Identification and cell lineage of individual neural precursors in the Drosophila CNS

Acknowledgements We thank Drs Kazuki Yasuda, Karen Raynor, Haeyoung Kong and Lei Yu, and membersof our laboratories for their contributions to these studies. Thiswork wassupported by grants from the US Public Health Service and by the Howard Hughes Medical Institute.

13, 185-192 7 Evans, C., Keith, D., Morrison, H., Magendzo, K. and Edwards, R. (1992)Science 258, 1952-1955 8 Kieffer, B., Befort, K., Gareriaux-Ruff, C. and Hirth, C. (1992) Proc. Natl Acad. Sci. USA 89, 12048-12052 9 Yasuda, K. et al. (1993) Proc. Natl Acad. Sci. USA 90, 6736 -6740 10 Fukuda, K., Kato, S., Mori, K., Nishi, M. and Takeshima, H. (1993) FEBS Lett. 327, 311-314 11 Lutz, R. and Pfister, H. (1992)J. Receptor Res. 12,267-286 12 Tallent, M., Dichter, M., Yasuda, K., Bell, G. I. and Reisine, T. (1993) Soc. Neurosci. Abstr. 19, 846 13 Chen, Y., Mestek, A., Liu, H., Hurley, J. and Yu, L. (1993) Mol. Pharmacol. 44, 8-12 14 Dohlman, H. G., Throner, J., Caron, M. G. and Lefkowitz, R. J. (1991)Annu. Rev. Biochem. 60, 653-688 15 Mello, N. and Mendelson, J. (1980) Science 207, 657-659 16 Lamberts, S., Krenning, E. and Reubi, J. C. (1991) Endocrine Rev. 12,450-482 17 Maurer, R., G~hwiler, B., Buescher, H., Hill, R. and Roemer,

D. (1982) Proc. Natl Acad. Sci. USA 79, 4815-4817 18 Pelton, J., Gulya, K., Hruby, V., Duckies, S. and Yamamura, H. I. (1985) Proc. Natl Acad. Sci. USA 82,236-239 19 Koob, G., Maldonado, R. and Stinus, L. (1992) Trends Neurosci. 15, 186-191 20 Loh, H. and Smith, A. (1990) Annu. Rev. Pharmacol. 30, 123-170 21 Nestler, E. (1993) Crit. Rev. Neurobiol. 7, 23-39 22 Childers, S. (1991) Life Sci. 48, 991-1000 23 Benovic, J., DeBlasi, A., Stone, W., Caron, M. and Lefkowitz, R. (1989) Science 246, 235-240 24 Hosey, M. M. (1992) FASEBJ. 6, 845-852 25 Kwatra, M. M. etal. (1993)J. Biol. Chem. 268, 9161-9164 26 Hausdorff, W., Caron, M. and Lefkowitz, R. (1990) FASEBJ. 4, 2881-2889 27 Silva, A. G., Stevens, C. F., Tonegawa, S. and Wang, Y. (1992) Science 257, 201-206 28 Kong, H. etal. (1993)J. Biol. Chem. 268, 23055-23058 29 Pieffer, A., Brantl, V., Herz, A. and Emrich, M. (1986) Science 223, 774-776

Identificationand cell lineage of individualneuralprecursors in the Drosophila CNS Chris Q. Doe and Gerhard M. Technau Chris Q. Doe is at the Dept of Celland StructuralBiology, and Neuroscience Program, University of Illinois, 505 S. Goodwin Ave, Urbana, IL 61801, USA, and GerhardM. Technauis at the Institut for GenetikZellbiologie, UniversitatMainz, Saarstrasse21, 55122 Mainz, Germany.

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The Drosophila CNS is co.n,plex enough to serve as a model for many of the molecular, cellular and developmental functions of the vertebrate CNS, yet simple enough for single-cell analysis. Recent advances have provided molecular markers that allow most Drosophila CNS precursors to be uniquely identified, as well as methods for determining the complete cell lineage of each precursor. A detailed understanding of wild-type neurogenesis, combined with existing molecular genetic techniques, should provide insight into the fundamental mechanisms that generate neuronal and glial diversity. Neurobiologists from the time of Ram6n y Cajal have reasoned that the study of neurodevelopment would contribute to an understanding of CNS function. While this may be considered a 'reductionlst' approach, it remains a daunting task: there are an estimated 1012 cells in the vertebrate brain, with a rich diversity of neuronal and glial cell types. How is neuronal and glial diversity generated during development? Do cell lineage relationships between neurons or glia foreshadow functional relationships? Our approach has been to study these questions in a relatively simple nervous system, the CNS of the embryonic fruitfly Drosophila melanogaster l''z. The early steps of insect neurogenesis are illustrated in Fig. 1. The CNS develops from the neurogenic region of the ectoderm 3. Individual cells enlarge and delaminate into the embryo to form a sub-ectodermal layer of neural precursors, called neuroblasts (NBs). As their name indicates, NBs are stem cells, dividing asymmetrically to 'bud off' a series of smaller ganglion mother cells (GMCs); subsequently, each GMC divides once to produce a pair of neurons or glia. Studies in grasshopper and Drosophila embryos show that NBs are determined by positional cues within the neuroectoderm 4'5, whereas GMC fate is correlated with its birth-order in the NB cell lineage, suggesting that GMC fate is regulated by reproducible cell inter© 1993,ElsevierSciencePublishersLtd,(UK)

actions or 'determinants' inherited from the parental NB (Refs 1, 6, 7). To identify the molecules controlling cell fate in the CNS, attention has turned to the Drosophila embryo. While many candidate genes have been identified, it has been difficult to interpret mutant phenotypes without markers for individual NBs, or a knowledge of their complete cell lineage. This article reviews recent progress on identifying individual NBs and elucidating their cell lineage, which provides a foundation for future molecular genetic analysis of neuronal determination in the CNS. Molecular markers allow identification of individual embryonic neuroblasts The first NB maps were generated by Bate in grasshopper 8, and Hartenstein and Campos-Ortega in Drosophila 3. These pioneering maps showed that NBs form in a stereotyped spatio-temporal pattern, and that individual NBs could be reproducibly identified by virtue of their position. However, the early maps had two limitations: NBs could be distinguished only by position and individual NBs could not be followed between developmental stages. Recently, it has been possible to overcome both of these limitations using antibody and enhancer-trap markers that label subsets of NBs (Ref. 1). NB formation occurs in five pulses (termed $1-$5), beginning about 30 minutes after gastrulation (Fig. 2). The ten $1 NBs form an incomplete 4 row × 3 column array, and each NB can be identified by its size, position and the genes it expresses. At approximately 40 rain intervals 9 a new group of NBs delaminates: there are five $2 NBs, five $3 NBs (and one glial precursor), five $4 NBs and six $5 NBs for a total of 31 NBs and one glial precursor per thoracic hemisegment (Fig. 2). The pattern of NBs in Drosophila is strikingly similar to that of the grasshopper embryo, where there are 30 NBs and one glioblast per thoracic TINS, Vol. 16, No. 12, 1993

cues? The segment-polarity genes have been attractive candidates for many years, because they are expressed in stripes of neuroectodermal cells coincident with NB formation n. The wingless (wg) segment-polarity gene encodes a secreted glycoprotein 13'14 which is expressed in row-5 neuroectoderm and is required for normal CNS development 15. In wg mutants, row-5 NBs form normally, but adjacent row-4 and row-6 NBs either fail to delaminate or are incorrectly determined (Fig. 3). For example, in 80% of the hemisegments NB 4-2 fails to delaminate, and in the remaining hemisegments it B delaminates but appears to be transformed to the NB 3-2 fate (based on expression of NB and GMC markers) 5. Two results show that the secreted wg -3 protein directly regulates neuroectodermal cell fate, leading to subsequent defects in NB formation and 3-2 determination. First, wg mutant embryos lack 5953 enhancer-trap expression in a cluster of neuroecto3-1 dermal cells at the 4-2 position prior to the formation of NB 4-2; and second, a temperature-sensitive wg mutation can be used to show that wg function is necessary and sufficient for NB 4-2 determination Fig. 1. Summary of D r o s o p h i l a CNS development. In all just before NB 4-2 formation s. These experiments panels the ventral midline is at the bottom; anterior is to the left. ( A ) Fate map of a gastrula-stage embryo shown in illustrate how a detailed array of NB markers can be lateral view. The CN5 develops from the neurogenic used to identify genes controlling NB fate or formation. Many NBs generate a different GMC with each regions: the procephalic NR (pNR) generates the brain; and the ventral NR (vNR; shading) generates the nerve division, and this can be correlated with alterations in cord. dEpi, anlage of the dorsal epidermis. (B) Schematic gene expression during the NB cell lineage 16. It may of the sub-ectodermal layer of neuroblasts (NBs) from an be that stereotyped changes in gene expression lead abdominal hemisegment. Names of the NBs are based on to alterations in NB identity at specific points in the their spatial homologue in the grasshopper embryo ~. lineage. For example, the ruing gene is expressed Abbreviation: MNB, median NB. (C) Each NB divides asymmetrically to make a daughter NB (left column) and a smaller ganglion mother cell (GMCs; middle column). Each GMC generates a pair of neurons or glia. The lineage of NB 1-1 is shown (position of progeny reflect lineage relationships, not position in the nerve cord; neurons, black; gila, shaded).

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hemisegment 1°. Both grasshopper and Drosophila NBs form in pulses, with each new population of NBs primarily arranged in columns; it may be that the mechanisms governing NB formation and determination have been conserved between the two organisms, despite ~300 Myr of evolutionary divergence and different modes of early embryogenesis. Several important questions can now be addressed using molecular markers for individual NBs. (1) Segmentation genes are expressed in subsets of the neuroectoderm 11 - do they specify NB fates? (2) Do proneural or neurogenic genes, which regulate NB formation 12, also play a role in NB determination? (3) Do early forming NBs play a role in the determination of later-forming NBs? (4) Does gene expression change in a single NB during its cell lineage, and does this lead to differential GMC determination? Many experiments are now waiting to be done; in the next section we describe a few experiments where NB markers are used to investigate the mechanisms of NB and GMC determination. Using molecular markers to assay NB and GMC determination Ablation experiments in grasshoppers suggest that positional cues in the neuroectoderm control NB determination 4. What genes encode these positional TINS, Vol. 16, No. 12, 1993

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Fig. 2. Molecular markers for individual embryonic NBs 1. Single hemisegments are shown; anterior, top; ventral midline, dashed. NB stages ($1-$5) and embryonic stages [early 9 (eg) - late 11 (111)] are indicated above each pattern. NB names are based on their spatial homologue in the grasshopper embryo1'4; GP, glial precursor (also called the glioblast3°,31); MNB; X, precursor with no known grasshopper homologue. Protein patterns of fushi tarazu (ftz), achaete (ac), engrailed (en), runt, gooseberry-early (gsb-e), prospero (pros), and wingless (wg) as well as fl-galactosidase patterns in enhancer-trap lines seven-up (svp), ming, 1912 and 5953, are shown. Note that 5953 is expressed in NB 4 - 2 one stage earlier than previously reported 1. 511

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Fig. 3. The secreted wg protein regulates NB formation and specification. Each panel illustrates one hemisegment; anterior, top; ventral midline, dashed. The neuroectoderm and NBs are subdivided into four rows (row numbers given to the left); molecular markers are 5953, wg, runt, seven-up (svp) and engrailed (en). (A,B) Wild-type embryo. (A) Neuroectoderm before the formation of $2 NBs; NBs 4 - 2 and 6 - 2 have not yet delaminated, wg Protein is synthesized in row 5 and secreted anteriorly and posteriorly (small arrows). 5953 Is expressed in subsets of rows 2/3, 4 and 5, whereas runt is expressed only in row 2/3. (B) $2 NB stage, wg Is expressed in NBs of row 5, 5953 is detected in NBs 2-3 and 4-2, and runt is observed in the NBs in row 2/3 and NB 5-3. (C,D) Embryo lacking wg function. (C) Neuroectoderm before the formation of $2 NBs; NBs 4-2 and 6 - 2 have notyet delaminated. Nonfunctional wg protein (T-symbol) is synthesized in row 5 neuroectoderm, but does not trigger expression of 5953 in row 4 neuroectoderm (other domains of 5953 expression are normal); runt is expressed ectopically in row 4; expression of en in the ectoderm fades away later in development. (D) 52 NB stage. NB 4 - 2 appears to be transformed to NB 3-2 (in 20% of the hemisegments) or fails to form (in 80% of the hemisegments); NB 6 - 2 never forms. See Ref. 5 for details.

throughout the NB 6-1 lineage, but is first expressed in the NB 7-4 lineage only after several GMCs are born 16. Are reproducible changes in NB gene expression triggered by intrinsic cues within the NB lineage (perhaps linked to transition of a specific number of cell cycles) or due to exogenous signals received by the NB? NB markers could be applied in two ways to help answer this question: existing cell cycle mutations 17-19 could be used to determine whether altered NB gene expression is cell cycle dependent, or, in vitro culture 2°'21 of individual, identified NBs could be used to determine if modulation of NB gene expression requires external cues. The observed stereotyped alterations in gene expression during NB lineages suggest a hypothesis for how GMCs are uniquely specified ]6. Modulated NB gene expression results in GMCs inheriting 512

different combinations of gene products, depending on their birth-order. Most of these gene products are transcription factors 22. Thus, each GMC may inherit a unique combination of transcription factors that could specify the fate of the GMC. This hypothesis has been virtually impossible to test, because GMC fate is best revealed by the fate of its daughter neurons, and the lineage of very few GMCs is currently known. Ideally, an entire NB lineage should be assayed following addition or deletion of a particular gene product from an identified GMC; transformation of GMC fates within the lineage could then be scored. If multiple NB lineages were fully known, transformations of GMC identity between lineages might be recognizable. These experiments are now possible using recently developed techniques for studying cell lineage.

Analysis of neuroblast cell lineages: the complete lineage of NB 1-1 Several different strategies have been used to label Drosophila embryonic cells and to trace their fate. One is the generation of genetic mosaics 23. Enhancertrap lines 24 and antibodies provide an increasing number of cell-specific markers. Photoactivation of a caged fluorescein has been used to trace the development of epidermal precursors 25. Another approach makes use of the enzymatic lineage marker horseradish peroxidase (HRP) which can be directly injected into cells ~6 or single HRP-filled cells can be transplanted into an unlabeled host ~7'2s. For lineage analysis, most of these techniques have limitations. For example: the numbers of labeled precursors can be difficult to ascertain (genetic mosaics, dye injections); cells have to be punctured by a capillary (dye injections) or removed from their original environment (transplantation of labeled cells); expression of cell-specific markers is transient and in most cases it is not clonally restricted (enhancer-trap lines, antibodies); the morphology of the clonal progeny is obscure because the marker is localized subcellularly or expressed weakly (genetic mosaics, most enhancer-trap lines and antibodies, photoactivation of fluorescent dyes). Because of these limitations, only a few CNS cell lineages have been completely determined; these are some of the midline cells 29'3° and the lineage of a glioblast that gives rise to the longitudinal glia31. To overcome these limitations, a new method has been developed for lineage tracing in which individual cells are labeled with the lipophilic fluorescent tracer 1, l'-dioctadecyi-3,3,3',3'-tetramethylindo-carbocyanine perchlorate (dil) (Ref. 32). Cells are labeled in a non-invasive fashion while remaining in their original positions. The tracer stains the entire cell membrane homogeneously and is transferred to all embryonic progeny in the clone, disclosing their morphology in full detail. Development of labeled cells can be followed in vivo by fluorescence video microscopy or laser scanning microscopy and subsequently, following photoconversion of the dye, the composition of fully differentiated clones can be analysed in permanent whole-mount preparations. Recently, the complete cell lineage of NB 1-1 has been characterized in the embryonic Drosophila CNS (Ref. 2). It derives from a neuroectodermal cell located about two cell diameters lateral to the midline TINS, VoL 16, No. 12, 1993

cells. In vivo tracings of single A C neuroectodermal cells labeled with diI prior to the 14th cell cycle show that the NB 1-1 precursor does not divide before its delamination from the neuro-ectoderm. In abdominal segments, the NB 1-1 lineage includes both neurons and gila: the sibling aCC motoneuron and pCC interneuron, a cluster of four to six interneurons with an ipsilateral fascicle of posteriorly projecting fibers, and two or three B D subperineural glia (Fig. 4). The NB 1-1 lineage seems to be highly invariant since the identical clonal / : ii /i . . . . . . progeny can be observed in a large number of cases using three different techniques (diI labeling of ~:a P' individual neuroectodermal cells, injection of HRP into neuroectodermal cells and isotopic transplantation of single HRP-labeled neuroectodermal cells) 2. The aCC and pCC neurons have been pre"\ viously described in grasshopper and Drosophila as the first progeny of NB 1-1 (Refs 1, 33). Doublelabel experiments show that the subperineural gila in the NB 1-1 Fig. 4. Thoracic (A,B) and abdominal (C,D) variant of the NB 1-1 clone [camera-lucida drawings; the preparations are shown in lateral view (A,C) and dorsal view (8,D); anterior is to the left]. The clone include the previously de- dashed lines indicate the outlines of the neuropil; the outline of the ventral nerve cord is marked by scribed A- and B-glia 34. Thus, NB solid fines. Arrowheads in B and D point to the dorsoventral channels, which demarcate the 1-1 gives rise to both neurons and neuromere. The aCC/pCC neurons (medium shading) are lying dorsally, slightly posterior to the glia, and consequently has to be posterior commissure (p). The aCC sends an ipsilateral projection through the anterior root of the regarded as a neuroglioblast ~. The intersegmental nerve (long black arrow). The pCC projects anteriorly through the ipsilateral NB 1-1 lineage represents the first connective (short black arrow). A cluster of neurons lies ventrocaudally to the aCC/pCC and case indicating that, like in ver- produces a posterior intemeuronal fascicle (short open arrow). In thoracic clones, the cluster tebrates a5, neurons and glia in the comprises 8-14 cells, among them 1-2 motoneurons projecting through the segmental nerve Drosophila CNS can share a com- (long open arrow in A,B); these motoneurons are absent in the abdominal cluster. 5ubperineural gila (light shading) are part of the lineage only in the abdomen (C,D). Two of the gfial cells are mon neural precursor. on the dorsal surface of the CNS (A- and B-gila) and one is located at the ventral surface Although the NB 1-1 lineage is located (C). a, anterior commissure. Scale bar, 30#m. invariant within abdominal segments, there are clear segmentspecific differences between thoracic and abdominal their original dorsal positions. Thus, the identity of a NB 1-1 lineages 2. The thoracic NB 1-1 lineage lacks neural precursor with respect to the type of lineage it glial cells and contains a larger cluster of neurons (8-14 produces (for example, whether it behaves as NB 1-1 cells), among them an extra one or two motoneurons or NB X-X) is not irreversibly committed at the early (Fig. 4). Some common features of the NB 1-1 gastrula stage. Instead, the cell is able to respond to lineage are generated at early stages, such as the aCC positional cues in the neuroectoderm and adjust its and pCC (Ref. 1); it may be that the segment-specific identity correspondingly aT. These data, taken together differences occur late in the lineage, producing glia in with the NB-ablation studies in the grasshopper the abdomen and additional neurons in the thorax. embryos a and the wg studies 5, all suggest that NB fate is specified by positional cues within the neuroUsing cell lineage analysis to assay NB ectoderm. determination Neuroectodermal cells at the gastrula stage may be Cell transplantations and cell lineage analysis have receptive to positional cues that specify NB identity, been used to examine the timing of NB 1-1 de- but they are already firmly committed with respect to termination a6'aT. In one experiment, single neuro- segmental identity 36. A neuroectodermal cell taken ectodermal cells at the early gastrula stage were from the abdominal neurogenic region and transheterotopically transplanted along the dorsoventral planted into the thoracic neurogenic region gives rise axis of the neurogenic region (Fig. 5). Each cell was to the abdominal variant of the NB 1-1 clone, and vice taken from the most dorsal area of the neurogenic versa (Fig. 5). Moreover, the abdominal NB 1-1 region and transplanted ventrally. Under these con- lineage is altered in homeotic mutants; the A- and ditions, the cells developed according to their new B-glia (normally part of the abdominal NB 1-1 lineage) position. For example, several transplants gave rise are missing in A1-7 segments in Df 109 embryos to the NB 1-1 clone or to ventral midline cell clones which lack Ubx and abd-A function. An abdominal that would never have been produced by the cells in neuroectodermal cell from Df 109 mutant transplanted _

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using this knowledge to investigate neuronal determination with a combined molecular, genetic and cellular approach.

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5. NB segmental identity, but not NB fate, is firmly committed in the neuroectoderm. Lateral view of the early gastrula stage. The head neurogenic region (14; dark shading), thoracic NR (T; medium shading), and abdominal NR (A; light shading) are indicated. Arrows indicate isotopic (a,b) and heterotopic (c-e) transplantations of individual HRP-filled neuroectodermal cells into unlabeled gastrula-stage hosts. The composition of the lineages derived from the transplanted cells is shown in the table. Cell numbers are indicated for the clonal components aCC, pCC, neuronal cluster (cl), motoneurons in the cluster (ran) and glia. See text for details. Fig.

into a wild-type host will produce a thoracic-type NB 1-1 clone, showing that Ubx and/or abd-A are required cell-autonomously to generate the abdominaltype NB 1-1 lineage.

Concluding remarks

Acknowledgements We thankAndreas Prokopand KeiIto for help with the figures, and Quynh ChuLaGraf'f,Xuan Cui andAndreasProkop for criticalcomments on the manuscript. Supportedby the NIH, an NSF PresidentialYoung InvestigatorAward, and the Searle ScholarsProgram (C. Q D.); and the Deutsche Forschungsgemeinschaft (G. M. T.). 514

Those of us studying the generation of neuronal diversity in the Drosophila CNS have a vast array of molecular genetic techniques for manipulating gene function, but the usefulness of these tools has been limited by our lack of detailed information about wildtype CNS development. Recent advances have resulted in molecular markers for individual NBs and GMCs that can be used as probes to assay for altered NB and GMC determination. Furthermore, novel celllineage techniques make it feasible for the first time to determine the entire lineage of every embryonic neuroblast. The NB 1-1 lineage can be taken as an example of how the methods available now allow for efficiently combining lineage analysis with experimental manipulations and genetics. For example, cell transplantations have provided insight into the timing of NB 1-1 determination 36'37. Genes expressed in the NB 1-1 lineage include engrailed, ruing, seven-up, 1 16 30 38

Ultrabithorax, f u s h i tarazu, and even-skipped '

'" '

;

Selected r e f e r e n c e s 1 Doe, C. Q. (1992) Development 116, 855-863 2 Udolph, G., Prokop, A., Bossing, T. and Technau, G. M. (1993) Development 118, 765-775 3 Hartenstein, V. and Campos-Ortega, J. A. (1984) Roux Arch. Dev. Biol. 193, 308-325 4 Doe, C. Q. and Goodman, C. S. (1985) Dev. Biol. 111, 206-219 5 Chu-LaGraff, Q. and Doe, C. Q. (1993) Science 261, 1594-1597 6 Taghert, P. H. and Goodman, C. S. (1984) J. Neurosci. 4, 989-1000 7 Raper, J. A., Bastiani, M. J. and Goodman, C. S. (1984) J. Neurosci. 4, 2329-2345 8 Bate, C. M. (1976) J. Embryol. Exp. Morphol. 35, 107-123 9 Hartenstein, V., Rudloff, E. and Campos-Ortega, J. A. (1987) Roux Arch. Dev. Biol. 196, 473-485 10 Doe, C. Q. and Goodman, C. S. (1985) Dev. Biol. 111, 193-205 11 Hooper, J. E. and Scott, M. P. (1992) Results Prob. CellDiffer. 18, 1-48 12 Campos-Ortega, J. A. and Knust, E. (1990) Annu. Rev. Genet. 24, 387-407 13 van den Heuvel, M., Nusse, R., Johnston, P. and Lawrence, P. (1989) Cell 59, 739-749 14 Gonzalez, F., Swales, L., Bejsovec, A., Skaer, H. and Martinez-Arias, A. (1991) Mech. Dev. 35, 43-54 15 Patel, N. H., Schafer, B., Goodman, C. S. and Holmgren, R. (1989) Genes Dev. 3,890-904 16 Cui, X. and Doe, C. Q. (1992) Development 116, 943-952 17 Edgar, B. A. and O'Farrell, P. H. (1990) Ceil 62, 469-480 18 Hime, G. and Saint, R. (1992) Development 114, 165-171 19 Lehner, C. F. (1992) J. Cell ScL 103, 1021-1030 20 Huff, R., Furst, A. and Mahowald, A. P. (1989) Dev. Biol. 134, 146-157 21 Luer, K. and Technau, G. M. (1992) Development 116, 377-385 22 Goodman, C. S. and Doe, C. Q. (1993) in The Development of Drosophila (Bate, M. and Martinez-Arias, A., eds), pp. 1131-1206, Cold Spring Harbor Press 23 Garcia-Bellido, A., Ripoll, P. and Morata, G. (1973) Nature 251-253 24 245, O'Kane, C. J. and Gehring, W. J. (1987) Proc. NatlAcad. Sci. USA 84, 9123-9127 25 Vincent, J-P. and O'Farrell, P. H. (1992) Cell 68, 923-931 26 Technau, G. M. and Campos-Ortega, J. A. (1985) RouxArch. Dev. Biol. 194, 196-212 27 Technau, G. (1987) Development 100, 1-12 28 Prokop, A. and Technau, G. M. (1993) in Cellular Interactions in Development: a Practical Approach (Hartley, D., ed.), pp. 33-57, Oxford University Press Klarnbt, C., Jacobs, J. R, and Goodman, C. S. (1991) Cell 64, 29 801-815 30 Doe, C. Q., Hiromi, Y., Gehring, W. J. and Goodman, C. S. (1988) Science 239, 170-175 31 Jacobs, J. R., Hiromi, Y., Patel, N. H. and Goodman, C. S. (1989) Neuron 2, 1625-1631 32 Bossing, T. and Technau, G. M. (1993) in Gene, Brain,

Behaviour (Proceedings of the 21st G6ttingen Neurobiology Conf.) (Eisner, N. and Heisenberg, M., eds), p. 744, Georg

Thieme

33 Goodman, C. S., Raper, J. A., Ho, R. K. and Chang, S. (1982)

in Developmental Order: Its Origin and Regulation (Subtelny, S. and Green, P, B., eds), pp. 275-316, Alan R. Liss 34 Kl~mbt, C. and Goodman, C. S. (1991) Gila 4, 205-213 35 Cameron, S. and Rakic, P. (1991) Gila 4, 124-137 36 Prokop, A. and Technau, G. M. (1993) in Gene,

Brain, Behaviour (Proc. of the 21st G6ttingen Neurobiology Conf.) (Eisner, N. and Heisenberg, M., eds), p. 743, Georg

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in the future, mutations in these genes can be assayed 37 Udolph, G., Prokop, A., Bossing, T., Sohn, J. and Technau, G. M. (1993) in Gene, Brain, Behaviour (Proc. of the 21st for changes in cell fate in the context of the entire G6ttingen NeurobioloEy Conf.) (Eisner, N. and Heisenberg, NB 1-1 lineage, rather than in single neurons in M., eds), p. 742, Georg Thieme isolation3°'38. Challenges of the future include linking 38 Doe, C. Q., Smouse, D. and Goodman, C. S. (1988) Nature 333, 376-378 each complete lineage to a specific NB in the map, and TINS, Vol. 16, No. 12, 1993