Development 115, 267-276 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

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Dynamic expression of the cell adhesion molecule fasciclin I during

embryonic development in Drosophila

LINDA MCALLISTER*, COREY S. GOODMAN

Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA

andKAI ZINN

Division of Biology 216-76, California Institute of Technology, Pasadena, CA 91125, USA

'Present address Department of Internal Medicine, University of California, San Francisco, CA 94143

Summary

A number of different cell surface glycoproteins ex-pressed in the central nervous system (CNS) have beenidentified in insects and shown to mediate cell adhesionin tissue culture systems. The fasciclin I protein isexpressed on a subset of CNS axon pathways in bothgrasshopper and Drosophila. It consists of four homolo-gous 150-amino acid domains which are unrelated toother sequences in the current databases, and is tetheredto the cell surface by a glycosyl-phosphatidylinositollinkage. In this paper we examine in detail theexpression of fasciclin I mRNA and protein duringDrosophila embryonic development. We find that fascic-lin I is expressed in several distinct patterns at differentstages of development. In blastoderm embryos it isbriefly localized in a graded pattern. During the germband extended period its expression evolves through twodistinct phases. Fasciclin I mRNA and protein areinitially localized in a 14-stripe pattern which corre-sponds to segmentally repeated patches of neuroepi-

thelial cells and neuroblasts. Expression then becomesconfined to CNS and peripheral sensory (PNS) neurons.Fasciclin I is expressed on all PNS neurons, and thisexpression is stably maintained for several hours. In theCNS, fasciclin I is initially expressed on all conunissuralaxons, but then becomes restricted to specific axonbundles. The early conunissural expression pattern isnot observed in grasshopper embryos, but the laterbundle-specific pattern is very similar to that seen ingrasshopper. The existence of an initial phase ofexpression on all commissural bundles helps to explainthe loss-of-commissures phenotype of embryos lackingexpression of both fasciclin I and of the D-abl tyrosinekinase. Fasciclin I is also expressed in several nonneuraltissues in the embryo.

Key words: adhesion molecule, Drosophila development,axon guidance, neural development.

Introduction

Fasciclin I is a 72xlO3Afr glycoprotein that wasoriginally identified by a monoclonal antibody (mAb)screen for molecules expressed on subsets of CNS axonsin embryos of the grasshopper Schistocerca americana(Bastiani et al., 1987). Cell ablation experiments in thisorganism had provided evidence that CNS growthcones use specific axon bundles as guidance cues(reviewed by Goodman et al., 1984), and the screen wasdesigned to identify surface molecules whose ex-pression patterns correlated with a potential role inguidance. Two other molecules, fasciclin II (in grass-hopper) and fasciclin III (in Drosophila melanogaster),were also identified in these types of screens (Bastiani etal., 1987; Patel et al., 1987; Harrelson and Goodman,1988; Snow et al., 1988). The cloned sequences for

fasciclin I and II were used to isolate homologs inDrosophila (Zinn et al., 1988; Grenningloh et al., 1990,1991).

The sequence of fasciclin II is closely related to thatof the vertebrate neural adhesion molecule N-CAM(Harrelson and Goodman, 1988). All three fasciclinsare capable of mediating cell adhesion in tissue culturesystems (Snow et al., 1989; Elkins et al., 1990b;Grenningloh et al., 1990).

To gain insights into the functions of these moleculesduring neural development, mutations in the Dros-ophila genes encoding the three fasciclins have beenisolated (Elkins et al., 1990a; Grenningloh et al., 1991;T. Elkins and C. S. Goodman, unpublished results).Apparent null mutations in the fasciclin I (fasl) andfasciclin III (fasIII) genes are homozygous viable, whilea null fasciclin II (fasll) mutation causes lethality when

268 L. McAllister and others

homozygous. FasII mutations prevent formation of aspecific longitudinal pathway that expresses the protein(Grenningloh et al., 1991). The other mutations do notproduce alterations in the embryonic CNS that can bevisualized with currently available antibody reagents.Defects in individual pathways are usually very difficultto detect, however, because few axon-specific anti-bodies exist. For fasciclin II, it was possible to visualizethe affected longitudinal pathway in fasll mutants withthe 22C10 mAb, which labels a restricted set of axons instage 12 and 13 embryos (Grenningloh et al., 1991).

Analyses of antibody-stained embryonic CNS prep-arations from fasl and fasIII mutants do show that manyof the axons that express these proteins follow normalpathways in the mutant embryos, suggesting that if theyare involved in axon guidance their functions can besubstituted for by other molecules (Elkins et al., 1990a;T. Elkins, R. Jacobs, and C.S.G., unpublished results).This apparent functional redundancy correlates withobservations made on vertebrate cell adhesion mol-ecules in tissue culture systems, where it has been foundthat antibodies against several different molecules arerequired to block axon fasciculation or neurite out-growth (Tomaselli et al., 1986; Chang et al., 1987;Neugebauer et al., 1988).

In the case of fasciclin I, a requirement for thismolecule in embryonic neural development can beuncovered by constructing a double mutant stocklacking expression of both fasciclin I and of theDrosophila homolog of the abl tyrosine kinase, which isexpressed on CNS axons (Gertler et al., 1989). InfaslI abl mutant embryos, the commissural axon path-ways are often completely absent. The mutant combi-nation can also be shown to alter pathfinding byidentified neurons. The fasciclin I-positive RP1 moto-neurons normally extend growth cones across themidline and exit the CNS in the contralateral mterseg-mental nerve (ISN) pathway, but in mutant embryostheir axons usually fail to cross over and instead growout in the ipsilateral ISN (Elkins et al., 1990a). The ablmutation alone does not cause embryonic lethality, andno gross alterations in the CNS are observed in ablmutant embryos (Henkemeyer et al., 1987; Gertler etal., 1989).

The synergistic interaction between the fasl and ablmutations suggests that fasciclin I does not simplymediate adhesion, but participates in a signal transduc-tion pathway involved in growth cone extension orguidance. This pathway may function in parallel with apathway involving abl, so that elimination of onepathway does not cause severe defects, but removingboth pathways prevents correct formation of the CNSaxon array.

The sequence of the fasciclin I protein does notclearly define its function, as it is not related to othersequences in the current databases. It consists of fourhomologous 150 amino acid domains, and is linked tothe external cell surface by a glycosyl-phosphatidyl-inositol (GPI) linkage (Zinn et al., 1988; Hortsch andGoodman, 1990). Alternative splicing of two 6-9 bpmicro-exons located between the coding regions for the

second and third domains produces mRNAs encodingthree different isoforms of the protein in each species(McAllister et al., 1992). In the present study, we haveexamined in detail the pattern of expression of fasciclinI mRNA and protein during development of theDrosophila embryo. These results help to explain theorigin of the fasl/abl phenotype, and may provideinsights into the possible roles of this molecule inembryonic development.

Materials and methods

Immunocytochemistry with anti-fasciclin I mAbsImmunocytochemistry on whole-mount embryos was per-formed according to Patel et al (1987, 1989). Ascitespreparations (1:500) or cell supematants (1:2) of the anti-fasciclin I monoclonal antibody 6D8 (Hortsch and Goodman,1990) were used as the primary antibody Photographs weretaken using Nomarski optics on either a Zeiss Axiophot or aNikon compound microscope.

Whole mount in situ hybridization with fasciclin IprobesIn situ hybridization to whole mount embryos was performedessentially according to the protocol of Tautz and Pfeifle(1989). Non-radioactive probes were made by incorporatingdigoxigerun-dUTP (Boehringer Mannheim) either by primingwith random hexamers or by synthesizing anti-sense single-stranded DNA probe using a single primer PCR reaction.Digoxigenin was detected using a monoclonal antibodyagainst digoxigenin coupled to alkaline phosphatase. Thealkaline phosphatase reaction was carried out using NBT andX-phosphate as supplied in the Boehringer Mannheim Geniusdetection kit, or with a Vector Laboratories ABC kit.Photographs were taken as for antibody staining.

Results

Dynamic expression of fasciclin I mRNA and proteinduring blastoderm and gastrula stagesWe examined the expression of fasciclin I RNA duringdevelopment using the digoxigenin whole-mount in situhybridization method of Tautz and Pfeifle (1989). Thereare two major fasciclin I transcripts, of 3.0 and 5.0 kb,which differ only in their 3' untranslated regions. Weused both a coding region probe and a probe specific tothe large RNA. Identical RNA expression patternswere observed with the two probes (data not shown).Fasciclin I protein expression was visualized usinghorseradish peroxidase (HRP) immunohistochemistrywith a mAb raised against a bacterially expressedfasciclin I/protein A fusion (Hortsch and Goodman,1990).

Fasciclin I mRNA is maternally deposited in the egg,and the message is initially uniformly distributed. Atabout 1 h after egg laying (AEL) the nuclei, which haveundergone 7 synchronous divisions in the central regionof the egg, begin to migrate out to the periphery. Thefasciclin I RNA hybridization signal becomes localizedwith the nuclei, so that it forms a broad annulus (datanot shown). After the nuclei reach the periphery they

Embryonic expression of fasciclin I 269

divide three more times, and then cellularizationbegins. Just before cellularization, fasciclin I RNA isobserved in a ring at the level of the nuclei (Fig. 1A).

Fasciclin I protein is initially uniformly distributed onthe surface of the egg, and it first appears when theoutlines of the forming cell membranes become evi-dent. During the cellularization process there is aperiod of about 10-15 min in which fasciclin I protein isexpressed at a low level in the anterior third of the eggand at a higher level in the posterior two thirds. Thehighest levels of protein observed at this time are on themembrane at the extreme posterior end of the egg,adjoining the pole cells (Fig. IB). Pole cells are thegerm line precursors, and segregate away from the restof the embryo very early in development, at the time ofthe 9th cleavage division. The pole cells themselves donot express the protein at this time. After this stage,fasciclin I protein again becomes uniformly distributed,and at the beginning of gastrulation (2:50 h AEL, stage6 of Campos-Ortega and Hartenstein, 1985) it isexpressed at a high level on all cell membranes. Duringgastrulation (2:50-3:20h AEL), fasciclin I mRNA isapparently uniformly distributed (data not shown). Theprotein, however, is expressed at higher levels on theinvaginating cells of the ventral and cephalic furrows(Fig. 1C).

Immunohistochemical analysis of the progeny ofcrosses in which there is no maternal fasciclin Iexpression (fasITE/fasITE femalesx+/+ males), or inwhich 1/2 of the orogeny lack zygotic fasciclin Iexpression (+/fasI'E femalesxfasP^ /fasITE males)indicates that the transition between maternal andzygotic expression takes place at about the time ofgastrulation (data not shown).

Two phases of fasciclin 1 expression in germ bandextended embryosImmediately following gastrulation, the fast phase ofgerm band elongation begins. The germ band refers tothe segmented region of the embryo, which gives rise tothree gnathal, three thoracic, and at least nine abdomi-nal segments. During germ band elongation thesegmental pnmordia fold over the posterior end of theembryo and extend forward as far as 75 % egg-length(EL; measured from the posterior end). The embryoremains in the germ band extended stage for severalhours (3:40-7:20 hours AEL, stages 9-11). During thistime neuroblasts delaminate from the ectoderm anddivide asymmetrically to produce a chain of ganglionmother cells (GMCs). Each GMC then divides once,giving rise to two neurons. This process eventuallygenerates approximately 250 neurons per hemisegment,and these will form the segmental ganglia of the CNS.

In the 4-5 h old embryo fasciclin I RNA is localized in14 stripes, which correspond to segmentally repeatedbands of neuroepithelial cells and delaminating neuro-blasts (Fig. ID, stage 9). The protein has a similardistribution, although it appears slightly later and seemsto be expressed at lower levels in the epithelial layer(Fig. IE, stage 10). These stripes of neuroblasts andneuroepithelial cells are localized to the posterior

compartment of the segment, or the anterior region ofthe parasegment (Martinez-Arias and Lawrence, 1985;data not shown). This was determined by doublestaining with anti-fasciclin I mAb and antibodies against/S-galactosidase in a transgenic line containing a/3-galactosidase gene under the control of the promoterof the fushi tarazu gene, which is expressed in 7 stripesat the anterior border of alternating parasegments(Hafenet al., 1984).

In later germ band extended embryos the hybridiz-ation signal moves inward into the embryo and becomesa single longitudinal stripe running the entire length ofthe segmented region (Fig. IF, stage 11). Immunocyto-chemistry with the mAb shows that the protein has asimilar distribution (data not shown). This stripecorresponds to the neuronal layer, in which many cellsexpress fasciclin I. These cells are localized in bothcompartments of the segment, so a banded pattern is nolonger observed. We do not know if the early neuronsexpressing fasciclin I are primarily derived from thefasciclin I-positive neuroblasts.

Stable fasciclin I expression in the peripheral nervoussystemEach thoracic and abdominal segment of the embryocontains five classes of peripheral sensory neuronslocalized in three clusters: the ventral, lateral, anddorsal clusters (Campos-Ortega and Hartenstein, 1985;Ghysen et al., 1986). The external sensory (es) neuronand chordotonal (ch) neuron classes both have singledendntes, and there are three classes of neurons withmultiple dendntes (md neurons). The precursors ofthese PNS neurons undergo their final mitoses at about6:30h AEL (Bodmer et al., 1989). The dorsal clusterappears first, followed closely by the lateral and ventralclusters. These cells then extend axons into the CNS,and the mature pattern of PNS neurons appears byabout 11 h AEL.

At the beginning of germ band retraction (about 7 hAEL) small, segmentally repeated clusters of fasciclin IRNA-expressing cells can be visualized under thedorsal ectoderm (data not shown). These are the dorsalcluster neurons. As germ band retraction proceeds, twoother clusters of hybridizing cells appear, and all threeclusters change shape and grow as the embryo develops(Fig. 1G). This embryo is at about stage 12/5 of Klambtet al. (1991). These authors have subdivided stage 12into three substages, 12/5, 12/3, and 12/0, where thesecond number corresponds to the number of segmentsstill on the dorsal side of the embryo. Fig. 2A shows insitu hybridization to neurons of the ventral and lateralclusters in a stage 14 embryo. Fig. 2B shows the lateraland dorsal clusters. All of the PNS neurons appear toexpress fasciclin I RNA, as do many of their supportcells.

The early cells of the PNS also express fasciclin Iprotein. This staining is less obvious than in in situhybridization experiments, because the surroundingectoderm also stains with the antibody (Fig. 3C,D;stage 12/0). This ectodermal staining could be due tosoluble fasciclin I which is bound to these cells (Hortsch

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and Goodman, 1990), as we do not see clear expressionof fasciclin I RNA in the ectoderm at this stage by in situhybridization.

Later in development fasciclin I protein is expressedon the surfaces of all cell bodies and axons in the PNS(Fig. 3B). The ectodermal expression disappearsshortly after germ band retraction is completed.Fasciclin I protein is also observed on the surfaces ofglial cells that enwrap the PNS axon bundles (Fig. 3A).In addition to the segmentally repeated PNS cells, thereare several specialized sensory structures in the headand tail. Most or all of the cells in these organs alsoexpress fasciclin I protein (Zinn et al., 1988). Fasciclin IRNA and protein are retained by some or all of the PNScells, albeit at reduced levels, until the embryo developscuticle and it is no longer possible to do whole-mountstaining (about 15 h AEL; Fig. 1H shows in situhybridization to a stage 16 embryo).

Fasciclin I is initially expressed by all commissuralaxons but then localizes to specific bundlesCNS axonogenesis begins in stage 12/5 with theformation of the commissural tracts. The posteriorcommissure is established first, followed rapidly by theanterior commissure. The commissures are initiallyfused at the midline (see Fig. 2D), but then becomeseparated by the midline glial cells (Klambt et al.,1991). By stage 15, a complete orthogonal axon scaffoldhas formed, with anterior and posterior commissures, abilaterally symmetric pair of longitudinal axon tractswhich extend the length of the embryo, and twoperipheral nerve roots per hemisegment (the ISN andthe segmental nerve (SN)), where motor-neuron axonsexit and sensory neurons enter the CNS.

Most or all of the initial commissural axons expressfasciclin I protein at approximately equal levels (Figs2D, 4A). The ventral unpaired midline neurons(VUMs), whose growth cones and axons lie at thejunction between the fused anterior and posteriorcommissures at stage 12/0, express fasciclin I on boththeir cell bodies and axons at this stage (Fig. 4A). Thisexpression is transient, and disappears shortly after theseparation of the commissures. Other early neuronsexpressing fasciclin I protein on their cell bodies includethe aCC and RP1 cells, and a large laterally locatedcluster of neurons. In contrast to the transient VUMexpression, these cells continue to express the proteinfor several hours. aCC and RP1 are motoneurons andextend axons in the ISN pathway. Fasciclin I isexpressed on this pathway at high levels throughoutdevelopment.

Shortly after the commissures have separated, theexpression of fasciclin I on commissural axons becomesrestricted to a single large bundle at the posterior edgeof the posterior commissure, adjacent to the aCC cellbodies, and a smaller bundle in the middle of theanterior commissure (Fig. 4D,E). The protein disap-pears from the rest of the commissural axon bundles.The axons of the lateral cluster neurons continue toexpress fasciclin I, and most of these axons form a tightbundle which appears to join the fasciclin-I positive

Fig. 1. Timeline of fascichn I mRNA and proteinexpression in the developing Drosophila embryo. In situhybridization to embryos with fasciclin I cDNA probes wasperformed using the digoxigenin whole-mount procedure ofTautz and Pfeifle (1989). Fasciclin I protein expression wasdetected using the 6D8 mAb and HRPimmunocytochemistry. (A) Fasciclin I mRNA localizationin a late syncitial blastoderm embryo (stage 4). Thearrowhead indicates a nng of higher fasciclin I mRNAconcentration The localization of this RNA appearssimilar to that of the basally localized transcripts describedby Davis and Ish-Horowicz (1991). Anterior is to the left.(B) Protein localization in a stage 5 embryo. Anterior is tothe right, dorsal up. The large arrowhead on the rightindicates the border between low anterior and higherposterior expression of fasciclin I. This border correspondsapproximately to the posterior edge of the acron. The twosmall arrowheads on the left delimit the membraneadjoining the pole cells, which has very high levels offasciclin I protein (black staining) (C) Protein localizationin a gastrulating embryo (stage 6). The embryo is viewedfrom the ventral side, and anterior is to the left Fasciclin Iis expressed on all cell membranes, but at somewhat higherlevels on cells at the edges of the ventral furrow (axismarked by smaller arrowhead on the right) and cephalicfurrow (axis marked by larger arrowhead on the left). (D)mRNA localization in a stage 9 germ band extendedembryo. 14 stripes of expression are visible (one stripeindicated by arrowhead); these appear to extend throughthe whole depth of the germ band and therefore includeboth ectodermal and mesodermal cells Anterior is to theright, dorsal up. (E) Protein localization in a stage 10embryo. Segmental stripes or patches of high-levelexpression are visible (arrowhead). Unlike the mRNAstripes, however, they appear to be localized to theneuroectoderm layer of the germ band Ectodermal andmesodermal cells express fasciclin I at lower levelsExpression is also observed in the cells of the posteriormidgut and proctodeum. Anterior is to the left, dorsal up.(F) mRNA localization in a stage 11 embryo. Segmentalstripe expression is no longer visible; instead expression isprimarily restricted to the neuronal layer, and extends thelength of the germ band (large arrowhead) Segmentallyrepeated thickenings of this longitudinal stripe areobserved, however, indicating that subsets of neuronsexpress the mRNA at higher levels. The small arrowheadindicates the brain region, which also expresses fasciclin IAnterior is to the right, dorsal up. (G) mRNA localizationin an embryo undergoing germ band shortening (stage 12).Segmentally repeated clusters of PNS cells are observed.The arrowhead points to a developing dorsal cluster.Anterior is to the left, dorsal up. (H) mRNA localizationin a stage 16 embryo, after the beginning of nerve cordcondensation The left arrowhead indicates the brain (outof focus). The middle arrowhead points to the dorsalcluster of a PNS cell group It is possible that only a subsetof PNS cells express fasciclin I at this stage. The rightarrowhead indicates the gonad, which expresses fasciclin ImRNA and protein at high levels. Anterior is to the left,dorsal up.

bundle in the posterior commissure (Fig. 4D,E). Thereis also a second, smaller fascichn I-positive tractconnecting these neurons to the anterior commissure.The protein is also expressed on a single bundle at the

Fig. 2. FascicUn I mRNAand protein expression inthe PNS and CNS. mRNAwas detected as in Fig. 1.(A) mRNA localization inthe ventral and lateralclusters of the PNS in astage 14 embryo. Thesmall arrowhead at thetop indicates a thoraciclateral cluster, and thelarger arrowhead directlybelow is a largerabdominal cluster. Thesmall arrowhead on theleft points to a smallventral cluster of neurons,and the large arrowheadon the right to anabdominal dorsal cluster.The dark, out-of-focusstaining on the left is theCNS. Anterior is up,dorsal to the right. (B)mRNA localization in thedorsal and lateral clustersof the PNS in an embryoof a similar age. The largearrowhead at the topindicates a larger thoracicdorsal cluster, and thesmall arrowhead below ita smaller abdominal dorsalcluster. The largearrowhead on the leftpoints to an abdominallateral cluster. The cells ofthe dorsal epidermis,which express fasciclin ImRNA at high levels,form an out-of-focusvertical line at the right,indicated by the right-hand arrowhead. An tenor

is up, dorsal to the right. (C) mRNA localization within the CNS at stage 14. A ventral view, anterior up, is shown. Segmentallyrepeated clusters of expressing cells are visible (7 complete segments). Note the lack of expression along the midline at this stage.The large arrowhead indicates one of the CNS lateral clusters of neurons. The cells located medial and slightly posterior to thelateral cluster may include the RP1 neurons; posterior to these cells is another group which may include the aCC and U neurons.The small arrowhead at the left indicates a group of PNS cells The segmental PNS clusters can be seen extending to the right andleft of each CNS segment. (D) FascicUn I protein expression in the CNS of a stage 12/3 embryo. This embryo was dissected andstained with rat anti-fascicUn I antiserum (1:200) and fluorescein-conjugated goat anti-rat second antibody. Anterior is up. Fourcomplete segments are shown. Note that the commissures, which have developed to slightly different extents in the four segments,are completely fused at this stage (indicated by large arrowhead). The two small vertical arrowheads at the left indicate thedeveloping ISN pathways in two of the segments, these extend toward the periphery. The two small arrowheads at the top pointto the migrating RP1 ceU bodies, which are moving toward the midline and will eventuaUy Ue adjacent to each other (seeschematic diagram in Fig. 5). The pattern of axon staining at this stage is similar to that observed with the BP102 mAb, whichlabels aU CNS axons, indicating that most or all of the commissural axons express fascicUn I (see Klambt et al., 1991 for the earlyBP102 pattern).

Embryonic expression of fasciclin I 271

bd

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medial edge of the longitudinal tract. The changingpattern of fasdclin I protein expression in the CNS isschematically depicted in Fig. 5.

In situ hybridization shows that fasciclin I RNA isalways restricted to a subset of CNS neurons (Fig. 2C),and there is no evidence that this subset is larger at earlystages of CNS development. Thus, the early expressionof fasciclin I protein on all commissural axons mayindicate that a subset of neurons that express fasdclin IRNA (perhaps at low levels) pioneer the commissures,or that fasdclin I cleaved by an endogenous phospholi-

Fig. 3. Expression of fasciclin Iprotein in the developing PNS.The 6D8 mAb was used forimmunocytochemistry. In A,C, and D anterior is up, and inB anterior is to the left. (A) Aventral view of the anteriorend of a late stage 15 AELembryo The segmental andlntersegmental nerves are seenextending out to the periphery(arrow). They come togetherat the edge of the CNS, and attheir junction a fasciclin Ipositive glial (G) cell is seen(B) The mature embryonicPNS of three abdominalsegments in an earlier stage 15embryo Most or all of thecellular constituents expressfascichn I. The lateral clusterwith chordotonal (ch) andexternal sensory (es) organsand the ventral clusters withseveral strongly stainingmultiple dendnte cells (md)are in the focal plane. Alsovisible is the bipolar dendrite(bd) cell of the lateral clusterwhich expresses fasciclin I, asdoes the support cellassociated with this neuron(not shown here). (C)Expression in the PNS of stage12 embryo. Fasciclin I proteinis expressed on both early PNSand ectodermal cells, but thePNS neurons have higherlevels of expression. Thearrowhead indicates anabdominal dorsal cluster ofneurons, g, the developmggonad (out of focus). (D) Anembryo of a similar age to theone in C; abdominal lateralclusters are shown withchordotonal neurons (ch) andtheir support cells, called capcells (c), indicated. Bar, 25 /an(A); 12.5 jon (B-D).

pase (Hortsch and Goodman, 1990) can bind to axonsof nonexpressing cells.

Expression of fasciclin I in nonneural tissuesIn addition to the early nonneural expression describedabove, fasdclin I RNA and protein are also observed ona variety of other cells and tissues during development.The cells at the dorsal edge of the epidermis, whichgrow together over the amnioserosa during the processof dorsal closure, express fasciclin I RNA and protein athigh levels (Fig. 2B). This process is completed during

272 L. McAllister and others

B

Fig. 4. Expression of fasciclin Iprotein in the developing CNS.The 6D8 mAb was used forimmunocytochemistry.Embryos were cleared inmethyl salicylate and nervecords were 'cracked out' undercover slips. Anterior is up forall nerve cords. (A) shows 2.5segments of the dorsal surfaceof a nerve cord of a stage 12/0embryo, somewhat older thanthe embryo in Fig. 2D). Theanterior and postenorcommissures (the leftarrowheads) are still attachedby the VUM axons (the nghtarrowhead). Both thecommissural axons and theVUM axons express fascichn I.Few of the longitudinal axonshave extended between thesegments at this time Somefasaclin I-positive lateral cellsare seen near the leftarrowheads Four segments ofa stage 13 embryo are shownin (C). The fasciclin I-positivecommissures (nghtarrowheads) have separatedand the VUMs and their axonsno longer express the proteinCells of the lateral cluster (leftarrowhead) express fasciclin I.One and one-half segments ofa slightly older nerve cord areshown in (B). Thecommissural expression hasdecreased and the fasciclinI-positive RP1 cell body (leftarrowhead) and aCC cellbodies (lower arrowheads) canbe identified very near to theircharactenstic positions RP1 iseventually localized betweenthe commissures, and aCC justpostenor to the postenorcommissure. A single bundlein the postenor commissurehas begun to express slightlyhigher levels of fasciclin I thando the other bundles. Asshown in (D) and (E), the axonal expression of fasciclin I protein resolves to a single bundle (left arrowheads) in theposterior comissure of the later embryonic nervous system (stage 15, five segments shown). There is also a smaller fasciclinI-positive bundle in the anterior commissure; this bundle is out of the plane of focus The embryo in E is slightly olderthan that in D, and the difference in expression between the fasciclin I-positive postenor commissure bundle and the otherbundles is more pronounced. The fasaclin I-positive lateral cluster of cells (nght arrowheads) extends a tight bundle ofaxons, which also express the protein, into the posterior commissure. Bar, 18/im (A); 14/an (B-E).

stage 15, and shortly after dorsal closure these cellscease to express fascichn I.

The gonads express fasciclin I RNA (Fig. 1H) andprotein (data not shown), and this expression continuesuntil cuticle has formed and the embryo is no longeraccessible. We do not know precisely when thisexpression begins, as it is difficult to visualize the pole

cells prior to their incorporation into the gonad. Polecells do not express fasciclin I during the germ bandextended stage. In the coalesced gonad of the laterembryo, both pole cells and mesodermal support cellsappear to express the molecule.

Fascichn I is expressed by all three cell types(glandular epithelium, separate and common ducts) of

Embryonic expression of fascichn I 273

Fig. 5. Schematic diagram of the dynamic expression of fasciclin I protein in the developing CNS. The diagrams in (A) and(B) show a single segment of the Drosophila CNS. The thin gTay bars indicate fasciclin I-positive axon bundles. All the cellbodies shown (gray circles) are fasciclin I-positive; other cell bodies are not depicted. The segment in (A) is typical of astage 12/2 embryo, in which germ band retraction is not entirely completed The commissures have formed (light bars withfine lines) but there are very few longitudinal axons. The commissures are still fused at the midline. Fasciclin I is expressedon the VUM cell bodies (VUMs) positioned at the dorsal midline and on their axons, which form the medial tract (mt)and then bifurcate in the commissures. It is also expressed on most if not all of the early axons in both commissures(ac.pc). The fascichn I-positive RP1 cell bodies, initially located laterally, migrate toward the midhne while thecommissures are still fused. They eventually insert themselves between the two commissures. The aCC cell bodies, enrouteon their anterior migration from the next posterior segment, also express fascichn I. (B) A segment of the CNS in a stage15 embryo The commissures have separated, and the VUMs and their axons no longer express fasciclin I. The orthogonalCNS axon scaffold is indicated by the wide black bars, and the fascichn I-positive bundles by the thin gray bars. The RP1cells are situated between the commissures near the midline and both the cell bodies and their axons are fascichn I-positive The aCCs lie just posterior to the posterior commissure; their soma and axons also express fasciclin I. The aCCand RP1 axons extend together out the intersegmental nerve (isn). A smgle bundle in each of the commissures nowexpresses fascichn I. Many of the axons in these fasaclin I-positive bundles are likely to onginate in the lateral cluster(LC) situated outside the longitudinal connectives and between the commissures. Only one of the two bilaterally symmetricclusters is shown here. These LC neurons express fasciclin I beginning at stage 12 AEL and continue to do so until at leaststage 16.

the salivary glands. Sections of the posterior midgut andof the hindgut also express the protein. Finally, fasciclinI protein expression is observed on the anterior andposterior spiracles, which are the outlets of thetracheae. Both neural and nonneural expression offasciclin I protein is eliminated by the fasl mutation,showing that all of the mAb staining observed is due toauthentic fasciclin I (data not shown).

Discussion

Dynamic expression of neural adhesion moleculesduring embryogenesis in DrosophilaThe results described above show that the cell adhesionmolecule fasciclin I is expressed in a complex patternthroughout embryonic development. The protein isuniformly expressed on nascent cell membranes at theend of the synctitial blastoderm stage. It is brieflydistributed in a gTaded pattern in the cellular blasto-derm (Fig. IB), after which expression again becomes

uniform. In the gastrulating embryo all cells expressfasciclin I, but invaginating cells have higher levels ofthe protein on their surfaces (Fig. 1C).

During the germ band extended stage, fasciclin Iexpression evolves through two distinct phases. In stage9-10 embryos, fasciclin I is localized to 14 segmentallyrepeated stripes of neuroepithelial cells and neuroblasts(Fig. ID, E). During stage 11, fasciclin I RNA andprotein disappear from the neuroblast layer andbecome concentrated in a single continuous longitudi-nal stripe of CNS neurons (Fig. IF). The early cells ofthe PNS, which first become visible just prior to germband retraction, also express fasciclin I RNA (Fig. 1G)and protein (Fig. 3C, D). As germ band retractionproceeds, fasciclin I protein expression on the ectodermincreases, and later disappears during stage 14 (Fig. 3;compare B with C and D). Expression on the cell bodiesand axons of the PNS, in contrast, is stable; the surfacesof these cells retain fasciclin I protein until the embryois no longer accessible to antibody.

274 L. McAllister and others

The CNS expression pattern of fasciclin I duringaxonogenesis can be described as a combination oftransient and stable components. During the formationof the commissures (stage 12), fasciclin I protein isfound on most or all axons (Figs 2D, 4A-C). Thispattern of expression lasts for less than an hour. Afterthis time, fasciclin I is expressed at high levels only on asingle bundle in each commissure, on a bundle in thelongitudinal tract, and on the axons of the ISN and SN(Fig. 4D,E). The cell bodies of the VUM neuronsexpress fasciclin I during the commissural formationphase, but become fasciclin-I negative thereafter. Incontrast, the aCC, RP1, and lateral cluster neuronsmaintain fasciclin I expression on their cell bodies untilat least stage 16 (summarized in Fig. 5).

In addition to neural expression, the germ bandretracted embryo expresses fasciclin I in a variety ofnonneural tissues. These include the dorsal edge of theepidermis, the salivary glands, the gonads, sections ofthe hindgut and posterior midgut, and the spiracles.The duration of fasciclin I expression varies betweenthese different tissues. For instance, the dorsal epider-mis expresses fasciclin I only during the process ofdorsal closure. In contrast, expression in the gonads isvery stable.

The expression patterns of the two other Drosophilafascichns have many of the characteristics of thefasciclin I pattern described here. Fasciclins II and IIIare both dynamically expressed in neural and nonneuraltissues. In the CNS, fasciclin III is transiently expressedon 5 commissural bundles and on part of the ISNpathway. Like fasciclin I, it also appears on asegmentally repeated subset of neuroepithelial cells andunderlying neuronal lineages at the germ band ex-tended stage. At this stage, it is also expressed onpatches of epithelial cells near the stomodeal andproctodeal invaginations and on the visceral mesoderm.After germ band retraction, it is expressed on luminalcells of the salivary gland, and in a 14-stripe pattern onthe ectoderm. These stripes coincide with the segmentalgrooves (Patel et al., 1987). Fasciclin II is initiallyexpressed on a single longitudinal CNS pathway, andappears on several other pathways later in develop-ment. It is also expressed in patches of ectoderm fromwhich the spiracles arise, as well as in a section ofhindgut and in the malpighian tubules (Grenningloh etal., 1991).

These results suggest that the function of neuraladhesion molecules in Drosophila embryogenesis is notrestricted to nervous system development. Thesemolecules are likely to be used for morphogenesis andadhesion in a variety of different tissues. In some casesthey may form stable adhesive zones that are involvedin maintaining the structure of a particular organ. Intissues where they are only transiently expressed, theymay facilitate morphogenetic movements or cell signal-ing events.

Comparison of fasciclin I expression patterns ingrasshopper and DrosophilaAlthough grasshoppers and Drosophila develop to

adulthood in very different ways, the overall structureof the early embryonic CNS is very similar in the twoorganisms. This resemblance extends to the axontrajectories of certain identified neurons (Thomas et al.,1984). Comparison of the expression patterns offasciclin I in the two species might thus yield insightsinto its function.

In both grasshopper and Drosophila, fasciclin I isexpressed at an early stage on segmentally repeatedsubsets of neuroepithelial cells. Later in development,all PNS cell bodies and axons express fasciclin I in bothinsects. In the grasshopper CNS, fasciclin I protein islocalized on a single bundle in each commissure, on abundle in the longitudinal tract, and on the axons of theISN. This pattern is almost identical to the laterexpression in the Drosophila CNS, and this resem-blance extends to particular identified cells such as aCCand RP1, which express the molecule in both organisms(Bastiani et al., 1987; Zinn et al., 1988; this work).

The pattern of fasciclin I expression in grasshopperand Drosophila differs in two major ways. First,fasciclin I is localized to patches of epithelium in thegrasshopper limb buds. There are no counterparts tothese structures in fly embryos. Secondly, the early,transient phase of CNS expression in Drosophila is notobserved in grasshopper. In Drosophila, the molecule isinitially expressed on all commissural axons and laterbecomes restricted to particular bundles. In contrast,fasciclin I is localized to specific pathways from thebeginning of axonogenesis in grasshopper. This maycorrelate with differences in the way the commissuresare established in the two insects. In Drosophila, thetwo commissures are initially fused and then becomeseparated by insertion of a midline glial cell, MGM(Klambt et al., 1991). In the much larger grasshopperembryo, the anterior and posterior commissures arealways separate.

Fasciclin I CNS expression and the phenotype offasl/abl mutantsThe fasl/abl phenotype (absence of commissural axontracts) is clearly not due to a failure of pathfinding bythose axons that comprise the fasciclin I-positivebundles observed in later Drosophila embryos (Fig. 5),because all commissural axons are affected. Rather, itappears that there is a general defect in growth coneextension across the midline (Klambt et al., 1991). Thiscorrelates well with the early, transient expression offasciclin I on all commissural axons reported here.

We do not know what mechanism accounts for thefailure of commissural axons to cross the midline infasl/abl embryos. The specialized midline glia andneurons are essential for commissure formation, butthese seem to be in their normal positions in mutantembryos (Elkins et al., 1990a). One possibility is thatcommissural growth cones must recognize and interactwith the VUM cells, whose growth cones and nascentaxons he at the fusion point of the two commissures. Inorthodenticle mutants the VUM cells die and theposterior commissure does not form (Klambt et al.,1991). Since fasciclin I has been shown to be a

Embryonic expression offasciclin I 275

homophilic adhesion molecule in tissue culture (Elkinset al., 1990b), and the commissural axons and VUMcells both express the molecule at this stage, it ispossible that elimination of fasciclin I-mediated growthcone adhesion to the VUMs is a component of thefasl/abl phenotype.

Because the commissures are absent in fasl/ablembryos, we cannot determine whether fasciclin I alsohas a role in specific pathfinding by later growth conesthat might use the fasciclin I-positive bundles asguidance cues. This is analogous to the situation in slitmutant embryos, where the early nervous systemcollapse phenotype (due to the absence of midline slitexpression) prevents the observation of a possiblephenotype correlated with the later expression of slitproduct on CNS axons (Rothberg et al., 1990).

Fasl/abl embryos are essentially normal except fortheir alterations in the CNS axon array (Elkins et al.,1990a). This is probably because the CNS is the majortissue in which both fasciclin I and abl are expressed.The double mutant phenotype would thus be mostlikely to be observed in the CNS. Fasciclin I expressionoutside the CNS is likely to have a functional role,however, since its pattern has been conserved over the300 million years of evolution that separate Drosophilafrom grasshoppers. In order to uncover such a role, itwill be necessary to find mutations in other genes thatproduce a synergistic phenotype when combined withfasl mutations.

A possible developmental role that is consistent withthe early nonneural expression of these apparentlyredundant adhesion molecules could be to ensure thatmorphogenetic movements take place normally in allembryos. We have observed that in collections of 11-13 h old embryos from wild-type stocks, about 1% ofembryos are severely abnormal. The most commonphenotype observed in these embryos is a failure ofgerm band retraction; gastrulation/germ band exten-sion defects are also observed. In egg collections fromhomozygous fasl flies, the percentage of these abnor-mal embryos increases to 10-12%, and, in collectionsfrom homozygous fasl/fasIII stocks, to 30-35 %. Thisphenotype of fasl mutants can be rescued by P-elementtransformants containing the fasciclin I gene (K. Zinn,T. Elkins, and C. S. Goodman, unpublished results).

This result suggests that these and other cell adhesionmolecules could act as 'correctors' in morphogenesis.We hypothesize that although most mutant embryoslacking a particular adhesion molecule will be able todevelop normally, embryos in which cells are inabnormal positions may fail to develop because they areunable to correct these defects. The fraction of embryosin this class would increase still further if multiplecorrecting functions are removed by mutation, becausethe extent of tolerable abnormality in cell positionwould be decreased as each molecule was eliminated(the 'window of normality' would shrink in size). Thatsuch a correcting mechanism actually exists is illustratedby embryos derived from mothers having differentnumbers of copies (1, 3 or 4) of the bicoid gene, whichdetermines anterior cell fates. These embryos vary

greatly from normal in the positions of their cephalicfurrows and in the placement of pair-rule geneexpression stripes, yet they all develop into normallarvae and adults (Driever and Nusslein-Vollhard,1988).

Involvement in a correcting mechanism could pro-vide a selection for the evolution of genes encoding newcell adhesion molecules, even if they do not initiallyhave functions independent of those of previouslyexisting molecules. The new adhesion proteins couldthen evolve new roles and patterns of expression thatwould allow the development of greater complexity inthe nervous system.

We thank Shin-Shay Tian for performing some of thewhole-mount in situ hybridization experiments, MichaelHortsch for the anti-fascichn I mAbs and for help with initialimmunocytochemistry experiments usmg these mAbs, KarenJepson-Innes and Susan Parkhurst for help with the wholemount in situ technique, Dolores Ferres-Marco for help withimmunocytochemistry, Yash Hiromi for a stock carrying theftz-lacZ insertion on the X chromosome, Bruce Alberts forhelpful discussions concerning bicoid phenotypes, and NipamPatel for invaluable advice This work was supported bygrants from the NIH to C S.G. and K.Z.

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{Accepted 7 February 1992)