homeotic regulation of segment-speciﬁc differences in ...al98b.pdf · homeotic regulation of...

Homeotic regulation of segment-specific differences in neuroblast numbersand proliferation in theDrosophilacentral nervous system

Andreas Prokopa,*, Sarah Brayb, Emma Harrisonb, Gerhard M. Technaua

aInstitut fur Genetik-Zellbiologie, Becherweg 32, D-55128 Mainz, GermanybDepartment of Anatomy, Downing Street, Cambridge CB2 3DY, UK

Received 9 February 1998; revised version received 20 April 1998; accepted 20 April 1998

Abstract

The number and pattern of neuroblasts that initially segregate from the neuroectoderm in the earlyDrosophilaembryo is identical inthoracic and abdominal segments. However, during late embryogenesis differences in the numbers of neuroblasts and in the extent ofneuroblast proliferation arise between these regions. We show that the homeotic genesUltrabithorax andabdominal-Aregulate these latedifferences, and that misexpression of either gene in thoracic neuroblasts after segregation is sufficient to induce abdominal behaviour.However, in wild type embryos we only detectabdominal-AandUltrabithorax proteins in early neuroblasts. Furthermore, transplantationexperiments reveal that segment-specific behaviour is determined prior to neuroblast segregation. Thus, the segment-specific differences inneuroblast behaviour seem to be determined in the early embryo, mediated through the expression of homeotic genes in early neuroblasts,and executed in later programmes controlling neuroblast numbers and proliferation. 1998 Elsevier Science Ireland Ltd. All rightsreserved

Keywords:BrdU; Central nervous system;Drosophila; Homeotic genes; Proliferation; Transplantation

1. Introduction

The central nervous system (CNS) is composed of a hugevariety of cells, most of which are unique in their properties.In insects these individual neurons are arranged in stereo-typed patterns with reproducible differences between thesegments corresponding to the diverse regional require-ments of the CNS, such as the control of the locomotionapparatus in the thorax or the reproduction organs in theabdomen. It is thus important to understand how regionaldifferences are programmed during nervous system devel-opment. The ventral nerve cord in insects derives fromneural stem cells, the neuroblasts (NBs), which segregatein reproducible segmental patterns from the ventral neuro-genic region of the ectoderm (Bate, 1976; Hartenstein andCampos-Ortega, 1984; Doe, 1992). InDrosophila, indivi-dual NBs give rise to lineages of specific size and cell com-position (Udolph et al., 1993; Bossing and Technau, 1994;

Bossing et al., 1996; Schmidt et al., 1997). However,although the pattern of segregating NBs is identical in thor-acic and abdominal segments (Doe, 1992), reproducibledifferences occur between the thoracic and abdominal ver-sions of some NB lineages (Prokop and Technau, 1994a;Bossing et al., 1996; Schmidt et al., 1997). This is alsoreflected in the proliferation patterns in the CNSs whichshow marked differences between thorax and abdomen atstages 15 and 16 (Prokop and Technau, 1991).

Segment-specific differences are also evident in the num-ber of NBs that persist beyond the end of embryogenesis andproliferate during larval stages. At stage 17, all NBs havestopped dividing but can still be monitored by NB-specificexpression ofgrainyhead(grh) (Bray et al., 1989). Analysesof GRH expression patterns in the CNSs of wild typeembryos and of mutant embryos where cell death is sup-pressed strongly suggest that a number of NBs normally dietowards the end of embryogenesis (White et al., 1994). Thedegree of cell death shows segment-specific differences, inthat many more NBs die in the central abdomen than in thethorax and anterior abdomen. As a consequence, when NBs

Mechanisms of Development 74 (1998) 99–110

0925-4773/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reservedPII S0925-4773(98)00068-9

* Corresponding author. Tel.: +49 6131 394328; fax: +49 6131 395845;e-mail: [email protected]

resume proliferation as postembryonic NBs in the larvalstages, 47 NBs are detected in each thoracic, about 12 inthe two anterior abdominal neuromeres, but only six in cen-tral abdominal segments. Furthermore, postembryonic NBsin the thorax and anterior abdomen produce hundreds ofdaughter cells each, whereas those in abdominal neuro-meres A3–A7 give rise to only five to 15 cells (Trumanand Bate, 1988; Prokop and Technau, 1994b). In summary,there are three major factors regulating the segment-specificproliferation of NBs: (1) the period and frequency ofembryonic NB proliferation, (2) the number of NBs elimi-nated at the end of embryogenesis, and (3) the frequencyand period of postembryonic proliferation.

The development of segment-specific characteristics isprogrammed during development by the homeotic geneswhich encode homeodomain transcription factors and areconserved from nematodes to vertebrates, both with respectto their function and to their organisation within gene com-plexes (for reviews see Lewis, 1978; Duncan, 1987; Bea-chy, 1990; Kaufman et al., 1990; McGinnis and Krumlauf,1992; Morata, 1993). Homeotic selector genes are in prin-ciple expressed in those body regions in which they arerequired to select the developmental pathways specific foreach particular segment. InDrosophila, mutations in any ofthe homeotic genes result in the transformation of the mor-phological characteristics of the segments, where they arenormally expressed, into those of other (in general moreanterior) segments. Following these principles, segmentidentities in the head and anterior thorax ofDrosophilaare controlled by genes of theAntennapedia-complex,whereas the posterior thorax and the abdomen are controlledby genes of thebithorax-complex, includingUltrabithorax(Ubx) andabdominal-A(abd-A).

The homeotic selector genes are expressed in a complexpattern in theDrosophila CNS (Doe et al., 1988) and thesegment-specific development of at least one embryonic NBlineage is regulated by homeotic genes (Prokop and Tech-nau, 1994a). Thebithorax-complex genes are thus likelycandidates for regulating the thoracic and abdominal pat-terns of NB behaviour. To investigate this possibility wehave analysed whether loss-of function mutations or mis-expression of homeotic genes have any effects on (a) pat-terns of bromo deoxyuridine (BrdU) incorporation whichindicates DNA synthesis and is used to give an indicationof NB proliferation (Truman and Bate, 1988; Prokop andTechnau, 1991), and (b) late embryonic patterns of NB-specific GRH expression, which labels the persisting NBsat that stage (White et al., 1994; see above). Our analysisindicates thatabd-AandUbx regulate the differences in cellnumber between thoracic and abdominal neuromeres thatare seen in the larval and adult CNS by altering both theamount of proliferation in embryo and larva, and the num-ber of NBs that are present in post-embryonic stages.Although segment-specific characteristics of BrdU andGRH patterns are first observed towards the end of embry-ogenesis, we only detect the expression of ABD-A and UBX

in NBs during earlier embryonic stages. Furthermore, trans-plantation experiments indicate that determination of thor-acic versus abdominal characteristics occurs prior to NBsegregation, before the homeotic proteins themselves areexpressed. We propose therefore that segment-specific dif-ferences in neuroblast behaviour are determined during theearly patterning of the embryo which results in specificexpression of homeotic genes in early NBs. This conferslater programmes of proliferation control, which no longerrequire the activity of homeotic genes.

2. Results

2.1. Segment-specific differences in late embryonic NBproliferation are controlled by abd-A and Antennapedia

Individual NBs in theDrosophilaCNS undergo differentpatterns of division depending on their position (Prokop andTechnau, 1994a; Bossing et al., 1996; Schmidt et al., 1997).Patterns of replicating cells can be detected through theirincorporation of BrdU which in late stages of embryogen-esis reveals scattered cells in the abdominal neuromerescompared with regular incorporation patterns in the moreanterior regions of the ventral nerve cord (Fig. 1B,C). In thesubesophageal ganglion approximately 20 groups of two tofour cells in ventral to ventrolateral locations are labelled,and on either side of each thoracic neuromere one to threegroups of two to four cells are detected in ventrolateral/lateral (but not in ventral) positions. Given that the initialnumber and organisation of NBs is the same in thorax andabdomen (Doe, 1992), the observed replication patternsindicate that equivalent NBs behave differently in the thoraxand the abdomen; in the thorax they continue replicatingwhereas in the abdomen they do not.

In order to determine whether homeotic genes regulatethese differences between thoracic and abdominal DNAreplication, BrdU was injected into homeotic mutantembryos at stage 16/17. In mutant embryos lackingUbxand abd-A function (Df109), BrdU incorporation appearsnormal in the subesophageal ganglion and in the thorax.However, in the abdominal neuromeres (except for theterminal region) lateral cells incorporate BrdU in patternsreminiscent of the thoracic region (not shown, but see Fig.1E). Mutant embryos lackingUbx function alone, show noobvious changes in the pattern of BrdU incorporation (notshown) in spite of a clear presence of the homeotic cuticlephenotype (Lewis, 1978). In contrast, embryos lackingabd-A function had defects reminiscent of embryos carrying thedeficiencyDf109 (Fig. 1E) with extra lateral NBs incorpor-ating BrdU in abdominal segments. The regulation byabd-Abut notUbx suggests that the NBs lie in the posterior com-partment of the segment, because the effects are also seen inA1 whereabd-Ais only expressed in the posterior compart-ment (see Fig. 6A). The posterior localisation of the lateralthoracic NB in wild type embryos was confirmed by double

100 A. Prokop et al. / Mechanisms of Development 74 (1998) 99–110

labelling with antibodies against BrdU and ENGRAILED, amarker for the posterior compartment (Fig. 1D). In embryoslacking theAntennapediagene, thoracic BrdU incorpora-tion associated with ventrolateral/lateral NBs is normal,however, additional staining is detected in ventral positionsresembling the ventral BrdU patterns of the subesophagealganglion (Fig. 1F). Taken together, these results demon-strate that in late embryosabd-A function is needed torepress DNA replication in some lateral NBs of abdominalneuromeres, andAntennapediafunction is required torepress DNA replication in ventral NBs of the thorax. Aspulse chase experiments have shown that the pattern ofBrdU incorporation in the thoracic neuromeres of late

embryos reflects NB proliferation (Prokop and Technau,1991) we believe that the DNA replication patterns weobserve in these experiments are indicative of effects onNB proliferation.

2.2. Homeotic genes also control the numbers andproliferation of postembryonic NBs

During the second larval instar, some of the embryonicNBs increase in size and resume proliferation as postem-bryonic NBs (pNBs). The thoracic neuromeres contain 47pNBs and the abdominal neuromeres A1 and A2 about 12pNBs, all of which proliferate extensively. In contrast, the

Fig. 1. Homeotic regulation of segment-specific BrdU-incorporation in the embryonic CNS. Late stage 17 embryos in lateral (A,B,J) or ventral view (C–H;anterior always to the left), labelled with antibodies against ENGRAILED (EN) and/or BrdU as indicated (bottom right). (A) Eachengrailedstripe indicatesone neuromere (T, thorax, delimited by black lines; Ab, abdomen; H, hemispheres; S, subesophageal ganglion). (B–D) White arrowheads point at lateralBrdU incorporation in the thorax, black arrows at ventral proliferation in the subesophageal ganglion, black arrowheads atengrailedpositive cells. Note thatBrdU labelled cells lie in one line withengrailedpositive cells, thus in the posterior neuromere compartment. (E–G) show mutant embryos as indicated (topmiddle), (H,J) are embryos with misexpression of UBX; white arrows indicate lateral BrdU incorporation comparable with wild type (see (B,C)), openarrowheads indicate ectopic lateral, open arrows ectopic ventral and a bent arrow loss of lateral BrdU incorporation. (K) Ectopic UBX expression appears inthe NB layer about stage 10/11 (white arrow), but not in the peripheral ectoderm (except intrinsic expression in T2 and T3; asterisks). Subesophageal(S) andthoracic (T1–3) neuromeres are indicated. Scale bar, 50mm (A–C, E–J); 20mm (D,K).

101A. Prokop et al. / Mechanisms of Development 74 (1998) 99–110

abdominal neuromeres A3–7 contain at most six pNBs (twosets of vm-, vl- and dl-pNBs), each of which divides only afew times (Fig. 2A,B; Truman and Bate, 1988; Prokop andTechnau, 1994b, 1995). In order to investigate a possibleinvolvement of homeotic genes in the regulation of thesepostembryonic segment-specific differences we analysedpatterns of BrdU incorporation or of pNB-specific toluidineblue staining (see Truman and Bate, 1988) in allelic combi-nations of homeotic genes that allow mutant animals tosurvive into larval stages (Lewis, 1978; Ghysen et al.,1985). Larvae carrying just one copy of theUbx andabd-A genes (Df109/+), show additional large clusters of BrdUlabelled cells in A3, A4 and sometimes also A5 (not shown,but see Fig. 2G). Most of these clusters are located in aventromedial position derived either from the vm-pNBs orfrom additional adjacent pNBs. Sometimes further BrdUlabelled large cell clusters are located in more dorsal posi-tions, adjacent to or originating from the vl- or dl-pNB.Larvae carrying just one copy of theUbx gene have slightdefects in the BrdU pattern in one-fourth of cases, with onelarger cell group in ventrolateral positions of A3 and/or anenlargement of the vm-cluster (normally about five cells) toup to 15 cells in A3 or A4 (not shown). However, thesedefects are less severe than in larvae heterozygous forDf109. In contrast, larvae lacking one copy of theabd-Agene, all show a BrdU pattern resembling the severe defects

observed in larvae carrying theDf109 deletion (Fig. 2G).This demonstrates thatabd-Ais the principle factor requiredto regulate segment-specific features of pNB proliferation inmost of the abdominal neuromeres, comparable with thesituation in the embryonic CNS.

This interpretation is supported by analyses of larvaecarrying one copy of theabd-Aallele Uab4 (Lewis, 1978)in combination with anabd-A null allele (Uab4/Df109 orUab4/abd-AMX1). Such larvae have about 12 pNBs in eachof the abdominal neuromeres A3–A7 (compared with sixpNBs in the wild type; Fig. 2A,D). Each of these surpluspNBs produces a hundred or more progeny, whereas in wildtype larvae the pNBs in A3–A7 produce at most 15 progeny(Fig. 2B,E; Truman and Bate, 1988; Prokop and Technau,1994b). Both the number and the proliferation behaviour ofthese pNBs in the central abdomen of the mutant larvae isreminiscent of the wild type pattern described for neuro-mere A2 (Truman and Bate, 1988). The NB pattern defectscan already be detected in the late embryo (stage 16/17)using the NB-specific markergrainyhead (grh): manymore GRH-expressing pNBs persist in the abdominal neu-romeres ofUab4/Df109 mutant embryos than in wild type(Fig. 2C,F). Thus,abd-Aregulates the proliferation of pNBsboth by controlling the number of NBs that are eliminated inthe late embryo and by regulating the number of divisionsthe persisting NBs undergo during larval stages.

Fig. 2. Effects of homeotic mutations on the pattern and proliferation of postembryonic NBs. (A,B,D,E,G) BrdU incorporation in nerve cords of late thirdinstar larvae exposed to BrdU during larval life. (C,F,H,I) Anti-GRH labelled nerve cords at late stage 17. Anterior is to the left. (A) In the wild typelarva,abdominal neuromeres A3–A7 (delimited by lines) have six clusters of BrdU labelled cells derived from two pairs of vm-, vl- and dl-pNBs, respectively(seehigher magnification in (B)), comparable with the abdominal NB patterns in late embryos (C); arrows in (A–C) indicate vm-pNBs. InUab4/abd-Aor Uab4/DF109 mutant larvae many more NBs persist in A3–A7 (D,F) and produce larger lineages (E). (G) Inabd-A/+ mutant larvae A3, A4 and sometimes A5 havefew large NB lineages (black arrowheads). Misexpression of UBX (H) or ABD-A (I) in all NBs removes most thoracic NBs (white arrowheads indicateremaining NBs; arrows indicate hemispheres). Scale bar, 20mm (F); 30mm (B,E); 35mm (C,H,I); 100mm (A,D,G).


2.3. Misexpression of bithorax-complex genes is sufficientto confer segment-specific behaviour on NBs

Impaired function or reduced levels ofbithorax-complexgenes affect NB behaviour in the domain where these genesare normally expressed, consistent with their direct require-ment for the regulation of abdominal NB characteristics. Inorder to test whether the presence of ABD-A or UBX issufficient to confer abdominal NB behaviour we analysedthe consequences of expressing these proteins in all NBsincluding those in thoracic neuromeres using the GAL4-expression system (Brand and Perrimon, 1993). When thetranscription of UAS-Ubx or UAS-abd-A was ectopicallyinduced in most or all segregated embryonic NBs andtheir progeny (but not in the neuroectoderm; Fig. 1K) withthe GAL4 driver lineMZ1407the overall shape of the CNSwas not perturbed indicating that most phases of prolifera-tion are not grossly abnormal. However, the pattern of BrdUincorporation at stage16/17 is altered with misexpression ofeither UBX or ABD-A abolishing the lateral replicatingclusters in the thoracic neuromeres (Fig. 1J; bent arrow).This is what normally occurs in the abdominal NBs suggest-ing that the thoracic expression of ABD-A or UBX after NBsegregation is sufficient to transform the characteristics ofsome thoracic lateral NBs into those of abdominal NBs.However, we also find ectopic sites of BrdU-incorporationin the ventral region of thoracic neuromeres (Fig. 1H,J). Thepattern appears like an extension of the subesophageal pat-tern and resembles the phenotype ofAntennapediamutantembryos (Fig. 1F vs. H,J; open arrows), as if ectopicbithoraxgene activity in the thoracic neuromeres repressesAntennapediafunction. Indeed ectopic UBX has beenshown to repressAntennapediagene expression (Gon-zalez-Reyes and Morata, 1990). Thus, our results indicatethat specific NBs respond differently to ectopic expressionof bithorax-complex genes, acquiring abdominal character-istics in some cases and subesophageal characteristics inothers. Similar results are obtained inPolycombmutantembryos, which have ectopic expression of ABD-A andUBX in the NB layer from stage 10/11 onwards (Simon etal., 1992; Prokop and Technau, 1994a). InPolycomb3

mutant embryos at stage 16, both the lack of lateral andgain of ventral BrdU incorporating cells are observed, simi-lar to the GAL4 experiments (Fig. 1G). Therefore, our dataare consistent with the hypothesis that expression ofbithorax-complex genes after NB segregation is sufficientto regulate the segment-specific replication of embryonicNBs. The homeotic genes do not directly interfere withthe cell cycle (e.g. block it completely upon misexpression),but they appear to determine which segmental proliferationschedule is carried out.

As only a few NB lineages have segment-specific BrdU-incorporation patterns in the embryo we wanted to investi-gate whether misexpression of ABD-A or UBX is also suf-ficient to alter the numbers of NBs at the end ofembryogenesis, which is a characteristic relevant to many

more NB lineages. Misexpression of eitherUbx or abd-Aleads to a reduction in the number ofgrh-expressing cells inthe CNSs of stage 17 embryos which we take to indicate thatthey affect the number of NBs that are eliminated (see Sec-tion 1; White et al., 1994). The effects are dramatic in thethoracic neuromeres where only four to eightgrh-expres-sing cells remain per neuromere at specific locations (Fig.2H,I). Thus, the effects of misexpressing ABD-A and UBXon the patterns of BrdU-incorporation and GRH expressionin the late embryo suggest that most, if not all, thoracic NBsare responsive tobithorax-complex genes, and that the pre-sence of ABD-A or UBX after NB segregation is sufficientto induce abdominal behaviour.

2.4. Expression of homeotic genes in NBs precedes theappearance of segment-specific features

To determine whenbithorax gene function is requiredduring embryogenesis to regulate NB behaviour, we inves-tigated when these proteins are present in the abdominalNBs and therefore likely to impose their regulation. Usinganti-GRH or agrh-lacZ reporter-gene (which recapitulatesthe NB expression ofgrainyhead) to mark the NBs wedetected no co-expression with ABD-A in the persistingNBs of stage 17 embryos (not shown). In stage 15 embryosprior to NB elimination, many more cells expressgrh. How-ever, even at this stage there are no cells which co-expressUBX and GRH and only rarely (one to two ventral cells perneuromere) are ABD-A and GRH expression detected in thesame cells (Fig. 3D,E). This suggests that neither ABD-Anor UBX are present in the NBs shortly before segment-specific differences in BrdU and GRH patterns occur.

During early stages of embryogenesis NB can be detectedeither by their position and shape or by expression ofasense. ASENSE is expressed in NBs shortly after theirsegregation from the neuroectoderm and so can be used asan early marker for NBs. UBX was detected in many NBs atstages 8–12 although there is wide variation between thelevels of UBX present in different NBs and a subset of NBscontain no detectable UBX (Fig. 3A,C). Similarly, ABD-Ais present in many NBs at early stages (Fig. 3B). Thus, bothUBX and ABD-A are expressed in embryonic NBs, but theirexpression fades before segment-specific differencesbecome detectable. To see whether we could define a pre-cise stage when homeotic gene function is sufficient to con-fer segment-specific NB elimination and proliferationpatterns we tested the effects of inducing ABD-A or UBXat different stages via heat inducible promoters. None of theheat shock regimes were able to confer homeotic changes onproliferation or GRH patterns (although these experimentsclearly caused homeotic effects in the epidermis), so wecannot identify the precise stage when the activity of thesegenes is required. Nevertheless the timing of ABD-A andUBX expression in NBs makes it unlikely that the homeoticgenes directly regulate those genes which mediate prolifera-tion cessation or NB elimination. Instead they seem to con-


trol regulatory pathways, which function subsequent to theexpression of thebithorax-complex genes.

2.5. Thoracic versus abdominal behaviour of NB isdetermined prior to NB segregation

The misexpression data indicate that homeotic genesdetermine segment-specific features in the CNS after NBshave segregated. However, the analysis of the NB1-1 line-age revealed that segment-specificity can be determined inthe peripheral ectoderm, upstream of homeotic gene func-tion (Prokop and Technau, 1994a). We therefore testedwhether the segment-specific postembryonic proliferationpatterns are also determined in the peripheral ectoderm, tosee whether this early determination could be a generalmechanism underlying CNS development. Single cells,labelled genetically with the gene encodingb-galactosidaseand cytoplasmically with HRP (horseradish peroxidase),were transplanted between the thoracic and the abdominalneuroectoderm at the early gastrula stage about 30 min priorto the onset of NB segregation. Asb-galactosidase is geneti-cally expressed by every cell of the clone, both the embry-onically and postembryonically derived cells will containb-galactosidase activity. However, only the embryonicallyderived cells will contain HRP, as the enzyme gets dilutedbelow detectable level when the NB takes up proliferationin the larva (Fig. 4B,D; for details see Prokop and Technau,

1991). The composition of the clones was analysed in thelate larval ventral nerve cord when the differences betweenabdominal and thoracic NB lineages are very distinctive.

We obtained 66 CNS clones from isotopic transplanta-tions in the thorax, 45 of which contained a pNB with a nestof daughter cells exclusively labelled byb-galactosidaseactivity indicating that they were postembryonic progeny(Figs. 4A,B and 5, T to T). This proportion of 68% is inaccordance with the number of thoracic NBs which arereactivated during larval life in relation to the total numberof embryonic precursors (47 pNBs versus 60 NBs and sevento eight midline precursors per neuromere in the embryo;Truman and Bate, 1988; Doe, 1992; Bossing and Technau,1994). Isotopic transplantations in the abdomen yielded 32abdominal CNS clones of which only two contained a largerperipheral cell (potential pNB) and a small cluster of post-embryonic progeny cells. This low rate of NB reactivation(6%) and their low proliferation rate reflect the typical beha-viour of abdominal pNBs (Fig. 5, A to A).

When cells were heterotopically transplanted from thor-acic to abdominal sites of the early gastrula neuroectoderm,67% gave rise to a large nest of postembryonic cells with apNB (Figs. 4C,D and 5, T to A) consistent with the char-acteristics of thoracic NBs. Conversely, when cells weretransplanted from abdominal to thoracic sites, all clonesfailed to express thoracic features and contained only(embryonic) cells strongly labelled by HRP activity (Fig.

Fig. 3. ABD-A and UBX are found in NBs only at earlier embryonic stages. ABD-A or UBX expression (as indicated top right) in embryos at stage 10/11(A–C) or stage 15 (D,E), or (F) in early third instar larval CNSs (L3; stages indicated bottom right). NBs were identified by position and shape (A,B,F) orexpression of ASENSE (ASE; (C)) or GRH (D,E). (A–C) NBs contain homeotic proteins at early stages (white arrowheads; orange colour in (C) indicatesdouble labelling). (D–F) NBs lack homeotic gene expression later on (white arrowheads; for L3 see also Truman et al., 1993). Scale bar, 15mm (A,B,F); 35mm (C–E).


4E and 5, A to T). Thus, NBs autonomously execute post-embryonic proliferation patterns according to their domainof origin in the neuroectoderm. This indicates that most oreven all neuroectodermal CNS precursors are determined atthe early gastrula stage with respect to their thoracic orabdominal identity. Since subsequent misexpression ofhomeotic genes can alter the behaviour of NBs, our findingssuggest that genetic mechanisms upstream of homeotic genefunction must initially determine the regional identity ofthese cells.

3. Discussion

3.1. Homeotic genes regulate proliferation and numbers ofNBs

The development of the nervous system involves the gen-

eration of the correct numbers of neural precursors, theirsubsequent division to generate a defined set of progenyand the acquisition by their progeny of individual neuralcell fates. These processes must be differentially modulatedalong the body axis so that the neural structures appropriatefor each region of the body develop. Previous studies inDrosophilahave emphasised the role of homeotic genes incontrolling the types of neural fate that are adopted by theprogeny of the NBs (Doe and Scott, 1988). Our resultsdemonstrate that the homeotic genes also regulate seg-ment-specific differences in NB numbers and NB divisions.This regulation is achieved both by controlling the prolif-eration period and frequency and by regulating whether NBspersist at the end of embryogenesis. Because of the latter,the number and pattern of postembryonic NBs depend onhomeotic gene function unlike the embryonic NBs wherethe pattern of segregating NBs is the same between thoracicand abdominal neuromeres (Doe, 1992). Thus in the larval

Fig. 4. Examples of late larval NB lineages originating from transplantations at the early gastrula stage. (A–D) Thoracically derived pNBs (arrowheads) withembryonic (brown cells) and postembryonic (blue cells) progeny can develop in the thorax (A,B) or abdomen (C,D). (E) Lineages without pNBs always lacklarge nests of purely blue cells. (F) Schematic representation of an embryo at the early gastrula stage indicating the modes of transplantation that led to thelineages in (A–E). Scale bar, 70mm (A,C); 20mm (B,D).


nervous system there are dramatic differences between thenumbers of pNBs present in the thoracic versus the abdom-inal neuromeres, where the homeotic genesUbx andabd-Aare expressed. The A1 and A2 neuromeres have an inter-mediate number of pNBs (Truman and Bate, 1988), and inthe absence ofUbx the A1 neuromere contains more pNBsindicating thatUbx regulates the pattern of pNBs within theA1 neuromere (Truman et al., 1993). Our findings of super-numerary NBs in larvae heterozygous forUbx suggest thatUBX may also influence the behaviour of pNBs in moreposterior neuromeres (similar transformations wereobserved in larval cuticles; Beachy, 1990). However, themain differences in the distribution and proliferation ofpNBs in more posterior abdominal neuromeres are regu-lated byabd-A, as is evident from the aberrant pNB prolif-eration seen in neuromeres A3–A7 inUab4 mutant larvaewhich is more reminiscent of the A2 segment and thus isanalogous to transformations observed in the cuticle (Lewis,1978). The regions where ectopic proliferation occurs whenabd-A function is reduced (e.g. in larvae hemizygous forabd-A) correspond to those which have been reported togive rise to abnormal neuropil structures under the sameconditions (Teugels and Ghysen, 1983; Ghysen et al.,

1985; Ghysen and Lewis, 1986). Thus, these morphologicalabnormalities may result from supernumerary progeny ofmisregulated NBs. Homeotic regulation of NB proliferationhas also been observed in the mothManduca sexta(Bookerand Truman, 1989; Miles and Booker, 1993) suggesting thatthis is a fundamental mechanism through which the devel-opment of the nervous system is regulated in insects andmost likely in other organisms.

The transition from the embryonic stages of neurogenesisto the postembryonic stages involves programmed celldeath. This transition can be seen in stage 17 embryoswhen the number ofgrh-expressing cells in the abdomendecreases dramatically, and it is prevented by mutations thatdisrupt the cell-death pathway (Bray et al., 1989; White etal., 1994). In abd-A mutant embryos (Uab4) more grh-expressing cells persist in the abdomen, already reflectingthe NB pattern which is later on detected by BrdU andtoluidine blue in the larval nerve cord. Conversely, whenABD-A or UBX are ectopically expressed, fewergrh-expressing cells are found, suggesting that the homeoticgenes are controlling whether or not these cells undergoprogrammed cell death. Interestingly, the numbers ofabdominal NBs are also reduced upon misexpression ofUBX or ABD-A (Fig. 2C vs. H,I). A possible explanationis that some NBs in A3–A7 (e.g. vm, vl and dl; Fig. 2A–C)never express functional amounts of UBX or ABD-A, sothat targeted expression of UBX or ABD-A in those NBscould trigger their elimination. If so, then regulation of theseNBs should normally be independent ofabd-A and Ubxgene function. For example these NB could have a basallevel of proliferation in all segments or they could undergosegment specific patterns of proliferation in response toother homeotic genes such asAntennapedia(see Fig. 1F).To distinguish these possibilities specific markers areneeded for the individual NBs so that they can be identifiedin all segments and studied throughout embryonic and post-embryonic stages.

3.2. Cellular memory is implicated in the regulation ofhomeotic gene expression and function

The segment-specific effects of homeotic genes on pro-liferation and pNB fates are first detected quite late inembryogenesis, at about stage 15, and continue to be man-ifest throughout larval development (Truman and Bate,1988; Bray et al., 1989; Prokop and Technau, 1991). Wetherefore expected that the late effect of homeotic mutationson NB behaviour would correlate with late expression ofUbx and abd-A in these cells. However, neither UBX norABD-A are present in NBs during stage 15–17 (Fig. 3D,E)nor during postembryonic stages (Fig. 3F; Truman et al.,1993), so they are unlikely to be directly controlling NBnumbers or proliferation. However, ABD-A and UBX arepresent in many NBs at earlier stages and their presence issufficient to induce abdominal regulation, as demonstratedby misexpression experiments. Likewise it has been shown

Fig. 5. Isotopic and heterotopic transplantation experiments. Schematicrepresentations of cell transplantations in embryos at the early gastrulastage (top), of late larval CNSs with schematic NB lineages (middle)and a table summarising the results (bottom). Numbers 1–4 indicate thetransplantation modes and are reflected in the table below (T, thorax; A,abdomen). Only modes 1 and 4 (explantation from the thorax) give rise tolarge nests of postembryonic cells (with pNB; white part in schematiclineage; about two-thirds of cases) regardless of whether they develop inthe thorax (1) or abdomen (4). Potential pNBs (?) in modes 2 and 3(explantation from the abdomen) are rare and have very small lineages.Only a third of thoracically derived lineages (1, 4) but about 90% ofabdominally derived lineages (2, 3) consist of embryonically born cellsonly (black in schematic lineage).


that the NB1-1 lineage requiresUbx and abd-A functionduring stage 10/11 of embryogenesis when the proteinscan be detected in this precursor (Prokop and Technau,1994a). Therefore, the activity of UBX and ABD-A at anearly stage must be able to determine the later proliferationand persistence of the NBs (Fig. 6C). This is contrary to theoriginal hypothesis, based on the requirements forUbx inthe epidermis (Morata and Garcı´a-Bellido, 1976), whichproposed that the expression of a homeotic gene wouldneed to be stably maintained in the progeny cells for them

to retain their identity. In the NBs there must be mechanismsdownstream of homeotic genes through which cells canretain a stable memory or imprint of having previouslyexpressedUbx or abd-A. This could involve the homeoticgenes acting as transcriptional repressors which initiate arepressed state of their target genes that can be maintainedeven after UBX or ABD-A have decayed. Alternatively,UBX or ABD-A may activate target genes which have thecapacity for autoregulation, so that the targets can maintaintheir own expression in the absence of homeotic proteins.

Fig. 6. Control of segment-specific proliferation and survival inDrosophilaneuroblasts. The scheme shows (A) the expression domains ofAntennapedia, Ubxandabd-Ain relation to (B) the phenotypes observed (prolif., prolonged proliferation of embryonic NB; NB1-1, segmental differences of the NB1-1 lineage,see Prokop and Technau, 1994a; elimin., late embryonic NB elimination; no diff., possible NBs without segment specific differences, e.g. vm-, vl-, dl-pNB).(C) The implementation of genetic programmes for the regulation of NB numbers and proliferation depends on earlier patterning events. Mechanismsregulating persistence and prolonged proliferation of thoracic NB (+ in square) appear to operate after UBX or ABD-A have faded in NBs. Only those NBsexpressing UBX (blue NBs) or ABD-A (red NBs) can prevent installation of these mechanisms (crossed out squares).Ubx (blue) andabd-A (red) areexpressed only in those NBs which contain lineage or cell specific activators of homeotic transcription (e.g. segmentation genes; green NBs), and which havenot expressed anterior gap genes (e.g. hunchback; NBs with grey gradients). The function of gap genes (gap, black) is maintained byPolycomb-group genes(Pc-group, grey) and both repress the NB-specific activation ofUbx or abd-A(bent green arrow). (D) Upon transplantation, precursors from the thorax (1)maintain their repressed state mediated by gap andPolycomb-group genes (grey gradient). Precursors from the abdomen (2) have not expressed inhibitorygap genes and can therefore express UBX and ABD-A (red/blue).


Although the activity of homeotic genes is required afterNB segregation we found that at the early gastrula stage theneuroectodermal cells are already autonomously deter-mined as to whether they will give rise to NBs with thoracicor abdominal proliferation behaviour, and whether they willpersist as pNBs (Figs. 4 and 5). Gastrula cells transplantedfrom the thorax to the abdomen display thoracic patterns ofproliferation, whereas cells transplanted from the abdomento the thorax retain abdominal characteristics. This is likelyto be the result of domain-specific repression of homeoticgenes mediated by the early expressed gap gene productssuch ashunchback, a known repressor ofUbx (White andLehmann, 1986). Once the proteins encoded by the gapgenes have decayed, the repressed state of the homeoticgenes is maintained byPolycomb group genes (Paro,1990), so that the regulatory sequences ofbithorax-complexgenes in cells derived from the thorax remain repressed,whereas homeotic genes in abdominal cells are accessibleto activation (Fig. 6C,D). Experiments inC. eleganshavealso demonstrated the importance of lineage rather thanlocal positional signalling in the regulation of homeoticgene expression (Cowing and Kenyon, 1996), suggestingthat there could be conservation of the mechanisms under-lying this regulation.

We conclude therefore that control of segment-specificdevelopment in the CNS involves a cascade of subsequentdetermination steps (Fig. 6C), in which first homeotic genefunction is regionally permitted or not, and subsequentlyhomeotic genes themselves select a programme, whichcan be maintained even after thebithorax-complex genescease to be expressed. Reproducible intrasegmental expres-sion patterns could spare some cells from homeotic geneexpression (e.g. the abdominal vm-, vl- and dl-NB) andthus add further complexity and regulatory potential tothis system.

4. Experimental procedures

4.1. Fly stocks

We used the following mutant strains:AntennapediaW10

(Wakimoto et al., 1984);Ubx6.28 (Kerridge and Morata,1982);abd-AMX1 (Sanchez-Herrero et al., 1985); theabd-Aallele iab3Uab4 (Lewis, 1978, referred to asUab4);Df(3R)Ubx109(Lewis, 1978; referred to asDf109) andPc3 (Lewis, 1978). For misexpression of homeotic genesin neural cells after NB segregation we used the GAL4system (Brand and Perrimon, 1993) with the GAL4 dri-ver-lineMZ1407(Sweeney et al., 1995; Urban et al., unpub-lished data) in conjunction with UAS-Ubx62.1and UAS-abd-A21.6 (Greig and Akam, 1993; Castelli-Gair et al., 1994).During misexpression experiments embryos were kept at25 or 29°C. For cell transplantations we used as hosts theb-Gal-1 straincq20(Knipple and MacIntyre, 1984), whichlacks endogenousb-galactosidase activity and therefore

shows no background staining. As donors we used P-lacZenhancer trap lines with strong expression ofb-galactosi-dase throughout the nervous system. Either we usedA45(O’Kane and Gehring, 1987) or the lineE;B1;Z1 carryingthree different P-lacZ insertions:E is an insertion into theelavlocus (Bier et al., 1989),B1an unpublished insertion onthe 2nd chromosome (Bier et al., 1989) and Z1 a 3rd chro-mosome insertion (unpublished data; kindly provided byJ.A. Campos-Ortega).

4.2. Generation of transgenic flies containing grh-lacZreporter gene

A 4-kb fragment (XhoI-XbaI) from the second intron ofthe grainyheadgene (Uv et al., 1997) was cloned intopBluescript (Strategene), excised using XbaI and KpnIand inserted into the XbaI-KpnI sites of the enhancer detec-tor P-element vector, HZ50PL (Hiromi et al., 1985) whichcarriesrosy as a selectable marker. The resulting plasmid(NBgrh-lacZ) was injected intocinnabar rosyembryos inthe presence of transposase using standard techniques(Rubin and Spradling, 1982) and transformants wereselected on the basis ofrosy+ eyes. At least four indepen-dent lines were analysed, all of which had expression in NBsas shown.

4.3. Bromodeoxyuridine (BrdU) labelling

For embryonic BrdU application 5–10 nl of a 15 mMsolution of BrdU (Sigma) in 0.2 M KCl were injected intoembryos about 45 min after the three-portioned midgutstage (stage16/17 according to Campos-Ortega and Harten-stein, 1997), following procedures described elsewhere(Prokop and Technau, 1991; Prokop and Technau, 1993).At late stage 17 embryos were mechanically removed fromthe vitelline membrane, washed in PBT, fixed in Carnoy’ssolution (6:3:1 ethanol/chloroform/acetic acid), rehydratedand their tips cut off to allow subsequent penetration ofantibodies. In larval stages BrdU was applied by feedinganimals with standard medium containing between 1 and10% of a BrdU-solution (33 mM BrdU in 40% ethanol;Truman and Bate, 1988). CNSs were removed, fixed inCarnoy’s solution, and rehydrated.

To stain for BrdU-incorporation we used monoclonalantibodies against BrdU (Becton and Dickinson, diluted1:100), HRP-coupled secondary antibodies (Dianova,diluted 1:500) or biotinylated secondary antibody (Dianova,diluted 1:500) followed by treatment with ABC elite kit(Vector Laboratories); blocking steps included 1% low fatmilk powder. Specimens were embedded in Araldite(Serva) in borosilicate capillaries (Hilgenberg; see Prokopand Technau, 1993).

4.4. Immunocytochemistry

Further antibody stainings were carried out on whole


mounts following standard procedures and using biotiny-lated secondary antibodies (1:300) and ABC elite kit (Vec-tor Laboratories) or fluorescent secondary antibodies fromJackson laboratories (1:200). Third larval instar CNSs werebriefly post fixed in methanol subsequent to formaldehydefixation. For detection ofgrainyhead(grh) expression, theCNSs were dissected from stage 17 embryos (approximately19–22 h after egg lay) and transferred to poly-l-lysinecoated coverslips (Bray et al., 1989). Embryos and larvalCNSs were embedded in Araldite (see above) or mounted incitifluor. The final dilutions of primary antibodies were1:1500 for rat polyclonal anti-ABD-A (Macıás et al.,1990); 1:20 for rabbit polyclonal anti-ABD-A (Karch etal., 1990), 1:20 for monoclonal anti-UBX (White and Wil-cox, 1984, 1985), 1:3 for monoclonal anti-ENGRAILED(Patel et al., 1989); 1:5 for monoclonal anti-GRH (Bray etal., 1989); 1:10 000 for rabbit anti-ASENSE (Brand et al.,1993); and 1:2000 for rabbit anti-b-galactosidase (Cappell).

4.5. Cell transplantation, preparation and staining ofdonors and hosts

For cell transplantations we used as donors enhancer traplines with lacZ gene expression throughout the CNS (seeSection 4.1). ThelacZgene is inherited upon cell division sothat embryonic and postembryonic progeny of transplantedcells produceb-galactosidase cell autonomously. In addi-tion, donors were injected with 5–10 nl of horseradish per-oxidase solution (HRP; 5–10% in 0.2 M KCl) at thesyncytial blastoderm stage. Transplantations of single cellswere carried out about 10 min after the onset of gastrulation(stage 7) in the ventral neurogenic region at either 30%(abdominal) or at 60% egg length (thoracic, Fig. 5; formore details see Prokop and Technau, 1991). Late larvalhost CNSs were dissected out in PBS (other tissues werediscarded), fixed in 1% solution of glutaraldehyde in PBS,stained forb-galactosidase and subsequently for HRP activ-ity. Specimens were dehydrated in alcohol, cleared inxylene, embedded in araldite (see above).

Acknowledgements

We thank Jose´ Campos Ortega, James Castelli-Gair, Ste-phen Greig, Rolf Reuter, Srikala Raghavan, SuzannaRomani, Ernesto Sańchez Herrero, Joachim Urban andRob White for providing fly stocks and antibodies, RobWhite and Ernesto Sańchez-Herrero for comments on themanuscript. A.P. is grateful to Michael Bate, in whoselaboratory part of the project was carried out. This workwas supported by a grant from the Deutsche Forschungsge-meinschaft to G.M.T. (Te 130/1-4), by a project grant fromthe Biotechnology and Biological Science Research Councilto S.J.B. and by a Human Capital and Mobility Fellowship(EU) and a research fellowship from the Lloyd’s of LondonTercentenery Foundation to A.P.

References

Bate, C.M., 1976. Embryogenesis of an insect nervous system. I. A map ofthe thoracic and abdominal neuroblasts inLocusta migratoria. J.Embryol. Exp. Morphol. 35, 107–123.

Beachy, P.A., 1990. A molecular view of theUltrabithorax homeotic geneof Drosophila. Trends Genet. 6, 46–51.

Bier, E., Vassin, H., Shepherd, S., Lee, K., McCall, K., Barbel, S.,Ackerman, L., Carretto, R., Uemura, T., Grell, E., Jan, L.Y., Jan,Y.N., 1989. Searching for pattern and mutation in theDrosophilagen-ome with a P-lac Z vector. Genes Dev. 3, 1273–1287.

Booker, R., Truman, J.W., 1989.Octopod, a homeotic mutation of themothManduca sexta, influences the fate of identifiable pattern elementswithin the CNS. Development 105, 621–628.

Bossing, T., Technau, G.M., 1994. The fate of the CNS midline progeni-tors inDrosophilaas revealed by a new method for single cell labelling.Development 120, 1895–1906.

Bossing, T., Udolph, G., Doe, C.Q., Technau, G.M., 1996. The embryoniccentral nervous system lineages ofDrosophila melanogaster. I. Theneuroblast lineages derived from the ventral half of theneuroectoderm. Dev. Biol. 179, 41–64.

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

Brand, M., Jarman, A.P., Jan, L.Y., Jan, Y.N., 1993.asenseis aDrosophilaneural precursor gene and is capable of initiating sense organ formation.Development 119, 1–17.

Bray, S.J., Burke, B., Brown, N.H., Hirsh, J., 1989. Embryonic expressionpattern of a family ofDrosophila proteins that interact with a centralnervous system regulatory element. Genes Dev. 3, 1130–1145.

Campos-Ortega, J.A., Hartenstein, V., 1997. The Embryonic Developmentof Drosophila melanogaster. Springer–Verlag, Berlin, 405 pp.

Castelli-Gair, J., Greig, S., Micklem, G., Akam, M., 1994. Dissecting thetemporal requirements for homeotic gene function. Development 120,1983–1995.

Cowing, D., Kenyon, C., 1996. Correct Hox gene expression establishedindependently of position inCaenorhabditis elegans. Nature 382, 353–356.

Doe, C.Q., 1992. Molecular markers for identified neuroblasts and gang-lion mother cells in the Drosophila central nervous system.Development 116, 855–886.

Doe, C.Q., Hiromi, Y., Gehring, W.J., Goodman, C.S., 1988. Expressionand function of the segmentation genefushi tarazuduring Drosophilaneurogenesis. Science 239, 170–175.

Doe, C.Q., Scott, M.P., 1988. Segmentation and homeotic gene function inthe developing nervous system ofDrosophila. Trends Neurosci. 11,101–106.

Duncan, I., 1987. The bithorax complex. Annu. Rev. Genet. 21, 285–319.Ghysen, A., Jan, L.Y., Jan, Y.N., 1985. Segmental determination inDro-

sophilacentral nervous system. Cell 40, 943–948.Ghysen, A., Lewis, E.B., 1986. The function ofbithorax genes in the

abdominal central nervous system ofDrosophila. Roux’s Arch. Dev.Biol. 195, 203–209.

Gonzalez-Reyes, A., Morata, G., 1990. The developmental effect of over-expressing aUbx product inDrosophilaembryos is dependent on inter-actions with other homeotic products. Cell 61, 515–522.

Greig, S., Akam, M., 1993. Homeotic genes autonomously specifyone aspect of pattern in theDrosophila mesoderm. Nature 362, 630–632.

Hartenstein, V., Campos-Ortega, J.A., 1984. Early neurogenesis in wildtype Drosophila melanogaster. Roux’s Arch. Dev. Biol. 193, 308–325.

Hiromi, Y., Kuroiwa, A., Gehring, W.J., 1985. Control elements of theDrosophilasegmentation genefushi tarazu. Cell 43, 603–613.

Karch, F., Bender, W., Weiffenbach, B., 1990.abd-Aexpression inDro-sophilaembryos. Genes Dev. 4, 1573–1587.

Kaufman, T.C., Seeger, M.A., Olsen, G., 1990. Molecular and genetic


organization of theAntennapediagene complex ofDrosophila melano-gaster. Adv. Genet. 27, 309–362.

Kerridge, S., Morata, G., 1982. Developmental effects of some newlyinduced Ultrabithorax alleles of Drosophila. J. Embryol. Exp.Morphol. 68, 211–234.

Knipple, D.C., MacIntyre, R.J., 1984. Cytogenic mapping and isolation ofmutations of theb-gal-1 locus ofDrosophila melanogaster. Mol. Gen.Genet. 198, 75–83.

Lewis, E.B., 1978. A gene complex controlling segmentation inDrosophila. Nature 276, 565–570.

Macıas, A., Casanova, J., Morata, G., 1990. Expression and regulation ofthe abd-Agene ofDrosophila. Development 110, 1197–1207.

McGinnis, W., Krumlauf, R., 1992. Homeobox genes and axial patterning.Cell 68, 283–302.

Miles, C.I., Booker, R., 1993.Octopod, a homeotic mutation of the mothManduca sexta, affects development of both mesodermal and ectoder-mal structures. Dev. Biol. 155, 147–160.

Morata, G., 1993. Homeotic genes ofDrosophila. Curr. Opin. Genet. Dev.3, 606–614.

Morata, G., Garcı´a-Bellido, A., 1976. Developmental analysis of somemutants of thebithorax system of Drosophila. Roux’s Arch. Dev.Biol. 179, 125–143.

O’Kane, C., Gehring, W.J., 1987. Detection of in situ genomic regulatoryelements inDrosophila. Proc. Natl. Acad. Sci. USA 84, 9123–9127.

Paro, R., 1990. Imprinting a determined state into the chromatin ofDrosophila. Trends Genet. 6, 416–421.

Patel, N.H., Martin-Blanco, E., Coleman, K.G., Poole, S.J., Ellis, M.C.,Kornberg, T.B., Goodman, C.S., 1989. Expression ofengrailedproteinsin arthropods, annelids and chordates. Cell 58, 955–968.

Prokop, A., Technau, G.M., 1991. The origin of postembryonic neuro-blasts in the ventral nerve cord ofDrosophila melanogaster.Development 111, 79–88.

Prokop, A., Technau, G.M., 1993. Cell transplantation. In: Hartley, D.(Ed.), Cellular Interactions in Development: A Practical Approach.Oxford University Press, Oxford, pp. 33–57.

Prokop, A., Technau, G.M., 1994a. Early tagma-specific commitment ofDrosophila CNS progenitor NB1-1. Development 120, 2567–2578.

Prokop, A., Technau, G.M., 1994b. Normal function of themushroombody defectgene ofDrosophila is required for the regulation of thenumber and proliferation of neuroblasts. Dev. Biol. 161, 321–337.

Prokop, A., Technau, G., 1995. BrdU incorporation reveals DNA replica-tion in non dividing glial cells in the larval abdominal CNS ofDrosophila. Roux’s Arch. Dev. Biol. 204, 54–61.

Rubin, G.M., Spradling, A.C., 1982. Genetic transformation ofDrosophilawith transposable element vectors. Science 218, 348–353.

Sanchez-Herrero, E., Vernos, I., Marco, R., Morata, G., 1985. Geneticorganization ofDrosophila bithorax-complex. Nature 313, 108–113.

Schmidt, H., Rickert, C., Bossing, T., Vef, O., Urban, J., Technau, G.M.,1997. The embryonic central nervous system lineages ofDrosophilamelanogaster. II. Neuroblast lineages derived from the dorsal part ofthe neuroectoderm. Dev. Biol. 189, 186–204.

Simon, J., Chiang, A., Bender, W., 1992. Ten differentPolycombgroupgenes are required for spatial control of theabd-AandAbd-Bhomeoticproducts. Development 114, 493–505.

Sweeney, S.T., Broadie, K., Keane, J., Niemann, H., O’Kane, C.J., 1995.Targeted expression of tetanus toxin light chain inDrosophilaspecifi-cally eliminates synaptic transmission and causes behavioural defects.Neuron 14, 341–351.

Teugels, E., Ghysen, A., 1983. Independence of the number of legs and legganglia inDrosophila bithoraxmutants. Nature 304, 440–442.

Truman, J.W., Bate, C.M., 1988. Spatial and temporal patterns of neuro-genesis in the CNS ofDrosophila melanogaster. Dev. Biol. 125, 145–157.

Truman, J.W., Taylor, B.J., Awad, T.A., 1993. Formation of the adultnervous system. In: Bate, M., Martı´nez Arias, A. (Eds.), The develop-ment of Drosophila melanogaster. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY, pp. 1245–1275.

Udolph, G., Prokop, A., Bossing, T., Technau, G.M., 1993. A commonprecursor for glia and neurons in the embryonic CNS ofDrosophilagives rise to segment-specific lineage variants. Development 118,765–775.

Uv, A.E., Harrison, E.J., Bray, S.J., 1997. Tissue-specific splicing andfunctions of theDrosophila transcription factor Grainyhead. Mol. CellBiol. 17, 6727–6735.

Wakimoto, B.T., Turner, F.R., Kaufmann, T.C., 1984. Defects in embry-ogenesis in mutants associated with theAntennapediagene complex ofDrosophila melanogaster. Dev. Biol. 102, 147–172.

White, K., Grether, M.E., Abrams, J.M., Young, L., Farrell, K., Steller, H.,1994. Genetic control of programmed cell death inDrosophila. Science264, 677–683.

White, R.A.H., Lehmann, R., 1986. A gap gene,hunchback, regulates thespatial expression ofUltrabithorax. Cell 47, 311–321.

White, R.A.H., Wilcox, M., 1984. Protein products of thebithorax com-plex in Drosophila. Cell 39, 63–171.

White, R.A.H., Wilcox, M., 1985. Distribution ofUltrabithoraxproteins inDrosophila. EMBO J. 4, 2035–2043.


homeotic regulation of segment-speciﬁc differences in ...al98b.pdf · homeotic regulation of...

Documents