s, scolopale cell, c, cap cell; a, attachme(see B) respectively. Anterior is always to the left and dorsal is up. (B) development), stained with the monoclonal antibody 22C10 (cytoplasmexpressing E7-2-36 enhancer trap line, nuclear staining). T, thoracic seghsp70-flp;Act-Draf-lacZ constructs (see Materials and Methods) that wclonally derived cells are revealed by staining with anti-β-gal antibodiebracket) and a mixed ectodermal and sensory clone (round bracket inditwo ectodermal cells).

with anti-β-gal staining). cut mutant embryos were recognized by thetransformation of dendritic caps into scolopales (determined with21A6 antibodies, see Bodmer et al., 1987).

RESULTS

Generation of small clones in the early embryousing the yeast flp/FRT methodThe PNS of Drosophila embryos is composed of numerous esand ch organs as well as md neurons that are arranged in asegmental, highly stereotyped fashion, allowing reliable identi-fication of all cellular components (Fig. 1A,B; Ghysen et al.,1986; Bodmer and Jan, 1987; Hartenstein, 1988). We havestudied cell lineages in the PNS by inducing random clones inthe early embryo using a site-specific recombination methodfrom yeast, the flp/FRT system (Golic and Lindquist, 1989;Struhl and Basler, 1993). Small clones of lacZ-positive cellswere generated randomly in embryos, before or during thedivision of PNS precursors (Fig. 1C, see also Materials andmethods). These clones were induced by activation of a site-

omenclature according to Ghysen and Dambly-Chaudiere (1986),uron with large dendritic arbors (diamond shape); td, tracheaehape); n, neuron; g, glial cell; th, thecogen cell; tr, trichogen cell; to,nt cell; v,v′,l and d refer to the two ventral, lateral and dorsal clustersLateral view of a stage 16 wild-type embryo (14-16 hours ofic staining) and a marker for multiple dendritic neurons (the lacZ-ments; A, abdominal segments. (C) Same stage embryo containing

as heat shocked for 30 minutes at 32°C at blastoderm stage. Nuclei ofs. This embryo contains two clones: a purely ectodermal clone (wavycates labelled cells of a single ch organ in lch5 and arrowhead points to

2926 R. Brewster and R. Bodmer

specific recombinase, flipase (flp), which fuses a constitutivepromoter (of actin) to the coding region of a lacZ reporter gene(constructs and transgenic flies of this system are described inStruhl and Basler, 1993). Prior to flipase expression, thesesequences are separated by two flipase recombination target sites(FRTs) in between which a stop codon is located. The lacZ geneproduct can therefore only be detected in the cells in which theintervening FRT/stop/FRT sequence has been recombined away.A heat-shock promoter-flp construct was used to control thetiming and the level of flp expression. A flp-mediated recombi-nation event can occur at any time after flp induction, thusmarking all or a subset of the cells that belong to a given lineage.

Fig. 3. lacZ-expressing clones in type I es and ch organs. Types of clonenuclear and 22C10 staining is cytoplasmic. (A,B) Two examples in whictormogen and trichogen) as well as a number of ectodermal cells (indica(note the neuronal dendrite between n/th and to/tr). (C) Only the thecogelabelled. Arrowheads point to the position of unlabelled es organ cells. (scolopale, neuron and ligament cell). (E) An ectodermal cell (ec) is also cell, described by Matthews et al. (1990) as an anchor point for ch organcell. (H) Clone composed of two cells: ch neuron and scolopale cell. Arr

We reasoned that comparison of larger with smaller clonesshould enable us to determine the sequence of cell divisions ofa given SOP. Since the SOPs of the embryonic PNS startdividing between 5 and 7 hours of development, we heat-shocked embryos at blastoderm (2-4 hours of development) tomaximize the labelling frequency of SOPs, or after gastrula-tion (4-6 or 6-8 hours of development) expecting to label ahigher proportion of SOP sub-lineages (Fig. 2). Embryos wereaged to 14-16 hours of development (stage 16, Campos-Ortegaand Hartenstein, 1985) and stained for lacZ expression. Allcells in clones that included PNS cells had approximately thesame level of staining of lacZ expression, which indicates that

Fig. 2. Variations of expected flp-induced clonesizes in a typical sensory organ lineage. The numberof labeled cells in a particular sensory organ lineagedepends on when during the lineage a recombinationevent took place. An early event at the precursorlevel or before is expected to label all cells that arerelated to a particular sensory organ. Later eventsshould produce progressively smaller clones, inwhich only the cells that are generated later in thelineage are labelled. Single cells can be labelled aswell.

s observed in es organs (A-D) and ch organs (E-H). β-gal staining ish all cells of an individual es organ are labelled (neuron, thecogen,ted by asterisk or line). In A, lesC is labelled; in B desD is labelledn and neuron and (D) only the tormogen and trichogen of desD areE,F) All cells of a single scolopidium of lch5 are labelled (cap,labelled that corresponds to the position of an ectodermal attachments. (G) Clone composed of three cells: ligament, neuron and scolopaleowheads point to unlabelled ch organ cells. Abbreviations as in Fig. 1.

2927PNS lineages in Drosophila

the flipase was not expressed preferentially in any given celltype (a representative embryo is shown in Fig. 1C). To helpidentify PNS clones, the embryos in some experiments weredouble labelled with the monoclonal antibodies 22C10 (aneuronal membrane marker) or 21A6 (which stains thedendritic caps of es organs and the scolopales of chordotonalorgans; Zipursky et al., 1984; see Bodmer et al., 1987).

External sensory (es) lineagesFirst we examined clones containing only es or ch organ cells,because the outcome of these studies could be compared withthe findings of previous lineage studies of the embryonic PNS(Bodmer et al., 1989). The cells of es organs tend to be tightlygrouped together. We chose to concentrate on the lineages oftwo dorsal es organs (desC/D) and one lateral es organs (lesC)since individual cells in these organs are spaced further apart(see Fig. 1A for exact location within a segment). The neuronwas easily recognized with the use of the 22C10 marker andthe neuron-associated glial cell, the thecogen, was localized onthe basis of its vicinity to the neuron along its dendrite. Thesupport cells, the tormogen and trichogen, were distinguishedbased on their position adjacent to the 21A6-positive dendriticcap, which is located at the tip of the dendrite at the base ofthe future cuticular structure associated with the sensory organ(Hartenstein, 1988).

Three types of clones were observed in desC/D and lesC(Fig. 3A-D; Table 1). 42 clones were composed of all es organcells: neuron, thecogen, tormogen and trichogen (Fig. 3A,B).This indicates that the cells within es organs in Drosophilaembryos are indeed clonally related. Many of these clones,

Table 1. Number oes organs

n, th, to, tr n, the to, tr n(s)

des C + D 29 10 10 0les C 13 3 0 0lesA + ldaA 1 2 0 0lesB + ldaB 0 1 0 0v′esB + v′ada 0 0 0 0v′es2 + v′pda* 1 1 5 17

ch organs

l, n, s, c l, n, s n, s

lch5 82c 25 34

ch organs

n, s, c** n, s s, c

vchA + v′td(v)*** 1 0 0

vchB + v′td(d)*** 1 12e 3

*v′es2 is a poly-innervated es organ (2 neurons).**The ligament cell was not identifiable in the ventral ch organs.***In rare cases (2% of the clones) the td neurons might have been confused daIn 1 or more cases other es organ cells might have been faintly stained.bIn 17 cases a group of cells in a ventro-lateral position to v′pda were also labecIn approx. 40% of the cases, an ectodermal cell dorsal to the cap cell was alsodOnly the neuron was labeled.eColabelling of n ans s, 3 cases; n alone, 4 cases; s alone, 5 cases.fIn 24 cases the neuron was colabeled with v′td(d).

however, also included a few labelled ectodermal cells(asterisks in Fig. 3) indicating that the recombination eventtook place in an ectodermal cell that underwent one or morerounds of cell divisions before the SOP was formed. 13 cloneswere composed of neuron/thecogen (Fig. 3C) and 10 cloneswere composed of tormogen/trichogen (Fig. 3D; Table 1).These findings are consistent with the previously reportedBrdU data (Bodmer et al., 1989) indicating that es organs aregenerated through two sets of cell divisions: neuron/thecogenderive from one SOP daughter cell, tormogen/trichogen fromthe other (Fig. 5A). Individually labeled es organ cells werealso observed, consistent with a late recombination event (seeFig. 2).

Chordotonal (ch) lineagesAn abdominal segment is composed of three single ch organs(v′ch1, vchB and vchA) and a cluster of five ch organs (lch5)(Fig. 1A). Unlike most es organs, the cells of ch organs areneatly arranged in a row. Whereas the ligament cells of the chorgans (scolopidia) in the lch5 cluster are clearly visible, thisis not always the case for the ligament cells in v′ch1, vchB andvchA. We therefore restricted our analysis of ch lineages to thelch5 cluster.

The majority of the clones that we observed in the lch5cluster (82 cases) were composed of all four cells within onescolopidium (ligament, neuron, scolopale and cap cell). Theseclones frequently included a few ectodermal cells, as was alsoobserved for es organ clones (Figs 1C, 3E,F; Table 1). Thisargues in favor of a clonal relationship between the cells ofindividual ch organs. 34 clones consisted of the neuron and

f lacZ + PNS clonesmd (da) neuron ± es organs

md + es (all) md + es (n + th) md + es (n) md alone

0 0 0 00 0 0 0

27 0 4a 221 2a 0 433 0 0 168b 12 78b 27

md (td) neuron + ch organs

c md + ch (n, s, c) md + ch (n, s) md alone

26c 0 0 0

c

2 13 + vpda 4 + vpda12 − vpda 1d − vpda

14 v′td9 1 + vpda 4 + vpda56 − vpda 29f − vpda

ue to variability in their position.

led. labeled.

66

66

2928 R. Brewster and R. Bodmer

Fig. 4. lacZ-expressing clones that include type II md neurons.(A-F) md/es clones. (G-J) md/ch clones. (K-M) Solo-md clones.(A-D) Two-segment-wide micrographs with anti-β-gal-labelledclones including all cells of an individual es organ (brackets indicateposition of labeled es organ cells and unlabeled es organ in adjacentsegment) as well as one md neuron (indicated by arrowhead, see Fig.1A for PNS map). (A) ldaA/lesA, (B) ldaB/lesB, (C) v′ada/v′esB and(D) v′pda/v′es2. (E,F) Two-segment-wide micrograghs consisting ofclones composed of v′es2 neurons with (E) and without (F) co-labelling of v′pda (arrowhead). This suggests that this md neuronderives from the same sublineage as the two v′es neurons. (G-I)Clones that include all cells of a ch organ. (G) Labelling of vchA,vchB (brackets), v′td2 (arrowheads point to dorsal (d) and ventral (v)cell and vpda (small arrow) due to an early recombination event).(H) Clone composed of all vchA cells, v′td(v) and vpda. (I) Clonecomposed of all vchB cells and v′td(d). (J) Clone composed of onlythe vchB neuron and v′td(d). (K) Clone composed of dbd neuron(black arrowhead) and associated glial cell (curved arrow). Anunstained dbd neuron in an adjacent segment is indicated by an opentriangle. (L) Two clones that include vmd5 neurons in two adjacentsegments. In the clone on the left three vmd5 cells are labelled, onthe right only one cell of vmd5 is labelled (position of the labelledmd neurons is indicated by arrowheads). (M) vpda is the onlylabelled cell in the right-hand segment (arrowhead). Open triangleindicates position of an unlabelled vpda in adjacent segment.

scolopale cell (Fig. 3H; Table 1) indicating that they are siblingcells. 25 cases clones were composed of three cells that con-sistently included neuron, scolopale and ligament cell (Fig. 3G;Table 1), which is indicative of a serial mode of ch SOPdivision: the cap cell is produced first then the ligament celland finally neuron and scolopale cell (Fig. 5D). In this model,the ligament/neuron/scolopale derive from a common secondorder precursor. BrdU-labelling studies also suggested a serialdivision pattern for ch SOPs; however, the ligament cell wasthought to be produced first. This sequence of cell divisionsimplies that cap/neuron/scolopale are generated from acommon second order precursor (Bodmer et al., 1989). Thesetwo contradictory observations can be reconciled in a modelwhere the first ch SOP division produces two second order pre-cursors, one that gives rise to the ligament/neuron/scolopalecell lineage, and the other that generates the cap and another(ectodermal) cell. This other, cap/ectodermal cell precursor,then replicates somewhat later than the first division of thesecondary precursor for the ligament, neuron and scolopale cell(see Fig. 5D). Consistent with this model is the observationthat an ectodermal cell, dorsal to the cap cells, was often co-labelled in clones that expressed lacZ in all cells of a scolo-pidium (ec-labelled cell in Fig. 3E). In 40% of the cases, whereonly the cap cell was labelled to the exclusion of other ch cells,this dorsal ectodermal cell was also labelled (Table 1). Indeed,two ectodermal cells have been identified in the position ofthese cap-related ectodermal cells as attachment cells for lch5(Matthews et al., 1990; see also Fig. 1A). In many cases,however, it seems the cap-related ectodermal cells fail to dif-ferentiate in this ectodermal position (perhaps because theydegenerate), thus escaping detection at the stage of analysis. Inaddition, we have observed one or two labelled ectodermalcells in embryos that have incorporated BrdU at the time ofPNS neurogenesis (R. B. and R. B., unpublished). These cellswere in the same position as the ch attachment cells and onlylabelled when cap cells were also labelled. These observationsfurther support the proposed model of ch lineages (Fig. 5D).

A considerable number of clones (29) observed in the lch5cluster consisted of two or more fully labelled scolopidia (indi-vidual ch organs) (data not shown). Since the frequency ofclones observed in the PNS is relatively low, most if not all ofthese larger clones are probably not due to independent recom-bination events. This raises the possibility that two or morescolopidia within the lch5 cluster derive from a commonprecursor. Alternatively, the labelled scolopidia may have orig-inated from a recombination event in an early ectodermal cell(at blastoderm stage), which, after further cell divisions, gaverise to independent ch SOPs (within the lch5 proneural region).Clones of less than four cells per ch organ were never observedwhen more than one scolopidium of lch5 was labelled.Therefore, it is unlikely that swapping of equivalent cellsamongst neighboring scolopidia occurs with appreciablefrequency.

Md cell lineages Md neurons constitute the third class of sensory neurons in theDrosophila PNS. The majority of these neurons have extensivedendritic arbors below the epidermis. Md neurons are easilyidentifiable by location and shape, using the 22C10 marker.Some md neurons are in close association with es or ch organs(see Fig. 1A). We explored the possibility that these md

neurons could be related by lineage to their neighboring typeI sensory organs. A few PNS cells have been shown to migrateaway from their point of origin. For example, lch5 originatesin the dorsal region and ends up more laterally (Bodmer et al.,1989; Salzberg et al., 1994), and v′ch1 originates in a ventralcluster and migrates dorsally (Ghysen and O’Kane, 1989).Therefore, we scored all subectodermal cells that were labelledclose to and at a distance from a labelled md neuron. Ourresults suggest that md neurons derive from three types oflineages (summarized in Fig. 5). One type of lineage probablygives rise to md neurons exclusively (termed ‘solo-md’lineage), whiles the others produce md neurons and es organs(‘md/es’ lineage) or md neurons and ch organs (‘md/ch’lineage).

Md/es lineagesSeveral md neurons of the da subclass are located adjacent toes organs (Fig. 1A). In practically every case (129 out of 131cases), when all cells of one of these es organs were labelled,the neighboring md neuron was also labelled (Table 1):lesA/ldaA (27 cases, Fig. 4A), lesB/ldaB (21 cases, Fig. 4B),v′esB/v′ada (33 cases, Fig. 4C), v′es2/v′pda (68 cases, Fig.4D). This strongly suggests that a subset of md neurons aredescendants of es SOPs. Since lesA, lesB and v′esB aremonoinnervated es organs and v′es2 is a polyinnervated esorgans, we will refer to these lineages as md/mono-es andmd/poly-es, respectively.

In order to determine the pattern of cell division of the cellsbelonging to the md/mono-es lineages, we scored clones inwhich only part of the md/es cells were labelled. Forv′esA/v′ada, 2 cases consisted of the es neuron, thecogen andmd neuron; and for ldaA/lesA, 4 cases consisted of the esneuron and md neuron (Table 1). This suggests that the mdneuron derives from a second order precursor, which alsogenerates the es neuron and thecogen, probably by an addi-tional division of the neuronal precursor. Due to the smallnumber of such clones, the order in which the md neuron, es

2929PNS lineages in Drosophila

K

2930 R. Brewster and R. Bodmer

Fig. 5. PNS lineages. (A-F) Proposed PNS lineages. Open symbols:precursor cells. Filled symbols: postmitotic cells. List of cellsbelonging to lineages A through F is given below lineage diagrams.Question marks indicate uncertainty about lineage classification. (A) Lineage of ‘pure’ es organs. (B) Lineage of md neurons relatedto mono-es organs. (C) Lineage of md neurons related poly-esorgans. (D) Lineage of ‘pure’ ch organs. (E) Lineage of md neuronsrelated to ch organs. vpda*: part of v′td(v)/vchA lineage only (Fig.4M) or together with vchA (Fig. 4H). Thus, vpda is likely to be asolo-md neuron that may be generated independently but in closevicinity of the vchA precursor. (F) Lineages of solo-md neurons.

neuron and thecogen cell are produced could not be determinedunequivocally.

The situation is similar for poly-es neurons (Table 1): in 12cases, we observed that the md neuron (v′pda) was co-labelledwith the thecogen cell and the two es neurons of v′es2. Thissuggests that, in md/poly-es lineages, the md neuron is alsogenerated from a second order precursor cell derived from thefirst division of the v′es2/v′pda precursors. In 78 clones, themd neuron and both es neurons co-labelled exclusively (Fig.4E). Co-labelling of the es neurons and the thecogen to theexclusion of the md neuron was almost never observed (Table1). This means that, in the case of md/v′es2, the md and esneurons are generated by additional divisions of the neuronalprecursor. The two v′es2 neurons were stained alone in 17cases (Fig. 4F) and the md neuron was labeled alone in 27 cases(Table 1), suggesting that the es neurons are siblings. In 5cases, we observed that only the tormogen and trichogen wereco-labelled suggesting that they are also siblings (Table 1). Weconclude that the first SOP division gives rise to thetormogen/trichogen precursor and the precursor for thethecogen cell and the md/es neurons. The latter secondaryprecursor then generates the thecogen cell and the precursorfor the md and es neurons. These lineages are consistent withthe BrdU-labelling studies, which show that thetormogen/trichogen precursor replicates first, followed by theprecursor of the thecogen and the neurons. In the case ofmd/v′es2, the precursor of the two es neurons replicates last. Itis likely that the division pattern for an md/poly-es SOP isessentially the same as the division pattern that generatesmd/mono-es, except that the precursor for the neurons of v′es2undergoes another division to generate two es neurons. Theproposed lineages for md/mono-es and md/poly-es are sum-marized in Fig. 5B,C.

Md/ch lineagesTwo types of clones that contained co-labeled md neurons andch organs were observed: one type of clone was composed ofthe ventral v′td2 (an md neuron belonging to the td subclass)and vchA; the other type of clone included the dorsal v′td2neuron and vchB (see Fig. 1A). The v′td2 neurons are locatedat some distance from vchA or vchB in the v′ cluster. Table 1(bottom half) shows the number and types of chordotonalorgan-associated md clones that we have observed. Since theligament cells of vchA and vchB were not easily visible in mostclones (too close to the ch neurons), we excluded them fromthe analysis. Clones composed of an md neuron and all cellsof a vch organ were observed in 82 cases (Fig. 4H,I; Table 1).These data suggest that a subset of md neurons derives fromch organ lineages. The other abdominal ch organs, lch5 andv′ch, do not seem to have lineage relations to md neurons. Insome embryos, we observed vpda, an md neuron belonging tothe da subclass, co-labelled with v′td/vchA clones or withclones including both v′td/vchA and v′td/vchB organs (Fig.4G,H). As discussed below, this probably means that an earlyrecombination event generated several neighboring PNS pre-cursors, similar to the situation with lch5.

In order to determine more precisely the relation betweenvpda and v′td2 to vch organs, we scored smaller clones. 29md/vchB clones were composed of the dorsal v′td2 and apartially labelled ch organ (i.e. the neuron and scolopale werelabelled to the exclusion of the cap cell). In most of these cases,

the ch neuron alone was co-labelled with the dorsal v′td2 (Fig.4J). In contrast, only 3 cases showed the vchB neuron andscolopale cell not co-labelled with the dorsal v′td2. Theseobservations suggest that the md/vchB lineage is likely to besimilar to other ch organ lineages, the only difference beingthat the ch neuron undergoes another cell division to generatean md neuron (Fig. 5E). The md/vchA lineage is most likelyidentical to the md/vchB lineage with respect to the td neuron(Table 1), but it is unclear if the other vchA-associated mdneuron, vpda, is indeed part of the md/vchA lineage. Only 13out of 25 cases show co-labelling of vpda with the othermd/vchA cells. This means that vpda is either generated firstin the md/vchA lineage or that it arises from an independentectodermal precursor in close proximity to the md/vchAprecursor (Fig. 5E). Consistent with the latter interpretation isthe observation that vpda is occasionally co-labelled with avchB clone (5 cases) and often by itself (40 cases) (Fig. 4M).We classify vpda tentatively as a solo-md neuron (see below).

Solo md lineagesA number of md neurons were not usually or consistently co-labelled with other sensory organ cells. These md neurons arethe dorsal bd neuron (dbd), the da neurons in the ventral vmd5cluster and possibly a few da neurons in the dorsal cluster (seeFig. 1A). 48 clones were observed that labelled the dbd neuronand/or its neighboring glial cell. 20 of these clones label boththe dbd neuron and the glial cell, excluding other PNS cells(Fig. 4K), which suggests that they are sibling cells and that

2931PNS lineages in Drosophila

they derive from their own ectodermal precursor (Fig. 5F, top).This is further supported by BrdU-labelling studies, whichshowed that these cells divide around the same time and arethus likely to be siblings.

The clones that include vmd5 neurons were quite hetero-geneous, varying between 1 and 4 labelled vmd5 neurons (Fig.4L), often in conjunction with ves organs. In 19 cases, vmd5neurons were labelled to the exclusion of other PNS cells and,in 19 other cases, vmd5 neurons were co-labelled with vesA,vesB or vesC. There did not seem to be a consistent pattern inwhich es organs were most frequently co-labelled with vmd5neurons. Although we can not rule out a complex lineage rela-tionship of ves organs and vmd5 neurons, it seems most likelythat the vmd5 neurons are generated from md-specific precur-sors, in the vicinity of ves SOPs (Fig. 5F). Some or all of thedorsal md neurons are probably generated by a similarmechanism although clones including these cells have not beenquantified (since they are difficult to identify individually).

Specification of md neurons is altered in numb andcut mutantsmd neurons in numb mutantsIt has been suggested that in numb mutants the first SOPdivision generates two identical second order precursors,which results in the overproduction of sensory organ supportcells (tormogen/trichogen) at the expense of neuron/thecogencells (Uemura et al., 1989; Rhyu et al., 1994). Our lineageanalysis suggests that md neurons related to es or ch organsare descendants of the neuronal second order precursors (Fig.5B,C,E). In numb mutants, we would thus expect that theseSO-related md neurons are absent. To test this hypothesis, wehave crossed an enhancer trap line, which expresses the lacZreporter gene exclusively in all md neurons (E7-2-36; Bier etal., 1989), into a numb mutant background. Embryos doublylabelled for all PNS neurons and for lacZ expression show thatthe sensory organ-associated md neurons are usually missingin numb mutants (Fig. 6A,B). Although this observation is con-sistent with a lineage relationship between a subset of mdneurons and sensory organs, one can not rule out that the mdneurons are affected independently by numb as well. This issupported by the observation that the solo-md neurons, vpdaand some vmd5 (and several dmds) were often missing innumb (Fig. 6B).

The transformation of the neuron and thecogen cells intotormogen/trichogen-like cells is frequently incomplete (seeUemura et al., 1989). We reasoned that, if the es-related mdneuron (md/es) and the es neuron derive from the same secondorder precursor lineage, both es and md/es neuron should eitherbe present (incomplete transformation) or absent (completetransformation). Embryos, doubly labelled for all PNS neuronsand for lacZ reporter gene expression in md neurons, show thatthis prediction was correct: both the es and md/es neuronseither did or did not express neural markers simultaneously(Fig. 6B). Similar observations were made in numb mutantembryos that were doubly labelled for md neurons andthecogen cells: every time an md/es neuron was present theneighboring thecogen cell was also labelled (Fig. 6C,D). Inter-estingly, two or three cells positive for the thecogen markerwere frequently present in the location of md neurons/md-related es organs (indicated by brackets in Fig. 6D). Thissuggests that the first SOP division appears to be normal in

some cases, but the secondary precursor for the thecogen andneurons now generates only thecogens (Fig. 6G, top panel).These results also indicate that numb not only acts in generat-ing asymmetry during the first SOP division but also duringboth secondary SOP divisions (see also Rhyu et al., 1994).Since our md marker is only weakly expressed in md-tdneurons associated with ch organs, we have not examined theirfate in numb mutants. Taken together, our analysis of numbmutants strongly supports the conclusions from the lineagestudies.

To determine if numb also affects other md lineages, weexamined the fate of solo-md neurons in these mutants. Weobserved only 2-3 neurons in vmd5 or the dorsal cluster. Dueto the lack of other markers, we do not know if a hypotheticalsolo-md precursor divides less or if the fate of its progeny isaltered. The md-bd neuron of the dorsal cluster is also absentin numb mutants (Fig. 6E,F). Our lineage studies suggest thatdbd is generated from a dbd-specific ectodermal precursor,which divides once to give rise to a glial cell and dbd (Fig. 6E).In numb mutants, instead of one dbd neuron and one glial cell,two glial cells are formed at the expense of the dbd neuron(Fig. 6F,G, lower panel). Therefore, numb not only affects thees and ch lineages but also other components of the PNS.

It had previously been shown that in numb mutants an excessof cap cells are produced but the fate of the ligament cell wasnot clear. Since lch5 neurons are rarely formed in numbmutants (Uemura et al., 1989), we expected that the secondorder precursor is often transformed into a cap-like precursor.Consistent with our proposed ch lineages, we find that in lch5not only neurons and scolopales are missing but also theligament cells (Fig. 6E,F).

cut mutant phenotypeThe da subclass of md neurons appears to be a morphologi-cally homogeneous population of cells (Bodmer and Jan,1987). The observation that these cells are generated bydifferent types of lineages (this study) and that the axonal pro-jections to the CNS are not identical for all da-md neurons(Merritt and Whitington, 1995), raises the possibility that thereare distinct subpopulations of da neurons. To address thisquestion and identify genes that may play a role in the speci-fication of md neurons, we sought for cell markers that differ-entially label subsets of da neurons. One such marker is the cutgene product. cut is first expressed in es SOPs and later in theirprogeny including the es-related md-da neurons (see Figs 5B,C,7C; Blochlinger et al., 1990). Since cut functions as a devel-opmental switch to specify the correct identity of es organs(Bodmer et al., 1987; Blochlinger et al., 1991), we wonderedif cut may also be involved in the specification of da neurons.In cut mutants, the morphology and position of md neurons isnot affected (data not shown). To test for more subtle changes,we used an enhancer trap line, E7-3-49 (Bier et al., 1989),which marks a subset of md neurons that is non-overlappingwith the Cut-positive da neurons: vpda, dbd and 3-4 dorsal daneurons (Fig. 7A,D). At least one of the cells marked with E7-3-49 (vpda) is atonal-dependent (Jarman et al., 1993).

The lacZ expression of the E7-3-49 enhancer trap line is dra-matically altered in cut mutant embryos. In addition to the cellsin which it is normally expressed, β-gal staining is nowobserved in the da neurons that are normally Cut positive: alldorsal cluster da neurons, ldaA&B, v′ada, v′pda and 3-4

2932 R. Brewster and R. Bodmer

Fig. 6. Cell fate changes of md neurons in numb mutants. (A-F) Two segments of wild-type (A,C,E) and numb mutant (B,D,F) embryos atstage 16 are shown. (A,B) Embryos double-labelled for nuclear md-specific lacZ expression of the enhancer trap line, E7-2-36 (Bier et al.,1989), and 22C10 (cytoplasmic labelling of PNS neurons). Arrowheads indicate md neurons. Dotted lines indicate vmd5. Open trianglesindicate lesA and v′esB neurons which are also occasionally present in numb mutant embryo (B). In B, left arrowhead points to v′ada and rightarrowhead to ldaA (in an adjacent segment); asterisks indicate missing md neurons. (C,D) Embryos double-labelled with thecogen/scolopale-specific anti-Prospero antibodies (black, Vaessin et al., 1991) and for md-specific lacZ expression of the E7-2-36 line (brown). Arrowheadsindicate md neurons. Open diamond indicates the Prospero-positive thecogen cell of v′esB. Asterisks indicate missing md/es neurons. Bracketsshow md/es organs with 2-3 Prospero-positive cells indicating transformation of the md and/or es neuron into thecogen cells. (E,F) Embryosdouble-stained with 22C10 antibodies (brown) and glia-specific anti-RK-2 antibodies (Campbell et al., 1994) (black). Arrowhead indicates adbd-md neuron and the curved arrow the dbd-related glial cell (black). RK-2 antibodies also label the ligament cells of lch5 (outlined by dottedlines in E), which are absent in numb (F). Note the transformation of the dbd neuron into an RK-2-positive glial cell in numb (F). (G) Diagrammatic representation of the lineage changes associated with the numb mutation. Upper panel: either the first SOP division producestwo equal secondary SOPs (a,a′) giving rise to only support cells (no neuron or thecogen cells are formed), or the secondary SOP (b) is formedbut gives rise to another thecogen precursor (b′) in addition to the normal thecogen cell. Lower panel: dbd/glia lineage in wild-type and numbmutants.

neurons of vmd5 (Fig. 7A-E). We conclude from these obser-vations that there are at least two subclasses of da neurons, Cut-negative and Cut-positive da neurons, and that cut is likely tobe involved in specifying the identity not only of es organs butalso of a subpopulation of da neurons (Fig. 7F).

DISCUSSION

Lineage relationships in the PNSThe analysis of patterns of cell division in a number of insectsand BrdU-labelling studies in Drosophila embryos and wingimaginal discs have provided evidence that the cells withinindividual es and ch organs are derived from single precursorcells (reviewed in Bate 1978; Bodmer et al., 1989; Hartensteinand Posakony, 1989). The pattern of BrdU incorporation in

embryos was suggestive of a cell division pattern that wasdifferent for es organs and for ch organs (Bodmer et al., 1989).Using the yeast flipase method to generate small clones in theembryo (Golic and Lindquist, 1989; Struhl and Basler, 1993),we have reassessed the lineages of type I sensory organs (esand ch) and examined the lineages of type II sensory neurons(md neurons) and their relationship to type I sensory organs,by scoring an excess of 5000 embryos.

es and ch organ lineagesWe scored clones that included cells of the dorsal most esorgans (desC/D) and the lateral ch organs (lch5). We observedthat a majority of clones were composed of all cells belongingto an es or ch organ, which indicates that es and ch organsderive from individual SOPs. The cellular compositions ofclones that included only a fraction (but more than one) oflabelled cells within an individual es or ch organ allowed us to

2933PNS lineages in Drosophila

Fig. 7. Identity changes of md neurons in cut mutants. (A,B) Two abdominal segments of stage 16 embryos stained for lacZ expression of theenhancer trap line, E7-3-49, which labels most md neurons that are Cut-negative (see C,D), and with 21A6 (Zipursky et al., 1984), which isspecific for es-associated dendritic caps and ch-associated scolopales. Segment boundary cells are also labeled in this enhancer trap line (arrowsindicate the position of the segment boundary). The wild-type pattern of β-gal-positive md neurons is indicated by arrowheads in one of thesegments of A and B. md neurons that ectopically express lacZ in cut mutants (pointed out by asterisks in B) are in the identical position of mdneurons that normally express the Cut protein (C, symbols in blue). A diagrammatic representation of the wild-type expression pattern of theE7-3-49 line is given in D (symbols in red indicate position of lacZ-positive mds). (E) The lacZ expression pattern of the E7-3-49 line isexpanded in cut mutants, to encompass virtually all md neurons, suggestive of a change in identity of the md neurons that are normally Cut-positive. The level of ectopic lacZ expression is variable. (F) cut mutant phenotype in the PNS: not only are the es organs transformed into chorgans (see Bodmer et al., 1987), but the subset of md neurons that is normally Cut-positive (mdes and mdsolo) is also transformed into E7-3-49-positive md neurons (mdE7) (see text).

infer the most likely lineage relationships. For es organs, weobserved co-labelling of either the neuron/thecogen or thetormogen/trichogen, confirming the previously proposeddivision pattern for es SOPs (Fig. 5A; Bodmer et al., 1989).For ch organs, we observed the following combinations oflabelled cells: ligament/scolopale/neuron, cap/attachment cellor scolopale/neuron. This suggests a division pattern for chorgans that is a modification of what had been previouslyproposed, but which is also consistent with the BrdU studies(illustrated in Fig. 5D). In this model, the two second orderprecursors replicate at different times: the ligament/scolopale/neuron precursor does so before the cap and attach-ment cell precursor. In agreement with the proposed ch lineagepattern is the finding of several clones in which the cap andattachment cell are co-labelled (Table 1), indicating that therecombination event took place in the immediate precursor ofthese two cells.

A relatively large number of multiple ch organs was labelledin lch5 (the lateral chordotonal cluster), suggesting that ‘superSOPs’ could give rise to more than one ch organ. It has previ-ously been suggested that there are at least two ch organ pre-cursors for lch5 that emerge during early stage 10 (Ghysen andO’Kane, 1989). Since BrdU-labelling studies have shown that

the precursors that give rise to lch5, divide in a graded fashion,always proceeding from anterior to posterior in each segment(Bodmer et al., 1989), we expected that clonally related chorgans would be adjacent to one another. However, the numberof adjacent scolopidia that were co-labelled was far lower thanthe number of non-adjacent clones (R. B. and R. B., unpub-lished). Moreover, the clones observed were composed ofrandom combinations of scolopidia (identified by their positionalong the anterior-posterior axis). This makes it unlikely thatmultiple scolopidia in lch5 are generated by a fixed lineagepattern but rather argues in favor of the existence of multipleprecursors (possibly five) for the lch5 cluster. We speculatethat these precursors emerge independently, in close vicinityto one another, in the posterior lateral region of each segment(Ghysen and O’Kane, 1989). One cannot rule out, however,that clonally related precursor cells rearrange themselvesrandomly after they have been generated.

Multiple md lineagesThree types of clones were observed that included md neurons:a subset of md neurons that were almost always co-labelled withclosely juxtaposed es organs (md/es), another subset of mdneurons that co-labelled with ch organs (md/ch) and a third

2934 R. Brewster and R. Bodmer

group of md neurons that did not co-label with other sensoryorgans in a consistent and reproducible pattern (solo mds). Weconclude from this clonal analysis that es- and ch-associated mdneurons are related to these sensory organs by lineage and thatthey share a common SOP (lineages are summarized in Fig. 5).

Clones that labeled all cells of the es organs, v′es2, v′esB,lesA and lesB, always included the associated md-da neuron,whereas the chordotonal organs vchA and vchB co-labelledwith one of the v′td2 md neurons. We inferred the patterns ofcell division from smaller clones where only a fraction of asensory organ was labelled (see Fig. 4; Table 1): SO-relatedmd neurons derive from secondary SOPs that also give rise tothe SO neuron(s).

The vpda md neuron often co-labelled with vchA and vmd5neurons often co-labelled with one of the ves organs. These mdneurons may also have lineage relations to sensory organs,possibly generated by an early ectodermal division (see Fig.5E). As we argued for co-labelling of multiple scolopidia oflch5, it is likely that the high frequency of co-labelledvpda/vchA or vmd5/ves simply reflects the close proximity ofindependently emerging SOPs, and is not due to a necessarylineage relation. This possibility is supported by the findingthat in mutations of the rhomboid gene, vchA is usually absentwithout the concomitant deletion of vpda (Bier et al., 1990).Therefore, we classify vpda and vmd5 as solo-md neurons. Thedorsal bd neuron and its sibling glial cell were never consis-tently co-labelled with SO cells and was therefore also classi-fied as a solo md neuron.

The putative es-related md neurons also happen to be amongthe md neurons that are missing in AS-C mutants (Dambly-Chaudiere and Ghysen, 1987). This suggests that genes of AS-C are required for the formation of precursors common to esorgans and a subset of md neurons. Similarly, the atonal geneseems to be required for the precursors common to vchA,vchB, the v′td2-md neurons and vpda (Jarman et al., 1993).

Fate of md neurons in lineage mutantsIn mutants of the numb gene, the second order precursor of esorgans which gives rise to the neuron and thecogen cell isusually transformed into its sibling, the tormogen/trichogensecond order precursor (Uemura et al., 1989). Our proposedmodel of md/mono-es lineages predicts that the fate of the mdneurons related to es organs may also be altered in numbmutants since these md neurons appear to have the samesecond order precursor as the neuron and thecogen cell. Thefinding that most md neurons are absent in numb mutantssupports this finding, but it could be argued that md neuronsand sensory organs are affected independently. Close exami-nation of partially transformed md/es organs (see also Uemuraet al., 1989) indicate that, whenever a thecogen cell and an esneuron is formed, the associated md neuron is formed as well(Fig. 6B). This strongly supports that es-related md neuronsderive from the same second order precursor as neuron andthecogen cell.

numb not only affects the lineage of es and ch organs in thePNS but also that of solo-md neurons, since most of them areabsent (Uemura et al., 1989). Although the fate of most solo-mdneurons in numb mutants could not be determined due to lackof specific markers, the dorsal bd neuron (dbd), is a notableexception. In numb mutants, dbd is transformed into its associ-

ated glial cell, consistent with them being siblings and requiringnumb for distinguishing between a neuronal and glial cell fate.

In poxn mutants, the poly-es organs are transformed intomono-es organs (Dambly-Chaudiere et al., 1992). At least forv′es2, the formation of the associated md neuron is not affectedin these mutants (C. Dambly-Chaudiere and A. Ghysen,unpublished data). This means that, although the identity of thewhole v′es2 has changed in poxn mutants, the only lineagedefect concerns the immediate precursor of the es neurons.

Md neurons have previously been classified into three broadcategories: da neurons characterized by large dendritic arrays,td neurons whose dendrites extend along trachea and bdneurons which have bipolar dendrites (Bodmer and Jan, 1987).Our clonal analysis of the PNS suggests that md neurons canbe further classified into different subtypes defined by lineage.Two markers, Cut and E7-3-49 label non-overlapping subsetsof md neurons. In addition to es organs, cut is expressed in thees-related da neurons and some of the potential solo da neurons(e.g. in vmd5). E7-3-49 expresses the lacZ reporter in a set ofda neurons that is essentially complementary to the cut-expressing da neurons (Fig. 7C,D). In cut mutant embryos, theE7-3-49 driven lacZ expression in md neurons is expanded toencompass virtually all da-md neurons including all es-relatedda neurons (see Fig. 7B,E). In contrast, when the Cut proteinis overexpressed via a heat-shock promoter (Blochlinger et al.,1991) during the time of PNS neurogenesis in embryos that arewild-type for cut, E7-3-49 driven lacZ expression is greatlyreduced or absent in those md neurons that normally expresslacZ (i.e. vpda and the dorsal cluster da neurons)(R. B. and R.B., unpublished observations). Therefore, cut seems to be anecessary and sufficient component for the specification of thecorrect identity of es-related da neurons, similar to its role ines organ development (Bodmer et al., 1987; Blochlinger et al.,1991). Since the cut gene product contains a homeodomain(Blochlinger et al., 1988), cut could act directly on downstreamgenes as a transcriptional activator and/or as a repressor. A ver-tebrate Cut-related protein, CDP/Clox/Cux1 (Neufeld et al.,1992; Andres et al., 1992; Valarche et al., 1993), has beenshown to function as a transcriptional repressor in co-trans-fection experiments (Andres et al., 1992; Valarche et al., 1993;Dufort and Nepveu, 1994; see also Skalnik et al., 1991). Apossible repressor function by Drosophila Cut is supported byour finding that E7-3-49 driven lacZ expression is apparentlysuppressed in md neurons that normally or ectopically expressCut. Thus, cut function seems not only required for the speci-fication of es organs but also for a subset of md neurons. Otherselector-type genes, which may function in parallel or down-stream of cut, are likely to be required to further specify thefate of different cell types, including es-related md neurons andes neurons.

How do axonal projections of md neurons correlatewith their lineage identity? Morphologically, md-da neurons look quite similar (Bodmeret al., 1987). However, the fact that md-da neurons derive fromdistinct lineages, raises the possibility that these cells formfunctionally distinct subpopulations, and the axonal projec-tions of different types of md neurons in the CNS may reflectthese differences. Merritt et al. (1993) have previously shownthat the central projections of es neurons are distinct from thoseof ch neurons and that cut is required for the correct projec-

2935PNS lineages in Drosophila

tions of es organs. Md neurons also have distinct central pro-jections (Merritt and Whitington, 1995). (1) Most md-daneurons form one class and project into a discrete longitudinalCNS fascicle. They are dependent on AS-C and virtually allexpress cut. (2) In contrast, the atonal-dependent v′td2 andvpda md neurons have a projection pattern that is differentfrom the majority of the md-da neurons. (3) The dbd-mdneuron and one of the dorsal md-da neurons do not depend ona known proneural gene and their projections seem to bedistinct from all others. This suggests that the central projec-tions of md neurons reflects their requirement for proneuralgenes (Merritt and Whitington, 1995).

How do the lineage relations of md neurons correlate witha particular projection pattern? The es-related da neurons andmost solo-da neurons seem to have an undistinguishable pro-jection pattern, but the ch-related v′td2 neurons and the dbd-md neuron have distinct projections. Although lineage rela-tionships may not be an absolutely reliable indicator of mdneuronal identity, md neurons of similar lineages also havesimilar projections in the CNS (e.g. all es-related md neuronsproject into the same fascicle). Thus, it seems that proneuralgenes, selector genes (cut) and lineage relationships, as well asother unknown factors, have influential roles in determiningthe identity of md neurons.

We are much indebted to Gary Struhl for generously providing uswith the flipase and FRT-lacZ transformant flies. We thank GregGibson, Alain Ghysen and David Merrit for critical reading of the man-uscript. We also thank Corey Goodman for the 22C10 antibody,Andrew Tomlinson for the RK2 antibody, Chris Doe for the anti-prospero antibody and Seymour Benzer for the 21A6 antibody. Wealso thank the Dr Yuh-Nung Jan and the Bloomington Stock Centerfor sending fly stocks. This work was supported by a grant from NIHto R. Bodmer.

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(Accepted 13 May 1995)