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In contrast, GSd misexpression using GAL4 , which drives expression in R8 only, did not produce any axon targeting defects not shown , suggesting that R cells that normally target to the medulla are refractory to the misexpression of GSd To begin to explore the mechanism through which misexpression of GSd in R cells causes an axon targeting defect, two antibodies were used to test the involvement of molecules known to influence R-cell axon guidance and whose mutant phenotypes are reminiscent of that caused by misexpression of GSd It has been hypothesized that the receptor tyrosine kinase Otk is part of the receptor complex controlling R-cell termination in the lamina, and that the transcription factor Bks regulates R1—R6 termination.

In addition, we also tested the possibility that GSd misexpression might upregulate the expression of Runt. However, Runt expression was not altered upon misexpression of GSd not shown. Mutations in the genes nonstop P oeck et al. To test whether GSd misexpression led to incorrect positioning of the glial cells in the lamina we studied the glial-cell-specific marker Reversed Polarity Repo X iong and M ontell ; R angarajan et al. In particular, the epithelial and marginal glial cells, which prefigure the lamina plexus and present a stop signal to incoming R1—R6 axons, formed neat rows that were aligned as in controls.

Runt expression is also unaffected. Thus, these known causes of axon mistargeting in the visual system cannot obviously explain the mechanism causing R-cell axons to be mistargeted by GSd Dorsal is characterized by an N-terminal Rel homology domain which possesses domains for dimerization, DNA binding, and interactions with its inhibitor Cactus.

In addition, it has a C-terminal transactivation domain. Two differentially spliced forms of Dorsal, known as Dorsal-A and Dorsal-B, can heterodimerize and act synergistically to enhance transcription from reporter genes in vitro G ross et al. These isoforms are identical in their N-terminal Rel homology domains amino acids 1— , but have divergent C-termini that encode unique transactivation domains I soda et al.

Dorsal-B may heterodimerize with Dorsal-A and thereby achieve nuclear entry G ross et al. Therefore, either one may be responsible for the R-cell axon mistargeting.

To examine axon targeting in the lamina more specifically, ro-tau-lacz was used as a reporter to visualize only the R2—R5 axons.

The effects of GSd are caused by misexpression of the dorsal gene. Anterior is left and posterior is right. Our findings suggested an important role for the dl gene in explaining the GSd misexpression phenotype, and raised several possibilities: 1 that dl -B is largely responsible for the axon targeting defects of the GSd misexpression phenotype; 2 that both Dorsal-A and Dorsal-B can mistarget axons, but that the UAS-dlB transgene is expressed at higher levels than the UAS-dlA transgene; or 3 that both dl -A and dl -B synergize to give rise to the full effect observed with GSd To distinguish between these possibilities, we performed a chemical mutagenesis screen for suppressors of GSd misexpression effects.

A total of 45, adult progeny of this cross were screened to identify two lines that showed suppression of this phenotype to a relatively normal eye structure Figure 3F. PCR primers were designed to span all coding sequences of the dl gene, both dl-A and dl-B isoforms. This mutation is identical to one previously reported which also displayed dominant female sterility: Arg lies in the DNA-binding domain of dorsal I soda et al.

It results in a truncation of the C-terminus of dl-A , in a region known to encode the transactivation domain. No other mutations in the dl gene were found in these lines. This antibody recognizes an epitope in the C-terminal half of Dorsal W halen and S teward , and is therefore specific for the Dorsal-A isoform and cannot recognize the Dorsal-B isoform.

For clarity, we continue to distinguish the isoforms of Dorsal as detected by this antibody. In R cells, the ectopic Dorsal immunoreactivity was found in the cell body and axons, through the optic stalk and into the brain. It is noteworthy that while UAS-dlA misexpression caused strong Dorsal expression in cell bodies and proximal axons, expression was negligible in distal axons in the lamina plexus and medulla Figure 4A. In contrast, Dorsal expression driven with GS and dl C was readily detectable along the entire length of axons Figure 4, B and C.

Dorsal and Cactus are expressed in the developing eye imaginal disc. E Confocal image of eye disc showing that Dorsal-A is expressed in the cell bodies of R cells in addition to cone cells and peripodial cells in wild-type WT, genotype: w eye discs. Anterior is upper left and posterior is lower right, with the beginning of the optic stalk marked by an arrowhead. The optical slice shown here largely cuts through a plane at the level of the R-cell nuclei.

In more anterior positions, Dorsal-A is also expressed in undifferentiated cells prior to their recruitment into ommatidia arrow. G, H In WT genotype: w eye discs at third instar stages, Cactus was expressed in the cell bodies of photoreceptor neurons G in addition to cone cells, glial cells of the optic stalk H , and peripodial cells.

We next analyzed whether Dorsal was endogenously expressed in the developing larval visual system. We used the anti-Dorsal antibody and fluorescence immunochemistry to stain wild-type animals. Confocal microscopy was used to analyze the fluorescently labeled tissues. Moderately intense staining was present in what appears to be many, if not all, cells of the developing eye disc and optic stalk of wild-type animals Figure 4E. Notably, Dorsal is localized to the cell bodies of R cells, cone cells, and peripodial cells.

There were traces of Dorsal immunoreactivity found in R-cell axons in the optic stalk or in the axon terminals in the optic lobe not shown. A large number of undifferentiated cells anterior to the morphogenetic furrow were also immunopositive Figure 4E. For R cells and cone cells, which have little cytoplasm, it is difficult to determine whether the staining was throughout the cytoplasm or in close proximity to the plasma membrane.

In peripodial cells, however, the staining was evident throughout the cytoplasm. None of the staining was found to be nuclear, suggesting the majority of Dorsal is held in the cytoplasm, perhaps bound in inactive complexes with Cactus, a known inhibitor of Dorsal and other Rel proteins. Previous studies have shown that Cactus is distributed at low levels throughout the brain and nerve cord of Drosophila larvae, and is notably enriched in the mushroom bodies C antera et al.

We confirmed the reported expression of Cactus in the brain data not shown and found that Cactus is also expressed in the R-cell bodies Figure 4G and in cone cells not shown. We also found strong Cactus labeling of the peripodial cells surrounding the eye disc and of the glial cells in the optic stalk Figure 4H.

Traces of Cactus were also evident in the optic lobe, though the diminished labeling there was difficult to characterize. Nevertheless, both Dorsal and Cactus are expressed in the developing visual system, and both are expressed in R cells.

We have provided evidence that misexpression of Dorsal resulted in R-cell axon targeting defects, that mutations of dl suppress those defects, and that Dorsal and its inhibitor Cactus are expressed in R cells. To test the hypothesis that Dorsal could function in R-cell targeting, we examined dl mutants.

No defects were observed relative to controls not shown. To use a more sensitive assay for R2—R5 axons, ro-tau-lacZ was tested in animals carrying heteroallelic combinations of five different alleles or deficiencies of dl , including dl C , dl D , dl 1 , dl 4 , and Df 2L TW However, none of the allele combinations showed R2—R5 axon targeting defects.

These findings suggested to us that dl alone is not required for correct axon targeting in the Drosophila visual system. Mutations of pll , though not other members of the dorsal signaling pathway, cause photoreceptor mistargeting. No R2—R5 axon targeting defects are observed in dl or Dif hemizygotes. L At higher magnification, many thickened axon bundles penetrate the lamina along its entire length.

Since Dorsal is most closely related to Dif, and because Dif has been shown previously to be expressed in the larval nervous system C antera et al. Several studies have reported that Dorsal and Dif exhibit partial functional redundancy in innate immunity and hematopoiesis, where double mutants of Dif and dl can be rescued by expressing either Dif or Dorsal L emaitre et al.

To test this possibility in the visual system, we examined the small deficiency Df 2L J4 , which deletes both genes the dl and Dif genes lie next to one another at the cytological location 36C Those that survive to third instar are exceedingly rare, presumably due to compromised innate immunity. In addition, we examined the projection pattern of Df 2L J4 -deficient mutant R-cell axons in an otherwise heterozygous or wild-type target region by using genetic mosaic analysis.

Together, the data indicate that neither Dif nor dl is required for axon targeting at these stages. Since misexpression of dl using GMR-GAL4 leads to axon guidance defects, we hypothesized mutations of cactus might mimic this effect. To test the requirement for Cactus in correct R-cell axon targeting, heteroallelic combinations of cactus mutations were analyzed with ro-tau-lacZ.

Several different combinations of cactus alleles and deficiencies were analyzed. It is therefore interesting to observe that, with respect to axon targeting, loss of cactus is not equivalent to the overexpression of Dorsal. We also found no targeting defects of R2—R5 axons in mutants of the receptor Toll or the adaptor protein Tube. In embryonic dorsal—ventral patterning the kinase Pelle plays a pivotal role in transmitting signaling from the Toll receptor to Dorsal. When they occur, these ectopic projections in control animals mostly penetrate through the center of an otherwise smooth lamina Figure 5I.

In contrast, over half of the hemispheres from pll mutants showed a mistargeting defect that was both qualitatively and quantitatively distinct from controls. Thickened bundles of axons along the entire length of the lamina were observed to penetrate into the medulla Figure 5, J and K , while the lamina itself was not smooth and somewhat irregular. Based on these criteria, and blind to genotype, we examined a larger number of animals and scored the number of phenotypically abnormal hemispheres in pll mutants and controls.

These results indicate that the IRAK family kinase Pelle is required for correct targeting of R-cell axons during larval development. We conducted a screen using the GAL4-UAS misexpression system to identify novel molecular mechanisms underlying axon guidance and targeting.

We generated a collection of lines that carry independent GS P -element insertions. GS is a particularly potent UAS-based vector, seven times more effective than related elements such as EP R orth , and each line in our collection was preselected for having lethal effects when misexpression was directed to the entire embryonic nervous system. During our prescreen we scored lethality at eclosion.

Therefore, it remains unclear whether embryonic neural misexpression is sufficient to explain the lethality in every GS line. Nevertheless, our screen for neuronal morphology using Fas2 would indicate that our prescreened collection of lines represents a significant enrichment for genes likely to disrupt neuronal morphology or function.

Compared with other misexpression screens whose hit rate was considerably lower A bdelilah -S eyfried et al. While providing added potency, the bidirectional nature of the GS element stipulates that genes located on either side of the insertion may be responsible for the misexpression effect, and that the culprit cannot be accurately predicted based on insertion site alone. In many cases it will take both molecular and functional analyses, such as those described here for GSd, to convincingly demonstrate the causal gene.

This is because many of the insertions sit between loci, and because GS can often initiate transcripts at considerable distances from annotated gene promoters.

In our screen we identified 42 genes that can influence patterning of the Fas2-positive axons. Many of these genes encode proteins thought to be secreted, membrane-associated, and cytoplasmic, though most encode nuclear proteins. Perhaps this is not surprising since nuclear factors may influence the expression of multiple downstream effectors, thereby increasing the probability they may elicit defects of nervous system development.

The identification of leak lea , otherwise known as roundabout2 robo2 , which encodes a transmembrane receptor involved in guidance of axons in response to the secreted guidance cue Slit, confirms that we can isolate genes involved in axon guidance with this technique. In addition, the screen also found genes that influence neurogenesis mam , H and neuronal differentiation ttk , pnt , mirr.

Determining whether any of the uncharacterized genes identified in our screen are also required for axon guidance or neural differentiation will require further experimentation. Here we have focused on the characterization of line GSd Misexpression of GSd caused the axons of motor neurons and R cells to bypass their correct termination sites.

In R cells, we have found that these targeting defects are a consequence of misexpression of Dorsal, and do not appear to result from changes of R-cell fate determination.

The EMS-induced mutation dl C provides evidence that the activity of Dorsal in mistargeting axons is likely to occur through its well-established role as a nuclear transcription factor, since dl C encodes a DNA-binding domain mutant. Interestingly, the number of mistargeted axons resulting from GSd was far higher than caused by misexpression of UAS-dl-A , and this correlated with elevated expression of Dorsal in distal R-cell axons and their terminals.

Perhaps the targeting of Dorsal to distal axons is simply a consequence of high levels of expression, but we do not favor this idea since Dorsal protein accumulation in cell bodies caused by misexpression of UAS-dl-A was comparable to that caused by GSd and should therefore have been readily detectable in axon terminals.

Alternatively, it is possible that the endogenous dl transcript driven by the GS insertion contains noncoding regulatory elements that promote expression or targeting in distal axons. In support of the idea that mistargeting correlates with the localization of Dorsal to distal axons, we found substantial targeting defects caused by misexpression of an epitope-tagged version of Dorsal-B UAS-dlB-HA , Figure 3, B and D , which has no nuclear localization sequence and can be readily observed along the entire length of R-cell axons E.

M indorff , J. Y ang and D. Misexpression of Dorsal has been shown to influence neuron number and axon growth and guidance in the mushroom bodies of the brain N icolai et al. In addition, misexpression of Cactus reduces the number of olfactory neurons in the fly antennal lobe Z hang et al. Despite our findings that Dorsal and Cactus are expressed in R cells, and that Dorsal misexpression has dramatic consequences on axon targeting, our studies of dl , Dif , and Relish mutations found no requirement for any of these genes in this process.

We also found that cactus mutants did not phenocopy the effect of Dorsal misexpression, suggesting that mutation of this cytoplasmic inhibitor is not sufficient to activate endogenous Dorsal and elicit mistargeting.

However, we did find that the effects of Dorsal misexpression can be enhanced in animals heterozygous for a cactus mutation E. M indorff and D. As yet, no direct role for the Dorsal pathway in regulating axon guidance has been shown in Drosophila.

In Toll and dl mutants, motor neurons innervate incorrect targets, although this effect is thought to result from cell non-autonomous functions of these genes in muscles or glia H alfon et al. It is interesting to note that both Dorsal and Cactus specifically localize to synapses of the larval neuromuscular junction, where immunoreactivity diminishes in response to either electrical stimulation or glutamate administration B olatto et al.

It is also notable that, in Drosophila, Dif and Cactus are expressed throughout the nervous system and at high levels in mushroom bodies, the learning and memory center of Drosophila C antera et al. At present, we can only speculate on the mechanism by which it influences R cells. It has been proposed that, during embryonic patterning, Pelle not only activates signaling to Dorsal but also provides negative feedback to the Toll receptor to dampen the signaling response T owb et al.

In R cells, perhaps Pelle provides negative feedback to prevent inappropriate activation through Dorsal signaling; this may explain why mutations of pll mimic the effects of Dorsal misexpression.

It is interesting to note that Pelle and Dorsal have been shown to physically interact in yeast two-hybrid assays E dwards et al. It is possible that misexpression of Dorsal sequesters Pelle from functional, physiological interactions with other proteins. The mechanism by which Pelle regulates layer-specific targeting of photoreceptor axons remains to be determined, and further analyses of candidate genes found in our screen will be required to identify additional novel regulators of nervous system development in vivo.

The authors thank John Thomas, in whose lab the misexpression screen was initially conceived and conducted while D. Read article at publisher's site DOI : Curr Biol , 29 17 Sci Rep , 8 1 , 23 Aug PLoS Genet , 11 10 :e, 16 Oct Neuroscience , , 07 Feb Cited by: 6 articles PMID: PLoS One , 9 5 :e, 15 May This data has been provided by curated databases and other sources that have cited the article.

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Abstract To identify novel regulators of nervous system development, we used the GAL4-UAS misexpression system in Drosophila to screen for genes that influence axon guidance in developing embryos. We found that one GS line drove misexpression of the NF-kappaB transcription factor Dorsal, causing motor axons to bypass their correct termination sites.

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Talk to a real person via phone, live chat, or email. Design and copy services based on consumer behavior. Print Almost Anything. Campuzano , T. Koda , J. Modolell and M. Barbacid , Dtrk , a Drosophila gene related to the trk family of neurotrophin receptors, encodes a novel class of neural cell adhesion molecule.

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Cell Biol. Xiong , W. Montell , tramtrack is a transcriptional repressor required for cell fate determination in the Drosophila eye. Montell , Defective glia induce neuronal apoptosis in the repo visual system of Drosophila. Neuron 14 : — Yang , J. Steward , A multimeric complex and the nuclear targeting of the Drosophila Rel protein Dorsal. USA 94 : — Petritsch , I. Clark , E. Gavis , L. Jan et al. Zhang , D. Zhou , C. Yin , W.

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Sign In. Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. Graduate Program in Neurological Sciences. Oxford Academic. Google Scholar. David D O'Keefe. The Salk Institute for Biological Studies. Centre for Research in Neuroscience.

Jennie Ping Yang. Yimiao Ou. Shingo Yoshikawa. Donald J van Meyel. E-mail: don. Select Format Select format. Permissions Icon Permissions. Abstract To identify novel regulators of nervous system development, we used the GAL4-UAS misexpression system in Drosophila to screen for genes that influence axon guidance in developing embryos. Figure 1. Open in new tab Download slide.

Fas2 bundles in the VNC were:. Motor nerves that were:. Closest gene. Missing or disrupted. Diffuse or less distinct. Misplaced within connective. Crossing the midline. Missing or thinned. Stalled or misrouted. Other observations. ISN, intersegmental nerve; SN, segmental nerve.

Open in new tab. Proposed site of protein activity. Molecular function. Demonstrated role in Drosophila neural development. Figure 2. Figure 3. Here you will find an overview of all cookies used. You can give your consent to whole categories or display further information and select certain cookies.

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