Regulation of Axon Guidance by Slit and Netrin Signaling ... · The following collections of wild-type embryos were used for RT–PCR analysis: 0–4 hr, 4–8 hr, 8–12 hr, and

Copyright � 2007 by the Genetics Society of AmericaDOI: 10.1534/genetics.107.075085

Regulation of Axon Guidance by Slit and Netrin Signaling in theDrosophila Ventral Nerve Cord

Krishna Moorthi Bhat,1 Ivana Gaziova and Smitha Krishnan

Department of Neuroscience and Cell Biology, University of Texas Medical Branch School of Medicine, Galveston, Texas 77555

Manuscript received April 26, 2007Accepted for publication May 30, 2007

ABSTRACT

Netrin and Slit signaling systems play opposing roles during the positioning of longitudinal tracts alongthe midline in the ventral nerve cord of Drosophila embryo. It has been hypothesized that a gradient ofSlit from the midline interacts with three different Robo receptors to specify the axon tract positioning.However, no such gradient has been detected. Moreover, overexpression of Slit at the midline has noeffect on the positioning of these lateral tracts. In this article, we show that Slit is present outside of themidline along the longitudinal and commissural tracts. Sli from the midline, in a Robo-independentmanner, is initially taken up by the commissural axon tracts when they cross the midline and istransported along the commissural tracts into the longitudinal connectives. These results are notconsistent with a Sli gradient model. We also find that sli mRNA is maternally deposited and embryos thatare genetically null for sli can have weaker guidance defects. Moreover, in robo or robo3 mutants, embryoswith normal axon tracts are found and such robo embryos reach pupal stages and die, while robo3 mutantembryos develop into normal individuals and produce eggs. Interestingly, embryos from robo3homozygous individuals fail to develop but have axon tracts ranging from normal to various defects:robo3 phenotype, robo phenotype, and slit-like phenotype, suggesting a more complex functional role forthese genes than what has been proposed. Finally, our previous results indicated that netrin phenotype isepistatic to sli or robo phenotypes. However, it seems likely that this previously reported epistaticrelationship might be due to the partial penetrance of the sli, robo, robo3 (or robo2) phenotypes. Our resultsargue that double mutant epistasis is most definitive only if the penetrance of the phenotypes of themutants involved is complete.

IN the Drosophila ventral nerve cord, there are �20longitudinal axon tracts on either side of the mid-

line. The signaling system from the midline mediatedby Slit (Sli) and its receptor Roundabout (Robo) ap-pears to prevent these tracts from projecting toward themidline (Kidd et al. 1999). Previous results indicate thatthere are at least three robo genes in Drosophila. It hasbeen postulated that a gradient of Sli emanating fromthe midline interacts with Robo receptors in a combi-natorial manner to specify the lateral positioning ofaxon tracts in the longitudinal pathways (Rajagopalan

et al. 2000; Simpson et al. 2000). In the Sli gradientscenario, the lowest level of Sli interacts with Robo andRobo2 to specify the lateral-most tracts, an intermediatelevel interacts with Robo and Robo3 to specify theintermediate tracts, and a high level interacts with Roboto specify the medial tracts (Rajagopalan et al. 2000;Simpson et al. 2000). However, a gradient of Sli ex-tending from the midline has not been detected byantibody staining. More importantly, overexpression ofsli at the midline does not alter the lateral positioning

of longitudinal tracts (Kidd et al. 1999; our unpub-lished results), arguing against the gradient model.Consistent with this result, ectopic expression of Sli infront of the growth cones of Robo-expressing neuronsdoes not alter the course of the projections of theseneurons (Bhat 2005). The only phenotype that we canobserve with a gain-of-function sli is the robo-like pheno-type when sli is ectopically expressed everywhere (Kidd

et al. 1999; our unpublished data), though the molecularbasis for this phenotype has not been determined.

There is an additional signaling system emanatingfrom the midline in Drosophila, which is the Netrin(Net) and its receptor Frazzled (Fra) system. This Net-Fra system appears to function as a midline attractant forthe commissural tracts (Harris et al. 1996; Kolodziej

et al. 1996; Mitchell et al. 1996). More recently, we haveshown that loss of function for Net or Fra activity resultsin the positioning of longitudinal tracts shifting fartheraway from the midline compared to wild type (Bhat

2005). Moreover, overexpression of Net at the midlinecauses a collapsing of the longitudinal tracts at themidline (Bhat 2005).

While there is no evidence of a gradient of Sli ema-nating from the midline, here we show that there ispresence of Sli in the commissural and longitudinal

1Corresponding author: University of Texas Medical Branch, 301 Univer-sity Blvd., Galveston, TX 30322. E-mail: [email protected]

Genetics 176: 2235–2246 (August 2007)

axon tracts. We show this presence of Sli in axon tractsusing two different Sli antibodies: one previously raisedagainst the C-terminal part of Sli and the other that weraised against the N-terminal portion of the protein. Wefurther show that the presence of Sli in commissures andconnectives is not due to an uptake of Sli from a gradientdiffusing from the midline; instead, it is taken up initiallyfrom the midline by commissural tracts when they crossthe midline. Furthermore, this transport of Sli is alsoindependent of Robo since embryos lacking the robogene still have Sli in commissural and longitudinaltracts.

Previous results indicate that the product of commis-sureless downregulates Robo to allow commissural axonsto cross the midline; however, in comm mutants, longi-tudinal tracts often cross the midline. Our results sug-gest that this is due to sequestration of Sli by the excessamount of Robo at the commissural tracts. Finally, ourprevious results indicated that the net phenotype isepistatic to the sli or robo phenotypes (Bhat 2005). Thisepistasis result, taken together with the failure of ectopicSli to repel Robo-positive growth cones, as well as resultsobtained with net loss-of-function and gain-of-functionanalyses, led to the proposal that the major function ofSli-Robo signaling is to neutralize Net-Fra attractant sig-naling during the positioning of longitudinal tracts(Bhat 2005). However, our more recent data indicatesthat this epistatic relationship between net and slit or robomutants may be due to embryos that are escapers for sli,robo, robo3 (or robo2, etc.) phenotypes. Such escaperembryos can be negative for the gene activity yet canhave the net phenotype. Since we did not observe a wild-type positioning of longitudinal tracts in these doublemutants, we must consider three possibilities: (1) Nethas a direct attractant role on longitudinal tracts; (2) italso partially neutralizes the Sli-Robo signaling; and (3)there is an additional attractant system at the midline.Our results also argue that double mutant analysis todetermine epistatic relationship between two mutants ismost definitive only if the phenotypes of both mutantsare fully penetrant. Our results also suggest that havinga balancer in the parents from which the embryos arederived can alter the penetrance and the severity of amutant phenotype.

MATERIALS AND METHODS

Fly strains and genetics: For the analysis of sli, we used thegenetically null allele, sli2. For the analysis of net, we used thepreviously described deficiency that eliminates the two netgenes, Df(1)RK2/FM7a. This deficiency also deletes severalother complementation groups; however, the specific defectsdescribed here are attributed to loss of netA and netB genes (seeHarris et al. 1996). For the analysis of robo, we used robo4, agenetically null amorph (the molecular nature of the mutationis not known), and for the analysis of robo3, we used thepreviously described loss-of-function allele robo31; the molecu-lar nature of the mutation is a G / A substitution in the splice

acceptor site of the sixth intron (Rajagopalan et al. 2000). Forthe analysis of fra, we used fra3 and fraGA957. The molecularnature of the fra3 mutation is a tryptophan to stop codon changeat amino acid position 1028 (W1028Stop). The nature of themutation in fraGA957 is not known. For the analysis of abelsontyrosine kinase (abl), we used abl4. For the second chromosomebalancer, we used the GFP-CyO: w1118; In(2LR)Gla, wgGla-1/CyO, P{GAL4-twi.G}2.2, P{UAS-2xEGFP}AH2.2. Homozygousembryos were identified either using the GFP-marked balanceror by lack of a positive staining with an antibody.

Generation and purification of the Sli N-terminal antibody:The most hydrophilic 142 aa (470–611 aa) of the N-terminalpart of Sli protein was selected for antibody production. The�430-bp product was amplified using forward primer 59-TGCATATGAATCCCATAGAGACGAGTG-39 (1659–1677 bp ofmRNA-A) containing a NdeI restriction site, which providesthe methionine start codon, and the reverse primer59-TGAGCTCTCACGATATCTCCTTGATCTTGTTCT-39 (2062–2084 bp of mRNA-A) containing a SacI restriction site and astop codon. This fragment was cloned into the expressionvector pET28a (Novagen). 6xHis-SliN fusion protein was ex-pressed in the Escherichia coli strain BL21-CodonPlus (Strata-gene) and purified on a Ni-NTA agarose column (QIAGEN)under native conditions. About 1 ml of the 6xHis-SliN fusionprotein (1 mg/ml) was used for rabbit immunization accord-ing to the standard protocol (Covance Immunological Serv-ices). The obtained polyclonal serum was affinity purified onthe AminoLink Plus coupling gel (Pierce) with immobilized6xHis-SliN fusion protein.

Immunohistochemistry and RNA in situ: Immunohisto-chemistry was performed using the standard protocols. Thedilution of anti-Sli-N antibody used for embryo staining was1:4000; for Fas II, the dilution was 1:5; BP102 was 1:6; Robo was1:3; and monoclonal anti-Sli (C555.6D) was 1:10. For wholemount RNA in situ staining, the standard procedure was usedwith digoxigenin-labeled anti-sense Sli probe. AP-conjugatedanti-digoxigenin (1:2000) and alkaline phosphatase reactionwas used to detect the staining. We used various fixationconditions to optimize the staining and avoid interference bytwo antibodies in double staining experiments. These mod-ifications can be obtained by request.

Western blotting: Drosophila embryos were extracted inSDS-loading buffer and the extracts from 15 embryos per lanewere analyzed on 10% SDS–PAGE. Blots were incubated withanti-Sli-N antibody (1:60,000) overnight at 4�. The secondaryHRP-conjugated anti-rabbit antibody ( Jackson Immunolabor-atories; 1:20,000) was visualized according to manufacturer’sprotocols using the ECL kit (Pierce). For the monoclonal Sli-C, we used 1:100 dilution.

Determination of sli maternal deposition—RT–PCR: Allembryo collections were done at 22�. w1118 virgins were aged forseveral days and then left to lay eggs for 24 hr. Total RNA fromthe dechorionated embryos was isolated using Trizol reagent(Invitrogen, Carlsbad, CA) and 1 mg of total RNA was used forthe first-strand cDNA synthesis in 20-ml reaction using Super-Script III reverse transcriptase (Invitrogen). sli mRNA fromwild type and sli2 mutants was amplified using forward primer59-AATCCCATAGAGACGAGTG-39 (1659–1677 bp of mRNA-A; exon 9) and reverse primer 59-GGTCAACGACTTGAGGTCC-39 (2756–2774 bp of mRNA-A; exon 15), which gives asingle product (1134 bp) in wild-type embryos and twoproducts (1188 bp and 1413 bp) in sli2 mutants. As an internalcontrol for various cDNA samples, the 335-bp product ofthe constitutively expressed mRNA for ribosomal protein 49(rp49; RpL32 in FlyBase) was amplified using forward primer59-GCTAAGCTGTCGCACAAATG -39 and reverse primer59-GAATCTTAAGCTTACTCGTTCTC-39.

2236 K. M. Bhat, I. Gaziova and S. Krishnan

The following collections of wild-type embryos were usedfor RT–PCR analysis: 0–4 hr, 4–8 hr, 8–12 hr, and 12–24 hr.Embryos that are 0–1 hr, 1–2 hr, 2–3 hr, and 3–4 hr old fromwild-type females crossed to sli2/Bal females were analyzed byRT–PCR to determine the zygotic expression of sli mRNA.

Mapping the sli mutation in the sli2 mutant: The molecularbasis of sli2 allele was previously reported as A /T transversionat the position 3321 bp (mRNA-A variant), which results inpremature termination of Slit translation (K / Stop) at posi-tion 1024 aa (Battye et al. 2001). To confirm this mutation insli2 chromosome, we amplified and sequenced the �450 bp ofexon 16 covering the mutation site using forward primer: 59-CGATGCTTGCTACGGAAATC-39 (3092–3111 bp of mRNA-A)and reverse primer 59-CGTATAGTCGTCTGGACAG-39 (3518–3536 bp of mRNA-A). sli2 homozygous embryos were distin-guished from their counterparts using the twist-GFP markedbalancer chromosome. Since we did not find the previouslydescribed mutation and that sli2 mutant embryos were nega-tive for Sli protein with anti-Sli-C monoclonal, in order todetermine the mutation in sli2 chromosome we first estimatedthe molecular weight of the truncated Sli2 protein. Usingour Sli-N antibody, we performed western blot analysis on sli2

embryos. Extracts from 15 12–24-hr-aged embryos of sli2 homo-zygotes and w1118 (wild-type control) were analyzed in in-dividual lanes. In sli2 homozygotes, instead of the two wild-typeSli bands, we observed a band of �75 kDa size. We amplifiedand sequenced the exons and introns from exon 11 to 14 ofthe sli gene from sli2 mutants; this region should cover theregion of Sli protein from 61 to 88 kDa. Forward primer 59-CCCGCATCCCTAACTCGC-39 (intron 10; 11,197–11,214 bpof slit gene) and reverse primer 59- AGCATTGCCAAGCAGAGAATC-39 (intron 12; 11,822–11,842 bp of slit gene) were usedto amplify exons 11 and 12 and intron 11. Forward primer 59-AAGAACCCTTTATCAACGCCG-39 (intron 12; 13,240–13,260bp of slit gene) and reverse primer 59-TTATTTCGTGGTTTCCGTGC-39 (intron 14; 13,869–13,889 bp of slit gene) wereused to amplify exons 13 and 14 and intron 12. The se-quencing of PCR products from three independent reac-tions revealed the single base pair substitution G / A in theposition of 11,460 bp of the sli gene, which corresponds to thefirst base pair of intron 11. A transition of G to A in the 59intron splice donor site is expected to result in an aberrantsplicing. To confirm this, we amplified this region from sli2

mRNA using RT–PCR. The RT–PCR from wild-type embryosusing forward primer 59-AATCCCATAGAGACGAGTG-39 (1659–1677 bp of mRNA-A; exon 9) and reverse primer 59-GGTCAACGACTTGAGGTCC-39 (2756–2774 bp of mRNA-A; exon15) gives a single product of size 1134 bp. However, RT–PCRfrom sli2 homozygous embryos gives two products, which aredifferent from the wild-type product. Sequencing of these twoproducts confirmed the aberrant splicing of intron 11. The1413-bp-long band represents the sli2 mRNA form in whichintron 11 is not spliced. The second sli2 mRNA that gives a1188-bp-long band is due to the use of an alternative splicedonor site in intron 11 located 54 bp downstream from thecorrect splice site in intron 11. In both sli2 mRNA forms, thestop codon is located immediately following the mutation andthus, Sli2 translation is prematurely terminated at aa position625 (Sli-A variant) and gives the product of �75 kDa size.

RESULTS

Slit is present outside of the midline in thecommissural and longitudinal tracts: While previouswork showed that Sli is expressed in the midline glialcells, we found that the commissures and longitudinalconnectives also have low levels of Sli (Figure 1b). The

presence of Sli at very low levels can be detected as earlyas stage 12 (�10.5 hr of development) in places wherethe growing commissural tracts intersect the nascentlongitudinal tracts (data not shown). The source of thisSli must be the midline since within the nerve cord, thesli gene is not zygotically transcribed outside of themidline (Figure 1a). In sli2 mutant allele, which isgenetically a null allele (Battye et al. 2001), the mutantprotein is not recognized by the monoclonal antibodyraised against the C-terminal portion of the protein; inthese embryos, the Sli staining of the commissural andlongitudinal tracts was absent (Figure 1c). These resultsindicate that the source of Sli in the connectives andcommissures is unlikely the background.

It is still possible that this extra-midline staining of Sliis an artifact associated with this particular monoclonalantibody. Therefore, we raised a polyclonal antibodyagainst the N-terminal portion of Sli (Figure 1, e–g).When embryos were examined with this antibody, thecommissural tracts and the connectives were also posi-tive for Sli (Figure 1, e and f). These results thereforeargue that the extra-midline staining of Sli is not anartifact but is due to the presence of Sli in these tracts.As suggested by previous work (Kidd et al. 1998), Sliappears to be processed into an N-terminal part and aC-terminal part and exists along with the unprocessedfull-length Sli. With Sli-N antibody, as shown in Figure1g, we observed the unprocessed �180 kDa band andthe processed N-terminal part (�135 kDa; we observedtwo to three additional bands with this antibody butthese are either degradation products or nonspecificbands). With the Sli-C antibody, we observed the unpro-cessed Sli and the C-terminal portion of Sli (Figure 1h).

We reasoned that the presence of Sli in axon tracts ofcommissures and connectives can be utilized to exam-ine the Sli gradient model. If Sli is present in a diffusiblegradient extending from the midline (Rajagopalan

et al. 2000; Simpson et al. 2000), the presence of Sli inconnectives and commissures is likely to be due to theuptake of Sli from the surrounding environment, re-sulting in the accumulation of Sli in these tracts. Al-ternatively, Sli in connectives and commissures mightbe due to a delivery of locally secreted Sli via the com-missural tracts given that these tracts are projectedacross the Sli-expressing midline glial cells.

We sought to distinguish between these two possibil-ities as follows. If Sli is transported via commissural tractsin embryos that lack most of their commissural tracts,as in loss-of-function commissureless (comm) mutantembryos (see Figure 2A; note that a few axons are stillprojected across the midline in comm mutant embryos,marked by arrows), the longitudinal tracts will haveno or reduced levels of Sli. If the Sli in longitudinalconnectives is due to accumulation of Sli from the sur-rounding environment, comm mutants will still havehigh levels of Sli in their connectives, as in wild type.When comm embryos were stained with Sli antibody, we

Slit-Robo and Netrin-Fra Signaling in the Drosophila CNS 2237

observed regions along the longitudinal connectivesthat lack Sli even when these regions are next to Sli-expressing midline cells (Figure 2, B and C, arrowheads).Whenever there is Sli staining in a stretch of longitudinalconnectives, the source can be traced through the fewcommissural tracts to the midline (Figure 2, B and C,arrows), indicating that Sli is transported away from thesource via these tracts.

We next determined if the transport of Sli from themidline is Robo-dependent. We examined embryos ho-mozygous for a deficiency that eliminates the robo genewith anti-Sli-C. In these embryos, we found that thecommissural and longitudinal tracts had Sli as in wildtype (Figure 2D). This indicates that the presence of Sliin these tracts is not Robo-dependent. This does notappear to be a redundancy problem with Robo2 orRobo3 since these Robo are reported to have narrowerdomains of expression (Robo3 is reported to be in thelateral and intermediate tracts; Robo2 is only in thelateral tracts) compared to Robo, which is in all longi-tudinal tracts (Rajagopalan et al. 2000; Simpson et al.2000). Besides, if there is such a redundancy, we wouldhave seen a narrower distribution of Sli in longitudinalconnectives, which was not the case. Moreover, Roboreceptors are downregulated in commissural tracts(Kidd et al. 1998; Myat et al. 2002) and thus unavailablefor binding of Sli in commissures. Therefore, an un-

known gene product must be involved in this transportof Sli via commissural tracts.

Longitudinal tracts inappropriately cross the midlinein commissureless mutants: Given the above results, wesought to determine if the Fas II-positive tracts weremisplaced in comm mutants. We found that the mediallongitudinal tracts often crossed the midline in embryosthat are loss of function for comm (Figure 3D; one or twosegments in�9% of embryos, n¼ 200 embryos; see alsoFritz and Vanberkum, 2000). In comm mutants, the lossof Comm protein from the commissural tracts causeselevated levels of Robo in these tracts (Kidd et al. 1998;Myat et al. 2002). It has been proposed that this ele-vated level of Robo leads to a repulsive interaction withmidline Sli and as a result these commissural tracts failto cross the midline, generating a commissureless phe-notype (Kidd et al. 1999). However, the inappropriatemidline crossing of longitudinal tracts was unexpectedfor comm mutants. One possibility is that the excessiveamount of Robo (due to deregulation of Robo) titratesout Sli, and as a result the longitudinal tracts are able tocross the midline. To test this possibility, we halved thedosage of sli in comm background (sli2/1; comm/comm)and examined the longitudinal tracts in these embryoswith Fas II antibody. As shown in Figure 3, E and F,this enhanced the midline crossing phenotype in commmutants significantly in both the number of segments as

Figure 1.—Sli is present in thecommissural tracts and longitudi-nal connectives. Anterior end is attop; midline is marked by verticallines. AC, anterior commissure;PC, posterior commissure; LC,longitudinal connectives. (a)Whole mount RNA in situ ofwild-type embryo with sli probe.sli is transcribed only in the mid-line cells in the nerve cord. (b)Wild-type embryo stained withanti-Sli-C antibody. Sli protein ispresent at very high levels in themidline, but it is also present atlower levels in the commissuraltracts and longitudinal connec-tives (arrow with asterisk). (c)sli2 mutant embryo stained forSli. This is a protein null allele.(d) Wild-type embryo stained withMab BP102 (BP). BP102 stainsboth the commissures and theconnectives, revealing a ladder-like structure of the nerve cord.(e and f) Wild-type embryosstained with anti-Sli-N antibody.Sli protein is present at high levels

in the midline, but it is also present at lower levels in the commissural tracts and longitudinal connectives. (g) Western blottinganalysis of wild-type embryo extract with anti-Sli-N; arrows indicate the unprocessed Sli and the processed N-terminal portion ofSli. This antibody does not recognize the processed C-terminal portion of Sli. (h) Western blotting analysis of wild-type embryoextract with anti-Sli-C; arrows indicate the unprocessed Sli and the processed C-terminal portion of Sli. This antibody does notrecognize the processed N-terminal portion of Sli. Note that the commissural and connectives staining of Sli is not discrete as Fas IIsince Sli stains all axon bundles in the nerve cord (�20), whereas Fas II stains only 3–4 bundles.


well as the number of embryos affected, although therewas variability between embryos. This result supportsthe above contention that high levels of Robo create acompetitive scenario for Sli, causing the longitudinaltracts to abnormally cross the midline. Finally, sli; commdouble mutants had the sli phenotype (data not shown).

Epistasis relationship between robo and fra loss-of-function mutants: In a previous article, we had proposedthat Net/Fra signaling is epistatic to Sli/Robo signalingand the repulsion mediated by Sli/Robo is indirect, i.e.,via neutralizing the attraction mediated by Netrin/FraSlit (Bhat 2005). However, we had not examined theepistatic relationship between sli/robo and fra; we hadexamined the epistatic relationship between frazzled-like( fral), a third chromosome mutant that we identifiedin a previous screen (V. Botta and K. M. Bhat, unpub-lished data; incorrectly identified in the discussion as frainstead of fra-l; see Discussion in Bhat 2005). Therefore,we sought to construct double mutants between robo andfra, as well as sli and fra, and examined the longitudinaltracts in these double mutants.

To visualize the longitudinal tracts, we first examinedembryos doubly mutant for robo and fra with Fas IIstaining. As shown in Figure 4, we observed a mixture of

phenotypes, ranging from robo phenotype (Figure 4e) toa fra phenotype (Figure 4, f–h) and an intermediatephenotype (Figure 4i), although the robo phenotype waspredominant. The double mutants were picked by thelack of their staining for GFP since the mutant chromo-some was balanced with a GFP-marked CyO.

Because of the predominance of embryos with a robophenotype among robo,fra double mutants, it appearsthat robo phenotype is epistatic to the fra phenotype.This conclusion is inconsistent with our previous resultthat Sli neutralizes attraction mediated by Net and theeffect of Sli on axon guidance is via Net (Bhat 2005).Therefore, we re-examined robo; net double mutantembryos with Robo and Fas II antibodies (we used adeficiency that eliminates the robo gene). We havepreviously found that having balancers in the parentalcross from which the embryos are derived for stainingcan often cause significant variability in terms of thepenetrance and the actual defect, and this was especiallytrue for axon guidance and RP2 lineage defects. There-fore, we make an attempt to avoid having a balancer inthe parental cross as much as possible and use antibodystaining or PCR to identify homozygous mutant em-bryos (as opposed to using LacZ or GFP balancers).Thus, we eliminated the balancer chromosomes fromthe parents from which the embryos were collected andused the lack of Robo staining and the axon guidancephenotype to identify the double mutants (we could notuse this same strategy when examining robo, fra doublemutants since the robo4 mutant allele we used was notprotein-null). As shown in Figure 5C, we observedembryos that are Robo-negative but with a net pheno-type (�2.75% of the total embryos were of this type;expected was �6.25% or 1/16 embryos; n ¼ 400). Thisnumber is less than expected but the same as we ob-served previously. It should be noted that often doublemutants are found in ratios less than expected due totrans-heterozygous interaction between two mutants orcaused by a background effect.

Given these above results, we entertained two possi-bilities. First, attraction of longitudinal tracts by Net isnot mediated via Fra; thus, it is possible to have differingepistasis results between robo; net and robo, fra. Second,robo mutant phenotype is partially penetrant and suchembryos in combination with net can be Robo-negativeyet have a net phenotype. To test this possibility, we bal-anced the robo4 mutant chromosome with a GFP-markedCyO balancer and selected GFP-negative embryos (n ¼572) and allowed these embryos to develop: we observed48% or 275 of the robo4 homozygous embryos hatchinginto first instar larvae, and 3% or 17 of the embryosdeveloping into pupal stage, but none of these pupaeeclosed into adults (on occasions, we have seen as muchas 7% of pupae that are robo4 homozygotes).

We also stained GFP-negative robo embryos with FasII antibody. As shown in Figure 6, we observed robo ho-mozygous embryos with more or less normal longitudinal

Figure 2.—Sli from the midline is delivered to the longitu-dinal tracts via commissural tracts. (A) comm mutant embryostained with BP102. Note the absence of commissures; how-ever, there are still axon tracts that project toward the midline(arrows). (B and C) comm mutant embryos stained with anti-Sli. Note the absence of Sli in the connectives adjacent to Sliexpressing midline cells (arrowheads). The low levels of Sli intracts elsewhere appear to be delivered via the few remainingcommissural tracts (arrows; outline of these commissuraltracts is visible by Nomarski optics). (D) robo deficiency em-bryo showing Sli in both the commissures and connectives.


axon projections (cf. Figure 6d). This was observed in3% of embryos (n ¼ 106). These results argue that asignificant number of embryos with guidance defectswould make it to pupal stages. These results alsoindicate that the robo; net double mutants that have thenet phenotype could be embryos that are ‘‘escapers’’ forthe robo phenotype. We have not examined if the es-

capers are due to a maternal deposition or redundancywith the other Robo receptors.

A significant number of escapers were found forrobo3 loss-of-function mutation: We next examined ifthe loss-of-function effects of robo-3 are also partiallypenetrant, which would explain again the presence ofrobo3; net double mutants with a net phenotype (Bhat

Figure 4.—Double mutant analysis between robo and fra. Embryos are stained with Fas II antibody. Anterior end is at top, mid-line is marked by vertical lines. (a) Wild-type embryo showing the Fas II positive longitudinal tracts. (b) robo4 mutant embryo; theintermediate and medial tracts are collapsed at the midline. (c) net mutant embryo; the tracts are positioned farther apart. (d) framutant embryo; tracts are also positioned farther apart. (e–i) Embryos double mutant for robo and fra. Phenotypes varying fromrobo-like to fra-like are observed. The results were similar with two different alleles of fra. The frequency 18% includes phenotypesrepresented in f–h.

Figure 3.—Longitudinal axon guidance defects in comm mutants and its enhancement by reducing the dosage of sli. Approx-imately 15-hr-old embryos are stained with Fas II antibody. Anterior end is at top, midline is marked by vertical lines.


2005). We transferred the robo3 mutant allele into theGFP-marked CyO balancer background and followedthe development of the GFP-negative robo3 homozygousmutant embryos (n ¼ 200). We found that 46% or 92/200 such embryos hatched to larvae; 29.5% or 59/200embryos reached the pupal stage; and 27.5% or 55/200embryos eclosed into functional normal adults. Consis-tent with this, when robo3 homozygous embryos fromrobo3/CyO-GFP parents were stained with Fas II, weobserved embryos that are either with robo3 phenotype(61%, n ¼ 31 embryos) or with normal Fas II-positivelongitudinal tracts (39%, n ¼ 31). It is most likely thatthese embryos that develop into viable normal adultshave normal axon pathfinding, and in combinationwith net, such embryos will give a net phenotype.

To explore this issue further, we collected embryosfrom these robo3 homozygous parents and stained theseembryos with Fas II. We found that these parentsproduced embryos that are completely normal (Figure7B) but also embryos that have the robo3 mutantphenotype (Figure 7, C and D), a sli-like phenotype(Figure 7E), and a robo-like phenotype (Figure 7F). Thesli-like or the robo-like phenotypes were not observedwhen the embryos were collected and stained from robo3heterozygous parents but were only among embryos

derived from robo3 homozygous parents. This resultargues that the axon guidance regulated by Robo3 (orthe other two Robo receptors) involves a much morecomplicated pathway(s) than what has been proposedpreviously (Rajagopalan et al. 2000; Simpson et al.2000). An alternative explanation is that there are mod-ifier mutations on the robo3 chromosome, or elsewherewithin in the robo3 strain, altering the phenotype.However, if such modifiers exist, we should see a similarvariability due to the modifier effect among embryosderived from robo3 heterozygous parents (robo3/1 3

robo3/1), which was not the case, and more importantly,the phenotypes in embryos derived from robo3 homo-zygous parents (robo3/robo3 3 robo3/robo3) have very spe-cific phenotypes: robo-like, robo3, slit-like, and, of course,wild type. Yet another possibility is that a Sli pathway-specific parental trans-suppressor exists on the CyObalancer (a balancer-mediated effect); this possibility isconsistent with our finding that a robo3 homozygousfemale can produce robo3 mutant embryos that reach allthe way to adulthood if that female parent is mated witha heterozygous male. We have lost the robo2 mutant lineand therefore have not re-examined robo2 to determineif the same above results also hold true for robo2, but itseems highly likely that a similar scenario exists for robo2as well.

Epistatic relationship between sli and fra: We nextexamined the axon guidance in sli, fra double mutantembryos with Fas II staining. As shown in Figure 8, weobserved embryos with the sli phenotype (Figure 8A;88%, n ¼ 400) but also embryos that have a phenotypesimilar to hypomorphic sli (Figure 8B; 3%) and embryosthat are closer to fra phenotype (Figure 8C; 9%). Giventhese results, we re-examined the previous results by

Figure 6.—Escaper embryos homozygous for robo havenearly normal longitudinal tracts. Embryos are stained withFas II antibody. Anterior end is at top, midline is markedby vertical lines. (a and b) robo4 embryos with the typical robophenotypes. Note the outward projection of longitudinaltracts in a (arrowhead). (c and d) robo4 mutant embryos withweak robo phenotypes; embryo in d is close to wild type.

Figure 5.—Double mutant analysis between robo and net.Embryos are double stained with Fas II and Robo. Anteriorend is at top. Midline is marked by vertical lines. (A) Wild-typeembryo; note the superimposed diffuse Robo staining, whichsomewhat masks the Fas II staining pattern. (B) net mutantembryo. (C) net; robo4 embryo. The Fas II tracts are fartherapart but broken as in net embryos. (D and E) Wild-type em-bryos stained with Fas II alone and Robo alone.


generating net; sli double mutants again and doublestaining these embryos with Sli and BP102. We avoidedhaving a balancer in the parental cross from which theembryos were derived. As shown in Figure 9, we observeddouble mutant embryos that were Sli-negative but hadthe net phenotype (Figure 9, C, D, and F). While weexpect 6.25% (or 1/16 embryos) will be double mutants

for both the chromosomes, we observed 1.9% of embryos(n ¼ 675) that were Sli-negative but with net phenotype.

These results were in line with the results from roboand robo3. Therefore, it is possible that sli mutant

Figure 8.—Double mutant analysis between sli and fra. sli,fra double mutant embryos are stained with Fas II antibody.Anterior end is at top, midline is marked by vertical lines.(A) The predominant sli phenotype in the double mutant.(B) This double mutant has a hypomorphic sli-like pheno-type. (C) This double mutant has a phenotype closer to fra.

Figure 9.—Double mutant analysis between sli and net. Em-bryos in A–F are double stained with BP102 and Sli. Anteriorend is at top. Midline is marked by vertical lines. (A) Wild-typeembryo, �15 hr old. (B) Approximately 15-hr-old net mutantembryo; note the midline Sli staining. (C and D) Two exam-ples of �15-hr-old net; sli2 double mutant, the BP102 tractsare farther apart but broken as in net embryos. (E) Approxi-mately 10-hr-old sli2 mutant embryo; note the collapse ofBP102 tracts at the midline as well as the appearance of theembryo itself. (F) Approximately 10-hr-old net; sli2 embryo.(G and H) Fas II stained sli2 mutant embryos, �14–15 hrold. Anterior is to the left. Note the weaker phenotype inthe embryo shown in H.

Figure 7.—Longitudinal axon tracts pheno-types in robo3 embryos derived from robo3 homo-zygous adults. Embryos are stained with Fas IIantibody. Anterior end is at top, midline ismarked by vertical lines. (A) Wild-type embryo.M, medial tract; I, intermediate tract; L, lateraltract. (B) robo3 embryo with normal axon tracts.(C and D) robo3 embryo with robo3 axon tractphenotype: the intermediate tract is collapsedat the medial tract. (E) robo3 embryo with a sli-likephenotype. (F) robo3 embryo with a robo-like phe-notype. (G) robo4 mutant embryo shown for com-parison to F.


phenotype is also partially penetrant and such embryosin combination with net can be Sli-negative but have a netphenotype. To test this possibility, we balanced the sli2

mutant chromosome with CyO balancer marked withGFP and stained GFP-negative embryos with Fas IIantibody. As shown in Figure 9H, we observed embryosthat are GFP-negative but had a weaker sli phenotype.This was observed in 0.7% of the GFP-negative embryos(n ¼ 300). This number is much less than what weobserved when the sli; net (or sli, fra) double mutantcombinations were examined. One likely explanation isthat the embryos in the sli; net double mutant combi-nation are derived from parents that have no balancersand therefore the partial penetrance of the defects/thevariability is much greater compared to embryos frombalanced parents. Additionally, genetic background inthe combination might alter the frequency. It is alsopossible that in some embryos, net and sli interact andnet may be epistatic to sli.

Maternal deposition of sli: We sought to determineif there is any maternal deposition of the sli gene pro-ducts. We performed RT–PCR of unfertilized embryosfor sli and found that there is maternal deposition of slimRNA to embryos (Figure 10A); however, there was nomaternal deposition of Sli protein (data not shown). Todetermine how long this maternal deposition lasts, weused sli2 embryos. It has been previously reported thatthe sli2 mutant chromosome carries a stop codon inexon 16 at aa position 1024 (Battye et al. 2001). Wethought that it is possible to make use of this change inthe sequence to determine the dynamics of maternaldeposition/initiation of zygotic transcription of sli.However, we did not find a stop codon in the reported

position upon sequencing of the sli gene in sli2 chromo-some. Therefore, we decided to examine the location ofthe mutation in the sli2 mutant. To narrow down thepossible mutation in the chromosome, we performedWestern blotting analysis of the sli2 homozygous mutantembryos using our newly generated antibody against theN terminus of the Sli protein (Figure 10B). As shown inFigure 10B, sli2 mutant embryos produced a truncatedprotein of�75 kDa (the predicted size for isoform A is 69kDa; often the SDS–PAGE can give a molecular weightgreater than the predicted one). We sequenced thecorresponding region (from exons 11 to 14 and introns11 and 12) and found that there is 1 change in the splicesite in sli2 mutant: the first base of intron 11 correspond-ing to isoforms A and B (which is intron 12 in isoform C)was changed from G to A (Figure 10C). This changealters the splice donor site sequence from GT to AT andtherefore would prevent splicing of intron 11 (which is279 bp in sli2 chromosome). If the splicing is prevented,the resulting mRNA will have a stop codon in the openreading frame immediately following the mutation.Additionally, there is a second splice donor site 54 bpdownstream and it is possible that this site is used; none-theless, since the stop codon occurs immediately follow-ing the mutation, both forms are expected to producethe same truncated protein.

To confirm the above results, we performed RT–PCRof sli gene products using primers flanking the mu-tant intron. As shown in Figure 10D, RT–PCR of sli2

homozygous embryos generated 2 products: a 1413-bp-long band representing the sli2 mRNA form in which theintron 11 is not spliced and a 1188-bp band from thesecond sli2 mRNA generated by the use of alternate

Figure 10.—Molecular characteriza-tion of sli. (A) Maternal deposition ofsli mRNA. RT–PCR analysis of wild-typeembryos showing the presence of sli tran-script in unfertilized embryos (Unf) aswell as in fertilized embryos from 0–24 hrof development. The expression of rp49was used as an internal control (bottom).(B) Western blot analysis of wild-type andsli2 homozygous embryos with anti-Sli-Nantibody. In wild type, the processed andunprocessed Sli can be observed, whereasa truncated Sli2 protein was detected as asingle band of �75 kDa in sli2 mutant em-bryos (arrowhead). (C) Schematic show-ing the normal splicing of sli in wild type(top diagram) and the aberrant splicingin sli2 embryos creating a termination ofthe Sli ORF (the two bottom diagrams;see text for details). (D) Detection of ab-errant sli2 transcripts using RT–PCR anal-ysis of sli2 embryos. Intron 11 does notundergo correct splicing in sli2 mutantsand only two defective splicing variants(asterisk) are present in sli2 mutant em-

bryos. (E) Zygotic expression of slit mRNA. RT–PCR analysis of embryos from wild-type females crossed to sli2/Bal males revealsthat sli2 transcript is present in embryos by 2–3 hr of age.


splice donor. These results are consistent with our aboveconclusion that the mutation generated a product thathas either the entire intron 11 or a part of the intron 11(when the downstream splice donor site is used).

Using this information, we examined when thezygotic activation of sli is initiated. RT–PCR of embry-onic sli mRNA derived from sli2/Cyo males and wild-typefemales (for the mutant bands) shows that the zygoticexpression can be seen in 2–3-hr-old embryos (Figure10E), much earlier than what has been previously thought.

Loss of function for abelson tyrosine kinase has axonguidance defects: We also examined embryos that aremutant for the gene abelson tyrosine kinase (abl), whoseproduct is thought to function downstream of Robo. Ithas been suggested that Abl phosphorylates Robo,whereas Enabled (Ena), a member of the proteinsknown to associate with the cytoskeleton, antagonizesAbl, allowing the hypophosphorylated Robo to mediateaxon repulsion (Bashaw et al. 2000). According to thishypothesis, loss of function for abl should have no con-sequence on axon guidance, as was reported (Gertler

et al. 1989; Bashaw et al. 2000). However, a subsequentarticle reported that abl loss-of-function mutants haveaxon guidance defects: the longitudinal tracts inappro-priately cross the midline in abl mutants (Wills et al.2002). We obtained abl mutant lines from the Goodmanlab and examined the embryos derived from parentsthat had the balancer and also embryos from parentsthat did not have the balancer. We observed strong axonguidance defects in longitudinal tracts in embryosderived from both sets of parents (Figure 11, A–D).Interestingly, embryos derived from parents that had thebalancer contained two classes of mutant embryos. Inone class, the longitudinal tracts were overall collapsedtoward the midline with medial tracts crossing themidline on an average of seven hemisegments perembryo with a lot more variability between embryos(n ¼ 7 embryos; Figure 11A; we calculate as hemiseg-ments because often tracts from only one side cross themidline) and a second class where no such collapsing ofthe tracts was observed but these embryos still had themedial tracts crossing the midline at slightly a lowerfrequency (Figure 11B; average five hemisegments perembryo). Embryos from the parents that had nobalancers, however, had only one class of embryos: therewas no collapsing of the tracts toward the midline, butthe medial tracts were crossing the midline (Figure 11, Cand D). The abnormal midline crossing of the medialtracts in these embryos was higher with an average ofnine hemisegmental crossings per embryo. In additionto the midline crossing of medial tracts in these embryos,we also observed an inappropriate outward projection oflateral tracts in these embryos (Figure 11, C and D; arrow-heads; average three to four projections per embryo).These results indicate that the events downstream ofRobo involving Abl and Ena, as proposed, are probablyincorrect and remain poorly understood.

DISCUSSION

Our results presented in this article show that theSlit/Robo signaling during axon guidance is muchmore complicated than previously thought. For exam-ple, it has been proposed that the Sli protein is secretedfrom the midline and a differing concentration of itinteracts with Robo receptors to specify the positioningof longitudinal tracts. We found that Sli is located alsoin the commissural and longitudinal connectives andis transported initially via the growing commissuraltracts. Furthermore, we found that a portion of embryosthat are mutant for the genetically null allele of robo havemore or less normal axon projections and the individ-uals develop until late pupal stages. A significantnumber of embryos mutant for robo3 have normalprojection pattern and go on to become viable adults.These robo3 homozygous individuals produce embryos,but these embryos exhibit normal axon projections toembryos with varying axon guidance defects: robo3, sli-like, and robo-like. All the embryos from robo3 homo-zygotes die as late embryo. Moreover, we also foundescapers among sli embryos as well, although in thiscase we did not find embryos that have a completely nor-mal projection pattern from a genetically null allele ofsli. This escaper phenomenon in these mutants thathas not been reported previously appears to be thelikely reason for the different epistatic results obtainedbetween sli/robo and net compared to sli/robo and fra.

In addition to these results, we find that sli mRNA ismaternally deposited and the zygotic transcription forsli begins as early as 2–3 hr of development. We alsoreport that the mutation in the sli gene in sli2 allele is achange in the splice-donor site, which is different fromthe mutational change reported previously. Finally, on

Figure 11.—Axon guidance defects in abl mutants. Em-bryos are stained with Fas II antibody. Anterior end is attop, midline is marked by vertical lines. (A–D) abl homozy-gous embryos. Note the outward projection of longitudinaltracts (arrowheads).


the basis of these results, we propose the following: (1) thecharacterization of a mutant/mutation for a gene mustinclude information on whether or not the defects inquestion are fully or partially penetrant; (2) any char-acterization of the double mutant phenotypes betweentwo mutants with opposing phenotypes must includeinformation on the penetrance or variability of the de-fects; and (3) all the phenotypes must be verified,whenever possible, by staining embryos derived fromparents that do not carry a balancer.

Slit is transported from the midline to the con-nectives via commissural tracts: It has been proposedthat Sli is secreted from the midline and it exists as adiffusible gradient extending away from the midline(Rajagopalan et al. 2000; Simpson et al. 2000). While nosuch gradient has been detected, we observed stainingof connectives and commissures with at least two dif-ferent antibodies, one the existing monoclonal and theother a polyclonal that we generated (see Figure 1). Ouruse of commissureless mutants indeed shows that theextra-midline Sli on the tracts was transported by thecommissural tracts and was not taken up by the tractsfrom the surrounding ‘‘environment.’’ Our results alsoshow that this transport is not via binding to Robo sincewe observed the staining of tracts for Sli in a deficiencythat eliminates Robo. Whether other Robo receptors,whose known expression pattern does not automaticallysuggest the possibility of a complementation for the lossof Robo, substitute for the loss of Robo is not known. IfRobo receptors are not involved, some other Sli trans-porter protein present on axon tracts must take up theSli from the midline glial cells and transport it across.

The major question, however, concerning the pres-ence of Sli in tracts is its functional significance: Doesthis Sli participate in any guidance process? This extra-midline Sli obviously is not altering the projectionpaths, although it is expected to encounter all threeRobo receptors. One possibility is that this Sli is in aninactive form and only midline Sli is functional. Perhapswe can also speculate that this is a mechanism to reduceSli at the midline. It seems like there is a fine balance forthe amount of Sli at the midline as indicated by ourresults with comm. Loss of function for comm causes a fewtracts crossing the midline; however, reducing the dos-age of sli in comm mutant background can dramaticallyenhance this phenotype (Figure 3). In comm mutantbackground, the Robo receptor is upregulated in thecommissural tracts. It seems likely that this excess Robothat Sli have to deal with titrates Sli out, reducing itslevel. Reducing the dosage of sli therefore would natu-rally enhance the phenotype. One result that is some-what inconsistent with these findings is that overexpressionof Sli at the midline using UAS-sli 3 single-minded-GAL4combination enhances the midline expression of Sli aswell as the intensity of Sli staining in the axon tracts;however, it does not cause any phenotype. We think thatfurther progress on this issue requires identification of

the Sli-transporter and functional dissection of the Sliprotein itself.

Which of the guidance signaling systems is epistatic,Net-Fra or Sli-Robo? We find a significant number ofescaper embryos reaching to adulthood and producingembryos in the robo3 mutant. A significant number ofthe mutant embryos had normal longitudinal axontracts. Intriguingly, a significant number of homozygousmutant embryos derived from homozygous parents hadmore severe phenotype than what has been reported forthis mutant. That is, embryos with a sli-like phenotypeas well as embryos with a robo-like phenotype wereobserved. On the other hand, homozygous embryosderived from robo3 heterozygotes showed either normalaxon guidance or the robo3 phenotype. One possibilityis that there is a partially penetrant maternal effect ofrobo3 mutation.

An important conclusion one can draw from theseresults on robo3 is that analysis of double mutants forepistasis between robo3 and any other mutation that hasan opposing phenotype (such as netrin) is basically mean-ingless since those robo3 mutant embryos that have nor-mal phenotype in combination with net mutant will havenet phenotype and the argument for epstasis relation-ship will then have to be based on frequency data. If thefrequency of ‘‘normal’’ mutant embryos is high, the con-clusion based on such epistasis results is not reliable.

We also found escaper embryos reaching the latepupal stages from a genetically null allele of robo. Onedifference between robo3 and robo is that the number ofsuch embryos in robo4 is much less than the numberobserved for robo3. Nonetheless, such embryos are suf-ficient to confuse, at the least, double mutant analysisbetween robo and mutants such as netrin, especiallyconsidering that these mutations are on different chro-mosomes and both the single mutant embryos will alsobe present in the collection. This problem, however, stillexists even if the double mutant analysis involves arecombinant chromosome between two mutations. Thisis indicated by our finding that embryos that are doublemutants for robo and fra can have a fra phenotype; thisis also consistent with the observation that robo; netrinembryos can have a netrin phenotype.

Why are there robo mutant embryos that have moreor less normal longitudinal axon tracts? One obviouspossibility is that it has redundancy with other Roboreceptors. The problem with this interpretation is thatother Robo receptors are not expressed in all tracts(Rajagopalan et al. 2000; Simpson et al. 2000). On theother hand, we observe robo3 homozygous embryos thatare sli-like or robo-like; therefore, these other Robo re-ceptors are likely to be expressed, perhaps at low levelsin all tracts.

The epistasis results are somewhat clearer with ourdetailed analysis of the sli2 mutant, a genetically nullallele of sli, and the analysis of sli and sli, fra doublemutants. However, the interpretation of the epistasis


results between sli, fra or sli; net still depends on thefrequency data since we found that sli2 mutants also giverise to embryos that have milder phenotype. That is,while we found no sli2 mutant embryos that have normalphenotype, we did find embryos that are similar tophenotypes found in sli hypomorphic alleles. Whetheror not a double mutant between this type of embryoand net or fra will have a net/fra phenotype is left toconjecture; we do observe a reasonable number ofembryos that have a net phenotype but lack Sli. However,it must be noted that sli; net mutants showing the netphenotype is greater than one would predict from theanalysis of the sli single mutant. This may be due to absenceof balancers in the parents from which the double mutantembryos are derived, as well as the genetic combination.Nonetheless, because the number of sli mutant embryosshowing the milder phenotype is very low, it is likely thatsli phenotype is epistatic to net phenotype.

Another important finding is that sli mRNA is mater-nally deposited to developing embryos, although thisdeposition appears to be exhausted at the latest by 5 hrof development, well before the requirements of Sli foraxon guidance. It may be that Sli is required in neuro-blasts or other early developmental processes.

If the Sli-Robo signaling is epistatic to the Net-Frasignaling in the guidance of longitudinal tracts, the mostone can suggest are the following two possibilities: (1)these two signaling pathways are independent systems,one repellent and the other attractant; and (2) Net-Frasignaling inhibits the Sli-Robo signaling. Since our pre-vious results indicate that Net-signaling has an attractanteffect on longitudinal tracts (Bhat 2005), this will be anadditional role for Net-signaling during the positioningof these tracts. With the available data, it is not possibleto distinguish with complete certainty whether we canexclude the second possibility. One can argue that if thetwo systems independently exert influence on the path-finding of longitudinal tracts, double mutants betweenthe two would have a normal phenotype. In the absenceof a normal phenotype in the double mutant, it must bethat the second scenario is correct. The problem withthis argument is that the repellent force exerted by Slisignaling on growth cones need not be equal to the at-tractant force by Net-signaling; the total attractant forcecan be mediated by Net signaling as well as a secondattractant pathway. In this scenario, although embryosare lacking both Sli and Net function, the second at-tractant signaling is still intact and is sufficient to attractgrowth cones to the midline. What is this attractant sig-naling that attracts the longitudinal tracts to the mid-line? We have not identified such an attractant system asyet. We expect that loss of function for such a system willhave the longitudinal tracts phenotype similar to the

loss of function for the net or fra mutants. Finally, ourresults with abl indicate that the proposed pathwaydownstream of Robo involving Abl and Ena is unlikelyto be correct (see Bashaw et al. 2000), and therefore,the events downstream of Robo remain obscure.

We thank the Goodman lab for the abl mutant lines, Guy Tear for thecomm mutants, Barry Dickson for the robo3 mutant allele, theBloomington Stock center for various stocks, and the Iowa Hybridomafacility for antibodies. This work is supported by grants from NIH-NIGMS and NIH-NINDS.

LITERATURE CITED

Bashaw, G. J., T. Kidd, D. Murray, T. Pawson and C. S. Goodman,2000 Repulsive axon guidance: Abelson and Enabled play op-posing roles downstream of the roundabout receptor. Cell 101:703–715.

Battye, R., A. Stevens, R. L. Perry and J. R. Jacobs, 2001 Repellentsignaling by Slit requires the leucine-rich repeats. J. Neurosci. 21:4290–4298.

Bhat, K. M., 2005 Slit-roundabout signaling neutralizes netrin-Frazzled-mediated attractant cue to specify the lateral positioningof longitudinal axon pathways. Genetics 170: 149–159.

Fritz, J. L., and M. F. VanBerkum, 2000 Calmodulin and son of sev-enless dependent signaling pathways regulate midline crossing ofaxons in the Drosophila CNS. Development 127: 1991–2000.

Gertler, F. B., R. L. Bennett, M. J. Clark and F. M. Hoffmann,1989 Drosophila abl tyrosine kinase in embryonic CNS axons: arole in axonogenesis is revealed through dosage-sensitive interac-tions with disabled. Cell 58: 103–113.

Harris, R., L. M. Sabatelli and M. A. Seeger, 1996 Guidance cuesat the Drosophila CNS midline: identification and characterizationof two Drosophila Netrin/UNC-6 homologs. Neuron 17: 217–228.

Kidd, T., C. Russell, C. S. Goodman and G. Tear, 1998 Dosage-sensitive and complementary functions of roundabout and commis-sureless control axon crossing of the CNS midline. Neuron 20:25–33.

Kidd, T., K. S. Bland and C. S. Goodman, 1999 Slit is the midlinerepellent for the robo receptor in Drosophila. Cell 96: 785–794.

Kolodziej, P. A., L. C. Timpe, K. J. Mitchell, S. R. Fried, C. S. Goodman

et al., 1996 frazzled encodes a Drosophila member of the DCC im-munoglobulin subfamily and is required for CNS and motoraxon guidance. Cell 87: 197–204.

Mitchell, K. J., J. L. Doyle, T. Serafini, T. E. Kennedy, M. Tessier-Lavigne et al., 1996 Genetic analysis of Netrin genes in Drosoph-ila: Netrins guide CNS commissural axons and peripheral motoraxons. Neuron 17: 203–215.

Myat, A., P. Henry, V. McCabe, L. Flintoft, D. Rotin et al.,2002 Drosophila Nedd4, a ubiquitin ligase, is recruited by Com-missureless to control cell surface levels of the roundabout recep-tor. Neuron 35: 447–459.

Rajagopalan, S., V. Vivancos, E. Nicolas and B. J. Dickson,2000 Selecting a longitudinal pathway: Robo receptors specifythe lateral position of axons in the Drosophila CNS. Cell 103:1033–1045.

Simpson, J. H., K. S. Bland, R. D. Fetter and C. S. Goodman,2000 Short-range and long-range guidance by Slit and its Roboreceptors: a combinatorial code of Robo receptors controls lat-eral position. Cell 103: 1019–1032.

Wills, Z., M. Emerson, J. Rusch, J. Bikoff, B. Baum et al., 2002 ADrosophila homolog of cyclase-associated proteins collaborateswith the Abl tyrosine kinase to control midline axon pathfinding.Neuron 36: 611–622.

Communicating editor: R. S. Hawley


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Regulation of Axon Guidance by Slit and Netrin Signaling ... · The following collections of wild-type embryos were used for RT–PCR analysis: 0–4 hr, 4–8 hr, 8–12 hr, and