Proc. Nati. Acad. Sci. USAVol. 91, pp. 5972-5976, June 1994Neurobiology

Tau-,B-galactosidase, an axon-targeted fusion proteinCHRISTOPHER A. CALLAHAN*t AND JOHN B. THOMAS**Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, P.O. Box 85800, San Diego, CA 92186; and tDepartment of Neurosciences,University of California at San Diego, La Jolla, CA 92093

Communicated by Dan L. Lindsley, March 4, 1994

ABSTRACT The most commonly used enzymatic reportermolecule, Escheichia col (-galactosidase (.3gal; -D-galacto-side galactohydrolase, EC 3.2.1.23), fails to readily diuse intoaxons; consequently, the morphologies of «-gal-labeled neu-rons cannot directly be determined. For analysis of neuronalpathfinding and synaptic connectivity, this information isessential. We have constructed an axon-targeted 1-gal reporterby fusing the cDNA encoding the bovine microtubule-bindingprotein, tau, to lacZ, the E. coli gene encoding 1-gal. Thisreporter labels cell bodies and axons when expressed bydeveloping and adult Drosophila neurons. It also reveals theentire cellular extent of nonneuronal cells such as muscle fibersand glia. To generate neuronal markers for studies of Dro-sophila neural development, we constructed a tau-3gal en-hancer-trap transposon. From 1500 independent lines gener-ated by mobilization of this transposon, we have isolated a setof useful markers for specific subsets of neurons, glia, andmuscles. Since the tau cDNA-acZ reporter utilizes bovine tau,it may also effectively target 1-gal in vertebrate neurons andprove to be a useful reagent for the analysis of vertebratenervous systems.

into axons, precluding their use in the analysis of neuronalpathfinding and connectivity.One possible means to target (-gal to axonal processes is

to fuse it with a protein that is itself associated with thecytoskeletal components of axons. This strategy was used byGiniger et al. (9) in their construction of a kinesin-gene-lacZfusion gene encoding the heavy-chain subunit of the kinesinmotor molecule fused to (-gal. This fusion protein is effi-ciently targeted to the terminal extensions of axons duringaxonogenesis, but except for rare cases of very high expres-sion levels, the reporter generally fails to give reproduciblestaining along the entire length of the axon. Since microtu-bules are abundant in axons, we felt that the fusion of (-galto a member of the microtubule-associated protein (MAP)family, tau, might target (3-gal more evenly throughout axons.In this report, we use promoter fusions to show that thetau-(-gal fusion gene product is indeed efficiently targeted toaxonal processes, permitting the tracing of neuronal projec-tions. Additionally, we present a set of useful markersgenerated in an enhancer-trap screen by using the bovine taucDNA-lacZ fusion gene.

During development, neurons become synaptically intercon-nected in a highly precise fashion. Identifying the mecha-nisms that underlie this specificity requires a detailed under-standing of both the pathway choices made by developingneurons as they grow to their synaptic target areas and thebehavior of neurons during final target selection. For mostnervous systems, such studies require either the use ofdiffusible dyes to label specific neurons or antibodies thatspecifically label the entire structure of the relevant neuronsthroughout development (1-5). Dye-labeling requires theneurons of interest to be easily identified and, in the case ofsoluble dyes, penetrable with microelectrodes. This is oftennot feasible because of the small size of many neurons andtheir inaccessible locations within the nervous system. Thislimitation, in addition to the limited number of suitableantibody probes, has prevented the detailed analysis ofall buta few central nervous system (CNS) neurons.

In Drosophila, where genetic analyses can be brought tobear on questions of neuronal development, the most effi-cient means to generate cell-specific markers is by enhancertrapping. In this method, a transposableP element containingthe Escherichia coli lacZ gene fused to a minimal promoteris mobilized within the Drosophila genome. When this con-struct inserts near a gene, the minimal promoter often comesunder the control of neighboring transcriptional enhancersand, as a result, directs the expression of the lacZ reporter ina pattern reflecting all or a portion of the transcriptionalactivity of the nearby gene. The vast majority of enhancer-trap experiments have used a lacZ reporter encoding eithera nuclear-targeted or a cytoplasmic form of (3-galactosidase((3-gal; (3D-galactoside galactohydrolase, EC 3.2.1.23) (6-8).Unfortunately, neither ofthese forms of (-gal readily diffuses

MATERIALS AND METHODSConstruction of pCftz/tau-lacZ and petau-lacZ. Standard

methods were used to construct all plasmids (10). The taucDNA-lacZ fusion gene was constructed by a three-wayligation of (i) a 1.2-kb HindIII-Rsa I fragment from pBT43-12, a bovine tau cDNA (11); (ii) a 4.2-kb Xma I-EcoRIfragment of cosPwhite (-gal (12) with the Xma I-cut endblunted with the Klenow fragment ofDNA polymerase I; and(iii) pBluescript KS (Stratagene) digested with HindIII andEcoRI. The resulting plasmid, pBKStau-lacZ, as confirmedby sequencing across the junction, contains an in-framefusion between the bovine tau cDNA and the lacZ gene atcodons corresponding to amino acid 383 of tau and aminoacid 5 of (3-gal. pBKStau-lacZ served as the source for allsubsequent tau cDNA-lacZ constructs. pCftz/tau-lacZ wasconstructed by ligating the blunt-ended 5.4-kb EcoRI frag-ment from pBKStau-lacZ to pCftz (supplied by D. VanVactorand C. S. Goodman) linearized with Not I and blunt-ended.pCftz is a P element vector that contains the ftz neuralenhancer (13) fused upstream of a minimal hsp7O promoter.petau-lacZ was constructed by ligation of the blunt-ended5.4-kb EcoRI fragment from pBKStau-lacZ to an 8-kb blunt-ended Kpn I-Not I fragment from pKZTRAP (9). The P[ry,ftz/lacC]-transformed line was generated by Y. Hiromi andcolleagues (13) and carries a transgene that consists of lacZfused in frame to codon 2 of the ftz gene plus 6.1 kb of 5'sequence containing the neural and zebra control elements.

Generation of Transformed Flies and Mobilization Scheme.Descriptions of Drosophila strains can be found in Lindsleyand Zimm (14). P[Cftz/tau-lacZ] and P[etau-lacZ] were in-troduced into the fly germ line by standard P element

Abbreviations: P-gal, /-galactosidase; CNS, central nervous system;X-Gal, 5-bromo-4-chloro-3-indolyl P-D-galactoside; MAP, microtu-bule-associated protein.

5972

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

May

13,

202

0

Proc. Natl. Acad. Sci. USA 91 (1994) 5973

transformation methods (15). For both constructs, multipleindependent transformants were obtained. A total of 1500autosomal insertions were generated by mobilization ofP[etau-lacZ] from the X chromosome essentially as described (6).Embryo collections were taken from an F3 intercross ofP[etau-lacZ] heterozygotes and screened for (3-gal enzymaticactivity. Lines of interest were then made homozygous ifviable and fertile or balanced over FM7c, CyO, or TM3.Rapid Screening for tau-3-gal Activity. Lines were first

assayed with 5-bromo4-chloro-3 indolyl f3-D-galactoside (X-Gal). Adults from each line were placed into 25-chambercollection blocks and allowed to lay eggs overnight. Embryoswere washed in H20 and dechorionated by placing the blocksin 50% bleach. After rinsing in H20, embryos were fixed for30 min in a 50:50 (vol/vol) mixture of 4% paraformaldehyde(in 0.1 M phosphate buffer, pH 7.4) and heptane. The blockswere blotted on paper towels to remove both aqueous andorganic phases and washed thoroughly in phosphate-bufferedsaline containing 0.1% Triton X-100 (PBT). After the wash,the blocks were placed in a standard X-Gal reaction mixture(16). Lines of interest were analyzed in more detail byimmunostaining of whole mounts and dissected embryos.Immunocytochemistry. Embryo collections, dechorion-

ation, and fixation were carried out as above for the X-Galscreen. Embryos were devitellinized by shaking the collec-tion blocks in 50:50 (vol/vol) methanol/heptane. After theywere blotted and washed in PBT, embryos were transferredto custom-made 25-well Plexiglas dishes and preincubated for2 hr in PBT containing 1% bovine serum albumin and 3%normal goat serum (PBTN). The embryos were incubatedovernight at 4°C with a rabbit anti-,B-gal polyclonal antibody(Cappel) diluted 1:10,000 in PBTN, 2 hr at room temperaturewith biotinylated goat anti-rabbit antibody diluted 1:250 inPBTN, and finally 1 hr with an avidin-biotin-horseradishperoxidase complex (Vectastain ABC elite kit, Vector Lab-oratories). Two-hour washes in PBT followed each incuba-tion. Embryos were processed for peroxidase immunohis-tochemistry. Stained lines were dehydrated in an ethanolseries, cleared in methyl salicylate, and mounted in Canadabalsam/methyl salicylate for examination. Embryo dissec-tions were performed as described by Thomas eta. (1), fixedin 4% paraformaldehyde, and processed through the aboveimmunohistochemistry protocol.

RESULTSTau cDNA-lacZ. Tau proteins constitute a heterogeneous

family of related gene products, generated by differentialsplicing and posttranslational modification, that range in sizefrom 40 to 120 kDa (17-22). Similar to many of their relativesin the MAP family, tau proteins are characterized by a seriesof small, homologous 18-amino acid repeats required forbinding to tubulin, separated by 13-14 amino acid linkingsequences (11). Tau proteins are expressed in both develop-ing and mature vertebrate nervous systems and are highlyenriched within the axons of expressing neurons (23-25),where they are thought to modulate the dynamic propertiesof the densely packed arrays of microtubules that lendstructural support to axons (26, 27).The tau cDNA-lacZ fusion gene used in this study was

created by fusing cDNA sequences encoding the first 383amino acids of a bovine tau protein (11) in-frame to codon 5of the bacterial lacZ gene (Fig. 1A). Contained within thisportion of tau are the amino acid repeats necessary formicrotubule binding.

Tau-3-gal Is Targeted to Axons. To test the axon-labelingcapability of tau-f-igal and to compare its behavior to that ofcytoplasmic (3gal, we examined reporter expression in trans-formed individuals carrying regulatory sequences ofthe fushitarazu (ftz) gene driving either lacZ (P[ry,ftz/lacC]), con-

bovine tau iacZA '''''-ml RIIMMM

microtubule binding domar

FIG. 1. Comparison of cytoplasmic (3-al and tau (3-gal. (A)Schematic ofthe tau (3-gal fusion protein, which contains the first 383amino acids of bovine tau (11), including its microtubule bindingdomain of four 18 amino acid repeats, fused to (3-gal. (B and C)Dissected 11-hr embryos carrying P[ry ftz/lacC] (B), from Hironu etaL (13), and P[Cftz/tau lacZ] (C) stained with antibodies to (3-gal. Inthe focal plane are the dorsal fascicles of the longitudinal connectivesplus a subset of expressing neuronal cell bodies, most of which liemore ventraily in the CNS. In the P~ryftz/lacC] individual, highlevels of staining are evident in the cell bodies of expressing neurons,but negligible levels of staining are seen in axons, the outlines ofwhich can be discerned with Nomarski optics. In contrast, theP[Cftz/tau-lacZ] individual shows axonal staining. Arrows point toan expressing neuron situated lateral to the longitudinal connective.In the P[Cftz/tau-lacZ] individual, an axon can be seen projectingtowards the midline, where it will fasciculate with its contralateralhomologue later in development (arrowheads).

structed by Hiromi et al. (13), or tau cDNA-IacZ (P[Cftz/tau-lacZJ). Both constructs express high levels of reporter inthe =:30 ftz-expressing neurons per hemisegment of thedeveloping embryonic CNS (13, 28). As shown in Fig. 1B,cytoplasmic (-gal in Poryftz/lacC] embryos fails to label theaxons of the ftz-expressing neurons, although high levels ofreporter are detected in neuronal cell bodies. This result isconsistent with the general lack of axonal staining seen inenhancer traps when using cytoplasmic (3-gal (8). In contrast,tau-(3-gal in P[Cftz/tau-lacZ] embryos (Fig. 1C) labels the cellbodies and axons of the expressing neurons. The full extentof the labeling can be seen more clearly by examiningtaul-f-gal-expressing motor neurons, which can be tracedthrough the periphery to their target muscles (Fig. 2B). Inaddition to its axon-targeting capability, taue-3-galalso retainsenzymatic activity and can readily be assayed with X-Gal(Fig. 2C), although immunohistochemical staining providessignificantly higher resolution.

Tau-f3-gal Does Not Mter Normal Neuronal Development.For its utility as a reporter, it is important that tau-(3-gal haveno effect on normal cellular growth and differentiation. Toaddress this, we examined the behavior of identified neuronsexpressing tau-3-gal. Among the ftz-expressing embryonicCNS neurons is the well-characterized aCC neuron (28).During embryonic development, the aCC neuron extends agrowth cone posteriorly and laterally along a stereotypedroute, pioneering a peripheral axon pathway, the interseg-mental nerve (ISN in Fig. 2) (1, 29). In embryos carrying upto four copies of the P[Cftz/tau-lacZ] transgene, aCC behav-ior is indistinguishable from wild-type (Fig. 2A). In addition,enhancer trap lines (see below) that ubiquitously express highlevels of tau-fP-gal do not show any lethality or observablealteration in neuronal development (data not shown). Thus,within our limits of detection, tau-(-gal appears to have noeffect on neuronal development.

Cell-Specific Markers. To generate a set of neuronal mark-ers for the analysis of wild-type and mutant development, weconstructed a tau cDNA-lacZ enhancer-trap vector, petau-

Neurobiology: Callahan and Thomas

Dow

nloa

ded

by g

uest

on

May

13,

202

0

5974 Neurobiology: Callahan and Thomas

A

C

-- . 22 ;4

SNa8

D

4SN

/

E F

N

FIG. 2. Tau-3-gal labeling of axonal projections. (A and B) Embryos carrying P[Cftz/tau-lacZ] stained with antibodies to (-gal. By 10 hr ofembryonic development (A), the aCC cell bodies within the CNS are labeled (arrows) and project axons posteriorly and laterally (arrowheads),pioneering the intersegmental nerve (ISN) in a normal fashion. Two adjacent segments are shown. In 13-hr P[Cftz/tau-lacZ] transformants (B),tau-p-gal-expressing motor neurons within the segmental nerve (SN) branch "a" (SNa) can be seen projecting to muscles 22 and 8. The projectionto muscle 8 is slightly out of the focal plane. (C) An adult from enhancer-trap line 3.358 processed for 3-gal activity with X-gal.Tau-p-gal-expressing motorneurons within the thoracic ganglion project towards the distal segments of the legs (arrow). (D) A 13-hr embryofrom line 3.538 stained with antibodies to 3-gal. Most of the 12 labeled neuronal cell bodies lie ventral to the focal plane. The arrow points toone of three clustered lateral motor neurons that project to muscles 5, 8, and 22. Its axon projects anteromedially to the longitudinal connectiveand then turns anterolaterally, exiting the CNS in the SN. The axons oftwo labeled motor neurons can be seen projecting out the ISN to muscles19 and 20. Six labeled interneurons project contralaterally via the anterior and posterior commissures and elongate axons in discrete fascicleswithin the longitudinal connectives (open arrow). (E) Embryonic CNS tau-p-gal expression in line 3.438 is restricted to five neurons perabdominal hemisegment. Within the focal plane, two lateral motor neurons (arrows) project to muscles 8 and 22 via the SN (out of the focalplane). A medial interneuron in each hemisegment (arrowheads) crosses the midline in the posterior commissure, fasciculating with itscontralateral homologue. A similarly projecting interneuron (open arrow) crosses the midline in the anterior commissure. (F) A sagittal viewof an 11-hr embryo from line 3.566A. The cell bodies (arrows) and axonal processes (arrowheads) of five midline neurons are labeled. The axonsproject dorsally within the CNS, then bifurcate, and extend laterally (arrowheads point to cross section of the bifurcated axons). Anterior isup for A, B, D, and E and is right for C and F.

lacZ. The petau-lacZ vector contains the tau cDNA-lacZfusion gene driven by the P promoter, the Drosophila mini-white gene as a genetic marker, plus a bacterial ampicillin-resistance gene and origin ofreplication for plasmid rescue offlanking genomic DNA (30). Independent insertions on theautosomes and the X chromosome were generated andscreened for expression in the developing CNS and bodywall. From a total of 1500 autosomal lines, r90 expressed

tau-3-gal predominantly in subsets of neurons, 50 showedglial expression, and 30 had somatic muscle patterns ofexpression. The range of different tissues expressing tau-,3gal was similar to that reported in previous enhancer-trapscreens using nuclear-targeted (3-gal (6, 7). However, weisolated a proportionally higher number of lines exhibitingglial and somatic muscle expression. The most probableexplanation for this result is that our ability to visualize the

Proc. Natl. Acad. Sci. USA 91 (1994)

Dow

nloa

ded

by g

uest

on

May

13,

202

0

Proc. Natl. Acad. Sci. USA 91 (1994) 5975

Table 1. Embryonic expression of tau cDNA-1acZ linesLink-

Line age CNS PNS Muscle Glia

3.067 III in

v, I

inin

ch, esin ch, es, mdin ch, es

in

in, sninin

in, sn, isnin, sn, isn

in, snininin

in, sn, isnin, sn, isnin, sn, isn

inin

in, snin, sn

inininin

inin, sn, isn

in

in, sn, isnin, sn

in, sn, isnin

es

eses, md

es

v, 1, d

v, 1, d

v, I

esch, esmd

ch, es

v, l, d

es

ch, esch, md

esv, I

v, 1, dmd I

v, 1, d

ch, es, mdv, 1, d

v, 1, d

chsn ch, es

inin, sn

ininin

in, sn v, I

entire profile of these cell types increased the likelihood oftheir identification. Based on their promise as the most usefulmarkers, we dissected embryos from 61 of these lines andexamined their expression patterns in detail (Table 1).Three examples of lines showing expression in relatively

small subsets of embryonic CNS neurons are shown in Fig.2. Line 3.538 expresses tau-f-gal in six motor neurons and sixinterneurons per hemisegment (Fig. 2D). CNS expression inline 3.438 is restricted to two motor neurons and threeinterneurons (Fig. 2E). Line 3.566 (Fig. 2F) expresses tau-(3-gal in five tightly-clustered neurons at the ventral midline,three of which are the previously identified VUM motorneurons (29, 32), while the other two represent an as yetunidentified class ofDrosophila VUM interneurons similar inmorphology to the grasshopper local DUM neurons (33).

In addition to labeling neuronal processes, the tau-3-galreporter also reveals the entire morphology of nonneuronalcell types. For example, line 3.301 displays tau-3-gal expres-sion in a restricted set of differentiated somatic muscles thatinclude muscles 5 and 25 (Fig. 3A), while line 3.619 displaysreporter expression in a different subset of muscle fibers,including muscles 14, 15, 16, and 17 (Fig. 3B). The cellularprofiles of glial and other support cells are also labeled whentau-,&gal is expressed (Fig. 3C).

DISCUSSIONThe tau-f3-gal reporter gene product efficiently labels theprocesses of neuronal and nonneuronal cells, and, by using itin an enhancer-trap screen, we have generated a number ofuseful cell-specific neuronal markers. In addition, promoterfusions of tau cDNA-lacZ to genes expressed in the CNS,such as ftz, allow the identification of expressing cells andany possible pathway relationships among them. One featureof tau-f3-gal expression we noted is that, although we wereable to follow the entire extent of axonal projections in manytau cDNA-lacZ-expressing neurons, when lower levels oftau-(-gal were present, the most distal portions oflong axonsfailed to label with the reporter. For example, it proveddifficult in many lines to follow the very distal projections ofmotor neurons that innervate the dorsal-most muscles. Itmight be possible to overcome this limitation by coupling thetau cDNA-lacZ reporter to the yeast GAL4 transactivationsystem (34, 35), which could provide an amplification of taucDNA-lacZ expression levels.

In vertebrate nervous systems, individual MAPs showcharacteristic subcellular distributions. For example, mam-malian high molecular weight MAP2 is highly concentrated indendrites (36-38), while tau proteins are enriched in axonsbut also can be detected in cell bodies and dendrites (39). Thecellular distribution of tau-,B gal in Drosophila is similar tothat of tau in vertebrates, in that neurons show stainingthroughout their cell bodies and dendritic processes as wellas in axons. However, we have no evidence that tau-t3-gal isactually distributed via a mechanism similar to that operatingin vertebrates. For instance, the tau-3-gal reporter maysimply be more stable than cytoplasmic (3-gal and subse-quently diffuse throughout the maturing cell.

Expression of tau--3-gal in neurons does not appear to altertheir normal differentiation. From our enhancer trap screen,

Ventral muscles (v) include muscles 15, 16, 17, 25, 26, 27, and 29;lateral muscles (1) include muscles 5, 6, 7, 8, 12, 13, 14, 21, 22, 23,24, 28, and 30; and dorsal muscles (d) include muscles 1, 2, 3, 4, 9,10, 11, 18, 19, and 20. Within the peripheral nervous system (PNS),expression is divided into the three major classes ofsensory neurons,the chordotonal neurons (ch), the external sensory neurons (es), andthe multiple dendrite neurons (md). Glial cells are subdivided intomidline glia (mg), CNS longitudinal glia (lg), exit glia (eg), andperipheral glia (pg) (31).

3.0833.1073.1223.1243.1653.1773.1873.1912.2343.2673.2783.2863.3003.3013.3033.3753.3793.3803.4003.4043.4193.4333.4383.4473.4483.4553.4933.5383.5543.5663.5813.6053.6123.6163.6193.6383.6443.6473.7213.7603.7653.7723.8143.8453.8603.8693.8803.8833.8943.8%3.9173.9203.9444.0024.0084.0404.0464.0594.0654.078

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIVIIIIIIIIIIIIXIIIIIIIIIIIIIIIIIIIIXIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

Expression patterns were assayed with anti-3gal antibodies indissected 11- to 15-hr embryos. Within the CNS, expression issubdivided into subsets of interneurons (in), segmental nerve motor-neurons (sn) that project to ventral and lateral muscles, and interseg-mental nerve motor neurons (isn) that project to dorsal muscles.

Neurobiology: Callahan and Thomas

Dow

nloa

ded

by g

uest

on

May

13,

202

0

5976 Neurobiology: Callahan and Thomas

A::./

Of- :,..

A .;

p,jkarir

,.kf

.|-4 imd, -. mSt.

.s

::.P.,-:

.4AF...

c

FiG. 3. Tau-P-gal reveals the morphology of nonneuronal cells.Thirteen-hour embryos were stained with antibodies to a-gal. (A)Line 3.301B expresses tau-p-gal in a restricted subset of somaticmuscles in abdominal segments A1-A7, including high levels ofexpression in muscle 5 (arrow) and lower expression levels in muscle25 (arrowheads). (B) Line 3.619 displays reporter expression in a

subset of somatic muscle fibers, including muscles 14, 15, 16, and 17.(C) Line 3.286 expresses tau-P-gal in a subset of cells associated withthe peripheral nervous system. The entirety of both of the chordo-tonal sensory organ sheath (s) and cap (c) cells is delineated, as arethe processes of the neighboring bipolar glia (g). Anterior is left forA and up for B and C.

we detected no aberrant morphology of any previouslyidentified neuron expressing the reporter. Although we can-

not formally rule out the possibility that a particular cell typemight be sensitive to tau-p-gal, our screen has yielded a largevariety ofcell-specific patterns without the obvious exclusionofany cell types. Finally, since tau-p-gal contains bovine tau,it may also label the axons of vertebrate neurons and thusprove to be a useful reagent for studying neuronal develop-ment in vertebrate nervous systems.

We thank E. Giniger, L. Y. Jan, Y. N. Jan, D. Van Vactor, C. S.Goodman, K. Butner, M. Kirschner, D. Buenzow, R. Holmgren, andY. Hiromi for providing reagents; M. G. Muralidhar, E. Dritsas, M.Frank, S. Lundgren, and E. Bier for assistance and valuable advice;and G. Lemke, M. Goulding, and members of the J.B.T. lab for

critical reading ofthe manuscript. This work was supported by grantsfrom the National Institutes of Health, a March of Dimes BasilO'Connor Scholar Research Award, and a Pew Scholars Award fromthe Pew Memorial Trusts to J.B.T. and a National Institutes ofHealth/National Institute of General Medical Sciences TrainingGrant PHSGM07198 to C.A.C., who is a student in the MedicalScientist Training Program at University of California at San Diego.

1. Thomas, J. B., Bastiani, M. J., Bate, C. M. & Goodman, C. S.(1984) Nature (London) 310, 203-207.

2. Zipursky, S., Venkatesh, T., Teplow, D. & Benzer, S. (1984) Cell36, 15-26.

3. Patel, N. H., Snow, P. M. & Goodman, C. S. (1987) Cell 48,975-988.

4. Sink, H. & Whitington, P. (1991) Development 112, 307-316.5. Halpern, M. E., Chiba, A., Johansen, J. & Keshishian, H. (1991) J.

Neurosci. 11, 3227-3238.6. Bier, E., Vaessin, H., Shepherd, S., Lee, K., McCall, K., Barbel,

S., Ackerman, L., Carretto, R., Uemura, T., Grell, E., Jan, L. Y.& Jan, Y. N. (1989) Genes Dev. 3, 1273-1287.

7. Bellen, H. J., O'Kane, C. J., Wilson, C., Grossniklaus, U., Pear-son, R. K. & Gehring, W. J. (1989) Genes Dev. 3, 1288-1300.

8. Smith, H. K. & O'Kane, C. J. (1991) Roux Arch. Dev. Biol. 200,306-311.

9. Giniger, E., Wells, W., Jan, L. Y. & Jan, Y. N. (1993) Roux Arch.Dev. Biol. 202, 112-122.

10. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) MolecularCloning: A Laboratory Manual (Cold Spring Harbor Lab. Press,Plainview, NY).

11. Butner, K. A. & Kirschner, M. W. (1991) J. Cell Biol. 115, 717-730.12. Pirrotta, V. (1988) in Vectors: A Survey of Molecular Cloning

Vectors and Their Uses, eds. Rodriguez, R. L. & Denhardt, D. T.(Butterworth-Heinemann, Boston), pp. 437-456.

13. Hiromi, Y., Kuroiwa, A. & Gehring, W. J. (1985) Cell 43, 603-613.14. Lindsley, D. L. & Zimm, G. G. (1992) The Genome ofDrosophila

melanogaster (Academic, San Diego).15. Rubin, G. M. & Spradling, A. C. (1982) Science 218, 348-353.16. Klambt, C., Jacobs, J. R. & Goodman, C. S. (1991) Cell 64,

801-815.17. Lee, G., Cowan, N. & Kirschner, M. (1988) Science 239, 285-288.18. Himmler, A., Drechsel, D., Kirschner, M. W. & Martin, J., D. W.

(1989) Mol. Cell. Biol. 9, 1381-1388.19. Himmler, A. (1989) Mol. Cell. Biol. 9, 1389-1396.20. Larcher, J. C., Boucher, D., Ginzburg, I., Gros, F. & Denoulet, P.

(1992) Dev. Biol. 154, 195-204.21. Goedert, M., Spillantini, M. G. & Crowther, R. A. (1992) Proc.

Natl. Acad. Sci. USA 89, 1983-1987.22. Cuochie, D., Mavilia, C., Georgieff, I. S., Liem, R. K. H., She-

lanski, M. L. & Nunez, J. (1992) Proc. Natl. Acad. Sci. USA 89,4378-4381.

23. Binder, L. I., Frankfurter, A. & Rebhun, L. I. (1985) J. Cell Biol.101, 1371-1378.

24. Matus, A. (1988) Annu. Rev. Neurosci. 11, 29-44.25. Tucker, R. P. (1990) Brain Res. Rev. 15, 101-120.26. Kanai, Y., Takemura, R., Oshima, T., Mori, H., Ihara, Y., Yan-

agisawa, M., Masaki, T. & Hirokawa, N. (1989) J. Cell Biol. 109,1173-1184.

27. Caceres, A. & Kosik, K. S. (1990) Nature (London) 343, 461-463.28. Doe, C. Q., Hiromi, Y., Gehring, W. J. & Goodman, C. S. (1988)

Science 239, 170-175.29. Goodman, C. S., Bastiani, M. J., Doe, C. Q., duLac, S., Helfand,

S. L., Kuwada, J. Y. & Thomas, J. B. (1984) Science 225, 1271-1279.

30. Cooley, L., Kelley, R. & Spradling, A. (1988) Science 239, 1121-1128.

31. Klambt, C. & Goodman, C. S. (1991) Glia 4, 205-213.32. Sink, H. & Whitington, P. M. (1991) J. Neurobiol. 22, 298-311.33. Goodman, C. S., Pearson, K. G. & Spitzer, N. C. (1980) Proc.

Natl. Acad. Sci. USA 77, 1676-1680.34. Fischer, J. A., Giniger, E., Maniatis, T. & Ptashne, M. (1988)

Nature (London) 332, 853-865.35. Brand, A. H. & Perrimon, N. (1993) Development 118, 401-415.36. Caceres, A., Binder, L. I., Payne, M. R., Bender, P., Rebhun, L.

& Steward, 0. (1984) J. Neurosci. 4, 394-410.37. DeCamilli, P., Miller, P. E., Navone, F., Theurkauf, W. E. &

Vallee, R. B. (1984) Neuroscience 11, 819-846.38. Huber, G. & Matus, A. (1984) J. Neurosci. 4, 151-160.39. Viereck, C., Tucker, R. P., Binder, L. I. & Matus, A. (1988)

Neuroscience 26, 893-904.

Proc. Natl. Acad Sci. USA 91 (1994)

Is 9ol (X

Dow

nloa

ded

by g

uest

on

May

13,

202

0