Molecular Neurobiology Group, MRC Centre for Developmental Neurobiology, Guys Hospital Campus, Kings College, London SE1 1UL, UK
*Author for correspondence (e-mail: guy.tear{at}kcl.ac.uk)
Accepted 22 March 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Commissureless, Drosophila, Midline, Axon guidance
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The differential expression of receptors defines how axons make their initial growth decision but fails to explain why the commissural growth cones then leave the intermediate target that they formerly found so attractive, in order to progress along their pathway. This change in behaviour is possible because the commissural growth cones can adapt their sensitivity to midline signals (Kidd et al., 1999; Kidd et al., 1998a
). Subsequent to reaching the midline commissural growth cones lose their sensitivity to attractive cues and become responsive to the midline repellent cues. This switch also serves to explain why commissural axons do not re-cross the midline as they now behave as ipsilaterally projecting neurones and remain away from the midline.
In Drosophila, the switch in sensitivity displayed by commissural axons is explained by the precise spatial regulation of the Robo receptor molecule (Kidd et al., 1998a; Kidd et al., 1998b
). Before crossing the midline, the commissural neurones express the robo mRNA but do not express high levels of Robo protein on their surface in contrast to the ipsilaterally projecting neurones, which do express the Robo protein. Upon crossing the midline, the Robo protein levels on the commissural neurones increases and they become responsive to Slit. Similarly vertebrate axons become sensitive to the repellents Slit and Semaphorin during crossing (Zou et al., 2000
). In addition, an increase in functional Robo levels silences the attractive response by commissural neurones to the midline cue netrin (Shirasaki et al., 1998
; Stein and Tessier-Lavigne, 2001
) through a direct interaction between Robo and DCC. Thus, regulation of Robo protein levels is crucial for correct axonal migration at the midline. The only molecule known to regulate Robo protein levels is the Drosophila transmembrane protein Commissureless (Comm) (Kidd et al., 1998b
; Tear et al., 1996
).
Comm and Robo activities are closely intertwined. In the absence of Comm commissural growth cones are unable to cross the midline, suggesting that they are particularly sensitive to the repellent molecules expressed at the midline (Seeger et al., 1993; Tear et al., 1996
). Overexpression of Comm in all neurones results in the downregulation of Robo protein levels which mimics the robo phenotype where axons are no longer repelled from the midline (Kidd et al., 1998b
). Comm is expressed at high levels at the midline where Robo protein levels are low, consistent with the ability of Comm to regulate Robo levels negatively. Once away from the midline, Comm is unable to affect Robo and its levels on the axon can increase so preventing the axon from returning to the midline. Previous work describing this process suggested that Comm transcript was only found in the midline glia (Tear et al., 1996
). Comm protein was proposed to move from these midline cells to accumulate on the commissural region of the commissural neurones, as the cell bodies of the commissural neurones did not appear to express high levels of comm transcript. This provided a mechanism whereby Comm could locally downregulate Robo levels to allow midline crossing. In the absence of Comm, there is no downregulation of Robo and the commissural axons are unable to cross the midline.
We describe further characterisation of Comm distribution and its mode of action. Re-expression of Comm in all midline cells in comm mutant animals cannot rescue the comm phenotype and we are unable to see the transfer of a tagged version of Comm from the midline cells to commissural axons. Using a targeted RNA interference strategy in combination with a re-analysis of Comm protein distribution, we have found that Comm is expressed and required in commissural neurones but not ipsilateral neurones. We suggest that Comm function in commissural neurones aids in the initial selection of commissural versus ipsilateral pathway choice. Comm protein is found to accumulate on the surface of commissural axons within the commissure where it could interact with Comm on the surface of the midline cells. This interaction may prevent Comm protein reaching the contralateral portions of the commissural axons so allowing Robo protein levels to increase and a switch in the responsiveness of the commissural axons to midline cues.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For rescue experiments, we used a transformant line carrying wild-type comm-coding sequence on the X chromosome (Kidd et al., 1998b) and also a line carrying a Comm-GFP fusion transgene on the second chromosome (constructed here), both under the control of the UAS promoter. P-element transformation was performed using standard procedures. For each UAS transgene, multiple lines were generated and checked for expression.
Re-expression of Comm at the midline in comm mutants was achieved by crossing flies heterozygous for comm (E39 or A490) and UAS-Comm to flies heterozygous for comm and either sim-GAL4 or slit-GAL4. The progeny from this cross will contain 25% comm mutant embryos. Twenty five percent of these comm mutants will possess both UAS-Comm and a GAL4 driver. When re-expressing two copies of Comm at the midline, flies heterozygous for comm, UAS-Comm and UAS-Comm-GFP were crossed to flies heterozygous for comm and slit-GAL4. In this case, 37.5% of comm mutant embryos will possess one or two copies of UAS-Comm and a slit-GAL4 driver.
DNA constructs
All manipulations of the comm cDNA are based on the published full-length cDNA clone (Tear et al., 1996). Comm-GFP was constructed by using the PCR on both comm and mGFP6 (Schuldt et al., 1998
) cDNAs to introduce compatible cloning sites ensuring that the entire coding sequence was in frame. The PCR products were then ligated into the pUAST vector (Brand and Perrimon, 1993
) and the construct sequenced on both strands. The entire open reading frame was excised using EcoRI and KpnI cloned into the pRmHa-3 vector for use in S2 cell assays.
The comm hairpin consists of an inverted repeat of comm sequence, separated by a linker region. A fragment of 375 bp of comm-coding sequence (positions 818 to 1192) and the linker region were synthesised by the PCR. The linker region consisted of AAGG (which provokes the loop) flanked by SphI and EcoRI restriction sites (linker region size, 16 bp). Two PCR products were created with either XbaI or XhoI at the 5' ends. comm 5'-3' plus linker was subcloned into the XbaI and EcoRI sites of pBluescript (Stratagene). comm 3'-5' plus linker was then inserted into the resultant construct using the XhoI and SphI restriction sites. This construct was amplified in Epicurian Sure Cells (Stratagene). The entire hairpin sequence was then cloned into pUAST using XbaI and XhoI sites.
S2 cell assay
Transfections were carried out as described (Di Nocera and Dawid, 1983). Twenty-four hours after transfection, protein expression was induced with 0.7 mM CuSO4. Sixteen hours post-induction, cells were processed for immunohistochemistry using mAb 13C9 (Kidd et al., 1998a
) and rabbit polyclonal
GFP (Molecular Probes). pRmHa-3-robo construct was provided by V. McCabe. pUAS-comm-HP was driven in S2 cells using the pMT-GAL4 construct, which was a kind gift from Kai Zinn (Caltech).
Comm antibodies and immunohistochemistry
cDNA corresponding to the extracellular region of Comm (amino acids 1 to 130) was amplified using the PCR and cloned into pQE30 (Qiagen) to generate QEEC9. The His-tagged protein was induced and purified using standard techniques and the protein injected into rabbits by a commercial company (Eurogentec). Rabbit serum was affinity purified using a column bearing the immunogen. Antibody was eluted from the column using 3M sodium thiocyanate and the eluate dialysed against PBS. The antibody was pre-absorbed against Drosophila embryos and used at a final concentration of 1:10. All immunostaining was performed using standard techniques (Patel, 1994). Anti-myc (9E10) was obtained from the Developmental Studies Hybridoma Bank, University of Iowa. Rabbit anti-Eagle was provided by J. Urban.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
The ability of one copy of comm at the midline to rescue commissure formation in commA490 embryos is very mild. Increasing the number of copies of comm expressed to two does not increase the average number of commissures formed per embryo (Table 1). The complete lack of any rescue of the commE39 null allele, even when two copies of Comm are re-supplied at the midline, suggests that comm expression is required in cells other than the midline cells.
We also attempted to rescue the comm phenotype by re-expressing Comm in all neurones in both commE39 and commA490. This resulted in the production of a robo-like phenotype in which axons misroute towards the midline (data not shown). The phenotype matches that seen when Comm is overexpressed in the wild-type embryo (Kidd et al., 1998b), suggesting that endogenous Comm at the midline is not necessary for this phenotype.
Comm-GFP driven at the midline does not transfer onto commissural axons
In addition to testing whether Comm at the midline is able to rescue the comm phenotype we investigated whether Comm in the commissural axons originates from the midline cells. To do this we expressed a C-terminal GFP tagged version of Comm (Comm-GFP) at the midline using sim-GAL4 and slit-GAL4 simultaneously (Fig. 2A). The fusion protein is fully functional, able to downregulate Robo and has the same subcellular localisation pattern as the wild-type protein, both in vivo and in vitro (M. G. and G. T., unpublished). We observed that Comm-GFP does not transfer from the midline cells to commissural axons but remains within the cells where the gene is expressed. Rather than transfer between cells we found that Comm-GFP can move to fill the cell where it is expressed. When large amounts of Comm-GFP are driven the protein is observed within the axons of neurones produced at the midline, such as the MP1s and the VUMs (Fig. 2B). However, no Comm protein is detected at the commissures. This suggests that the midline cells do not supply the Comm protein found on commissural axons.
|
Comm-hairpin specifically disrupts comm expression in vitro and has no effect on neuronal survival or general axon guidance in vivo
To verify that the comm-hairpin transcript (comm-HP) inhibits comm expression and that this inhibition is specific to comm, the hairpin was driven in S2 cells. When Comm-GFP is co-expressed with comm-HP, very few cells show GFP fluorescence in contrast to transfections where comm-HP is not co-expressed (Fig. 3A,B).
|
To confirm that comm-HP is able to disrupt Comm expression in the embryo, we expressed comm-HP in a subset of neurones using the eagle-GAL4 driver (MZ360) (Dittrich et al., 1997). The eagle (eg)-positive neurones were identified using an anti-Eg antibody and examined for co-expression of Comm. In wild-type embryos the eg-positive neurones express Comm protein (Fig. 3E); however, when comm-HP is expressed in these cells, the levels of Comm protein is reduced (Fig. 3F). This reduction in Comm expression occurs in most eg-positive neurones but not all suggesting that the comm-HP is not completely effective in vivo or that it is not correctly expressed in all cells where it is driven.
Disruption of comm function in either neurones or midline cells results in guidance defects at the midline
comm-HP was expressed in midline cells and/or neurones to identify if interference with Comm function in these cells affect axon outgrowth in the CNS. In order to look at specific neurones that cross the midline, we used the Sema2b-myc marker (Rajagopalan et al., 2000
). This marker labels the cell bodies and axons of two to three neurones per hemisegment within five posterior abdominal segments, A4-8. These laterally positioned neurones normally send their axons immediately across the midline at the anterior margin of the anterior commissure, then turn to project anteriorly within the contralateral longitudinal tract (Fig. 4A). Thus, in each segment, three Sema2b tracts can be scored, two longitudinal tracts and a commissural tract. Absence of a commissural tract will occur when Sema2b axons from both sides of the CNS fail to cross the midline, whereas a defect in a longitudinal tract will occur when some axons fail to extend across the midline to the contralateral longitudinal pathway. In the wild-type embryo, the Sema2b commissural and longitudinal axon tracts are rarely defective (five tracts affected in 34 embryos; i.e. 0.15 defective tracts per embryo). When comm-HP is expressed, either pan-neurally or at the midline, we observe an increased incidence of failures to form Sema2b tracts (Fig. 4B,C). These failures occur as the Sema2b axons now stall and fail to extend across the midline or do not fasciculate with one another appropriately. Counting the number of Sema2b tracts severely affected reveals an increase from an average of 0.15 defective tracts per embryo (n=34) in wild type to 0.78 tracts affected per embryo when comm-HP is expressed at the midline (n=40) or 1.96 tracts affected per embryo when comm-HP is expressed in neurones (n=27). This phenotype suggests a requirement for Comm in both midline cells and CNS neurones. If the observed defects were due to a limited inhibition of Comm function, we would expect the phenotype to be enhanced if Comm levels in the embryo are reduced by removing one copy of the gene. Indeed we find that the phenotype is enhanced when comm-HP is driven in a comm heterozygous background (Fig. 4D,E). In these embryos, an average of 2.9 Sema2b tracts are severely affected or absent per embryo (n=21), in addition many or all of the Sema2b tracts are reduced in thickness in the embryos. In all cases the same phenotypes are observed using several different UAS-Comm-HP inserts at various sites in the genome. The observed failure of axons to cross the midline and the genetic interaction with comm shows that comm-HP is indeed disrupting comm expression in vivo, as was observed in vitro.
|
Driving Comm-HP in specific populations of neurones
The widespread expression of comm-HP within the CNS can cause axonal outgrowth defects; however, comm-HP is mosaic in its action and axon outgrowth defects are best-observed using markers for subsets of neurones. Therefore we tested whether expression of comm-HP could cause axon outgrowth defects when it is expressed in small populations of neurones within the CNS. For these experiments, we used three GAL4 drivers: eg-GAL4, 15J2 and MZ465 (Dittrich et al., 1997; Hidalgo and Brand, 1997
). eg-GAL4 is expressed in four neuroblasts and their progeny. The neuronal progeny includes a group of lateral EL neurones that project within the anterior commissure and a smaller group of more medial neurones that extend via the posterior commissure (Fig. 5A), the commissural tracts they form are complete by stage 13 (Dittrich et al., 1997
; Dormand and Brand, 1998
; Higashijima et al., 1996
). eg-GAL4 was used to drive comm-HP and a tau-GFP reporter in these commissural neurones. The expression of comm-HP within the neurones causes the axon bundles in certain segments to fail to cross and in some cases appear to turn away from the midline (Fig. 5B). When driving one copy of the hairpin construct, 13% of hemisegments show midline crossing errors (n=368) (Fig. 5B). With two copies of comm-HP, the number of midline crossing failures is increased to 61% of hemisegments (n=160) (Fig. 5C). When driving one copy of comm-HP the disruption is limited to the more medial neurones; when driving two copies, the more lateral neurones now fail to cross in certain hemisegments (Fig. 5B,C). Interestingly, by late stage 15, the eg-positive neurone crossing errors are overcome and the staining pattern appears normal. If one copy of comm-HP is expressed in the eg-positive neurones and one copy at the midline the number of crossing failures remains similar to the level seen when driving one copy of comm-HP in the eg-positive neurones alone at 13% of hemisegments (Fig. 5D).
|
Comm protein is detectable in neuronal cell-bodies
Our rescue and targeted RNAi experiments both suggest a requirement for Comm function not only in the midline cells but also in the commissural neurones. This has led us to re-examine the Comm protein distribution in the embryo and to do this we developed a rabbit polyclonal antibody against Comm. This antibody is specific for Comm as no staining is observed in the commE39 embryos and has the same distribution as was reported previously (Tear et al., 1996). Using confocal microscopy, it is apparent that Comm is expressed on the commissural tracts and throughout the nerve cord (Fig. 6A,B). The staining within the neuronal cell bodies is qualitatively different from that seen at the commissure in that it appears punctate (commissural axons have what appears to be cell surface staining) (Fig. 1C). This punctate staining was previously suggested to result from transfer of Comm to the axons from the midline cells. We now believe this transfer does not take place and that the vesicular Comm is produced by the neurones themselves. Our data from expression of comm-HP suggests that Comm is required in commissural but not ipsilateral neurones. To identify whether all neurones express Comm, we labelled the Sema2b, eg-positive and 15J2 neurones and looked for colocalisation with Comm. Punctate Comm staining clearly co-localised with both the Sema2b and eg-positive cell bodies (Fig. 3F, Fig. 6A-D). However no obvious colocalisation was apparent when using the 15J2 driver to drive GFP in the vMP2 and dMP2 neurones (Fig. 6E,F). It therefore appears, from both the hairpin and co-localisation experiments, that these ipsilateral interneurones possess no Comm protein or RNA.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Comm protein does not transfer from the midline glia onto commissural axons
To allow commissural axons to cross the midline, Comm specifically downregulates Robo on these axons. Once across the midline, Robo levels increase, suggesting Comm acts only at the midline. It has been suggested previously that Comm acts specifically at the midline via a mechanism whereby Comm is supplied to axons by the midline cells (Tear et al., 1996). In this model, Comm transfers from the surface of midline cells to the commissural axons during cell/cell contact. If Comm were then unable to extend far along the axon it could act locally to downregulate Robo in the commissures and allow extension across the midline. This model predicts that Comm can transfer from midline cells to the commissural axons and that Comm expression is required only at the midline. We have shown that the expression of Comm protein at the midline alone is not sufficient for commissure formation. We were unable to generate any rescue of midline crossing when re-expressing wild-type Comm at the midline in comm-null mutant embryos. However, a mild rescue was observed when Comm was re-expressed at the midline in commA490 mutants. As no rescue is seen in null embryos and only partial rescue is seen in commA490 embryos, Comm is necessary in other CNS cells and we show Comm is also necessary within the commissural axons themselves. Thus, in the commA490 embryos, the truncated protein is expressed in commissural axons in addition to the midline cells and it retains some weak function. The re-expression of Comm protein adds function to the midline cells, thereby slightly increasing the number of axons able to cross the midline. Unfortunately we cannot rescue the comm phenotype by overexpression of Comm in all neurones as this results in the efficient downregulation of Robo and promiscuous midline crossing. However we were able to reveal a requirement for Comm in neurones using a targeted RNAi approach.
We also examined Comm transfer from the midline cells using a C terminal GFP-tagged Comm protein, Comm-GFP. The addition of GFP to the Comm protein allowed us to differentiate the re-expressed Comm from that expressed endogenously and to identify whether Comm could move from one cell to another in the embryo. Comm-GFP retains full function, as revealed by its ability to phenocopy the robo phenotype when overexpressed in all neurones (data not shown). Yet when Comm-GFP was expressed at the midline, we failed to observe transfer into commissural axons. The Comm-GFP did not appear to be tightly associated with any cellular component, as the protein was clearly able to move within the cells. Comm-GFP was visible within the axons of the neurones derived from the midline cells, e.g. MP1 and the VUMs. Despite clear labelling of these axons, no staining of commissural axons was visible. Thus, Comm is unable to move from the midline cells to the commissural neurones, confirming that Comm must function in cells other than or in addition to the midline cells to regulate commissural axon extension at the midline. Interestingly when Comm is driven in MP1 it does not affect its guidance, even though Robo is normally required in this neurone to keep it away from the midline (Seeger et al., 1993).
Our investigations examining rescue of the comm phenotype by re-expression of Comm at the midline and analysis of the ability of Comm-GFPs to move from midline cells leads to the conclusion that the Comm protein observed on the commissural tracts in wild-type embryos is not provided by the midline cells. It is possible that low levels of comm transcript are present in neurones and that Robo downregulation at the midline is due to neuronal Comm protein accumulating at the midline.
Comm is expressed and required in both commissural neurones and midline glia
To identify which cells in the CNS require Comm function, we made use of the technique of targeted RNAi. This method inhibits the production of Comm by expressing double-stranded RNA, in the form of a hairpin-loop (comm-HP).
We used a Drosophila S2 cell assay to test the ability of comm-HP to inhibit comm expression. Here, comm-HP caused a marked reduction in Comm expression, but did not affect the expression of Robo. Expression of comm-HP does not lead to general, nonspecific axon guidance defects on PNS axons. Furthermore, when comm-HP was driven in specific ipsilateral neurones, guidance of these neurones was unaffected. However, expression of comm-HP in commissural neurones does lead to midline crossing defects and these defects are enhanced when one copy of comm is removed. This suggests that comm-HP specifically inhibits Comm expression in vivo.
Several conclusions can be made from the targeted RNAi experiments. First, midline crossing errors were observed when Comm expression was inhibited specifically in neurones. Thus, comm transcript and Comm activity are present in neurones. Second, an identical yet milder phenotype was observed when driving the hairpin specifically at the midline. Comm function is therefore required in both neurones and the midline glia. The identical nature of the neuronal and glial phenotypes suggests that comm within both of these cell types is required for the same process, i.e. allowing a growth cone to cross the midline. Finally, the lack of any guidance errors in ipsilateral neurones, when driving the hairpin either throughout the nervous system or in specific neurones, suggests that Comm activity is not required in these ipsilateral neurones.
The CNS defects produced by the expression of Comm-HP in the CNS are fairly mild when observed with BP102, a reduction in the size of the commissural tracts and some longitudinal breaks can be observed. These phenotypes are more obvious when the outgrowth of a smaller population of neurones, the Sema-2b neurones, are examined. Here, there are clear defects in extension across the midline and stalling of some commissural axons. These axons also appear to fail to extend in the longitudinals. This affect is likely to be specific to Comm, as removal of one copy of comm enhances this phenotype. However, it is apparent that comm-HP is unable to fully inhibit comm in all cells. This might explain why the phenotype differs from that seen in comm loss-of-function embryos. In comm mutants, when all cells lose Comm function and all axons do not cross the midline, the default is to extend in the longitudinals. In the comm-HP mutants, a subset of commissural axons fail to extend and stall. This may affect the extension of neighbouring axons producing the longitudinal defects seen with BP102. Expression of comm-HP in neurones or midline cells gives rise to the same phenotype, suggesting that a slight reduction in Comm levels at the midline also leads to crossing failures. By contrast, driving large amounts of fully functional Comm-GFP at the midline has no effect on guidance. This would suggest that a certain threshold level of Comm is required at the midline.
Our results suggest that the Comm protein identifiable in neuronal cell bodies is not the result of transfer from midline cells. This punctate vesicular neuronal staining was seen to be restricted to commissural neurones, with no protein observed within ipsilateral neurones. Thus, the targeted RNA interference and protein localisation results suggest that Comm is not present in ipsilateral neurones. It is therefore possible that the presence or absence of neuronal Comm determines whether or not an axon crosses the midline. Indeed expression of Comm in the normally ipsilateral projecting Ap neurones (OKeefe et al., 1998) using an Ap-GAL4 driver causes all the Ap neurones to cross the midline (Bonkowsky et al., 1999
). Thus, individual ipsilateral neurones can be converted to midline crossing neurones by the introduction of comm activity, suggesting that the presence of neuronal Comm dictates axon pathway choice.
Comm protein accumulates on the commissural portion of the contralaterally projecting axons. The appearance of this staining is very different to the vesicular localisation seen in the cell body. Rather it appears that Comm protein can accumulate at the cell surface within the commissure. Perhaps Comm protein is transported from the cell bodies within the vesicles to be presented at the cell surface during commissure formation. Furthermore, Comm protein does not seem to extend beyond the midline region onto the contralateral segments of the axon. Comm thus appears to be targeted to the commissural tract or somehow sequestered in this region. This may either concentrate its activity in this region and/or serve as a device for preventing Comm activity spreading further along the axon. In this way Comm could act to prevent Robo-mediated sensitivity to the midline Slit signal in commissural axons prior to crossing, but be unable to act on Robo once the axon has extended beyond the midline. It is possible that an interaction between Comm at the midline and Comm in the neurone provides the means to trap or concentrate Comm at the commissure. An accompanying paper provides evidence of the ability of Comm to bind itself through its extracellular domain (M. G. and G. T., unpublished).
We reveal that comm activity is necessary in commissural neurones and midline glial cells. A similar requirement for Comm in neurones has been observed using cell transplantation techniques (Diana Cleppien, Gerd Technau and Barry Dickson, personal communication). We suggest the presence of Comm in the commissural neurones may encourage midline crossing. This tendency is promoted by Comm activity in the midline cells. The combined action is predicted to allow inhibition of Robo activity specifically in the commissural neurones, allowing growth across the midline. Comm protein accumulates at the axon surface within the commissural region, using a mechanism that is likely to involve Comm in the midline glia. However, Comm activity does not extend beyond the midline, allowing Robo levels to increase at the growth cone surface and initiate a sensitivity to the midline inhibitor Slit that encourages extension away from the midline and prevents re-crossing.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bonkowsky, J. L., Yoshikawa, S., OKeefe, D. D., Scully, A. L. and Thomas, J. B. (1999). Axon routing across the midline controlled by the Drosophila Derailed receptor. Nature 402, 540-544.[Medline]
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401-415.
Brose, K., Bland, K. S., Wang, K. H., Arnott, D., Henzel, W., Goodman, C. S., Tessier-Lavigne, M. and Kidd, T. (1999). Slit proteins bind robe receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96, 795-806.[Medline]
Colamarino, S. A. and Tessier-Lavigne, M. (1995). The role of the floor plate in axon guidance. Annu. Rev. Neurosci. 18, 497-529.[Medline]
Di Nocera, P. P. and Dawid, I. B. (1983). Transient expression of genes introduced into cultured cells of Drosophila. Proc. Natl. Acad. Sci. USA 80, 7095-7098.[Abstract]
Dittrich, R., Bossing, T., Gould, A. P., Technau, G. M. and Urban, J. (1997). The differentiation of the serotonergic neurons in the Drosophila ventral nerve cord depends on the combined function of the zinc finger proteins Eagle and Huckebein. Development 124, 2515-2525.
Dodd, J., Morton, S. B., Karagogeos, D., Yamamoto, M. and Jessell, T. M. (1988). Spatial regulation of axonal glycoprotein expression on subsets of embryonic spinal neurons. Neuron 1, 105-116.[Medline]
Dormand, E. L. and Brand, A. H. (1998). Runt determines cell fates in the Drosophila embryonic CNS. Development 125, 1659-1667.
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. and Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811.[Medline]
Harris, R., Sabatelli, L. M. and Seeger, M. A. (1996). Guidance cues at the Drosophila CNS midline: identification and characterization of two Drosophila Netrin/UNC-6 homologs. Neuron 17, 217-228.[Medline]
Hidalgo, A. and Brand, A. H. (1997). Targeted neuronal ablation: the role of pioneer neurons in guidance and fasciculation in the CNS of Drosophila. Development 124, 3253-3262.
Hidalgo, A., Kinrade, E. F. and Georgiou, M. (2001). The Drosophila neuregulin Vein maintains glial survival during axon guidance in the CNS. Dev. Cell 1, 679-690.[Medline]
Higashijima, S., Shishido, E., Matsuzaki, M. and Saigo, K. (1996). eagle, a member of the steroid receptor gene superfamily, is expressed in a subset of neuroblasts and regulates the fate of their putative progeny in the Drosophila CNS. Development 122, 527-536.
KeinoMasu, K., Masu, M., Hinck, L., Leonardo, E. D., Chan, S. S. Y., Culotti, J. G. and TessierLavigne, M. (1996). Deleted in Colorectal Cancer (DCC) encodes a netrin receptor. Cell 87, 175-185.[Medline]
Kennedy, T. E., Serafini, T., de la Torre, J. R. and Tessier-Lavigne, M. (1994). Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Cell 78, 425-435.[Medline]
Kidd, T., Bland, K. S. and Goodman, C. S. (1999). Slit is the midline repellent for the robo receptor in Drosophila. Cell 96, 785-794.[Medline]
Kidd, T., Brose, K., Mitchell, K. J., Fetter, R. D., Tessier-Lavigne, M., Goodman, C. S. and Tear, G. (1998a). Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily conserved guidance receptors. Cell 92, 205-215.[Medline]
Kidd, T., Russell, C., Goodman, C. S. and Tear, G. (1998b). Dosage-sensitive and complementary functions of roundabout and commissureless control axon crossing of the CNS midline. Neuron 20, 25-33.[Medline]
Klambt, C., Jacobs, J. R. and Goodman, C. S. (1991). The midline of the Drosophila central nervous system: a model for the genetic analysis of cell fate, cell migration, and growth cone guidance. Cell 64, 801-815.[Medline]
Kolodziej, P. A., Timpe, L. C., Mitchell, K. J., Fried, S. R., Goodman, C. S., Jan, L. Y. and Jan, Y. N. (1996). frazzled encodes a Drosophila member of the DCC immunoglobulin subfamily and is required for CNS and motor axon guidance. Cell 87, 197-204.[Medline]
Mitchell, K. J., Doyle, J. L., Serafini, T., Kennedy, T. E., TessierLavigne, M., Goodman, C. S. and Dickson, B. J. (1996). Genetic analysis of Netrin genes in Drosophila: Netrins guide CNS commissural axons and peripheral motor axons. Neuron 17, 203-215.[Medline]
OKeefe, D. D., Thor, S. and Thomas, J. B. (1998). Function and specificity of LIM domains in Drosophila nervous system and wing development. Development 125, 3915-3923.
Patel, N. H. (1994). Imaging neuronal subsets and other cell types in whole-mount Drosophila embryos and larvae using antibody probes. Methods Cell Biol. 44, 445-487.[Medline]
Rajagopalan, S., Vivancos, V., Nicolas, E. and Dickson, B. J. (2000). Selecting a longitudinal pathway: Robo receptors specify the lateral position of axons in the Drosophila CNS. Cell 103, 1033-1045.[Medline]
Schuldt, A. J., Adams, J. H., Davidson, C. M., Micklem, D. R., Haseloff, J., St Johnston, D. and Brand, A. H. (1998). Miranda mediates asymmetric protein and RNA localization in the developing nervous system. Genes Dev. 12, 1847-1857.
Seeger, M., Tear, G., Ferres-Marco, D. and Goodman, C. S. (1993). Mutations affecting growth cone guidance in Drosophila: genes necessary for guidance toward or away from the midline. Neuron 10, 409-426.[Medline]
Serafini, T., Kennedy, T. E., Galko, M. J., Mirzayan, C., Jessell, T. M. and Tessierlavigne, M. (1994). The Netrins Define a Family of Axon Outgrowth-Promoting Proteins Homologous to C-Elegans Unc-6. Cell 78, 409-424.[Medline]
Shirasaki, R., Katsumata, R. and Murakami, F. (1998). Change in chemoattractant responsiveness of developing axons at an intermediate target. Science 279, 105-107.
Stein, E. and Tessier-Lavigne, M. (2001). Hierarchical organization of guidance receptors: silencing of netrin attraction by slit through a Robo/DCC receptor complex. Science 291, 1928-1938.
Stoeckli, E. T. and Landmesser, L. T. (1998). Axon guidance at choice points. Curr. Opin. Neurobiol. 8, 73-79.[Medline]
Stoeckli, E. T., Sonderegger, P., Pollerberg, G. E. and Landmesser, L. T. (1997). Interference with axonin-1 and NrCAM interactions unmasks a floor-plate activity inhibitory for commissural axons. Neuron 18, 209-221.[Medline]
Tear, G. (1999). Axon guidance at the central nervous system midline. Cell Mol. Life Sci. 55, 1365-1376.[Medline]
Tear, G., Harris, R., Sutaria, S., Kilomanski, K., Goodman, C. S. and Seeger, M. A. (1996). Commissureless controls growth cone guidance across the CNS midline in Drosophila and encodes a novel membrane protein. Neuron 16, 501-514.[Medline]
Tessier-Lavigne, M. and Goodman, C. S. (1996). The molecular biology of axon guidance. Science 274, 1123-1133.
Zou, Y., Stoeckli, E., Chen, H. and Tessier-Lavigne, M. (2000). Squeezing axons out of the gray matter: a role for slit and semaphorin proteins from midline and ventral spinal cord. Cell 102, 363-375.[Medline]