Guanine nucleotides are key players in mediating growth-cone signaling during neural development. The supply of cellular guanine nucleotides in animals can be achieved via the de novo synthesis and salvage pathways. The de novo synthesis of guanine nucleotides is required for lymphocyte proliferation in animals. Whether the de novo synthesis pathway is essential for any other cellular processes, however, remains unknown. In a search for genes required for the establishment of neuronal connectivity in the fly visual system, we identify the burgundy (bur) gene as an essential player in photoreceptor axon guidance. The bur gene encodes the only GMP synthetase in Drosophila that catalyzes the final reaction of de novo GMP synthesis. Loss of bur causes severe defects in axonal fasciculation, retinotopy, and growth-cone morphology, but does not affect photoreceptor differentiation or retinal patterning. Similar defects were observed when the raspberry (ras) gene, encoding for inosine monophosphate dehydrogenase catalyzing the IMP-to-XMP conversion in GMP de novo synthesis, was mutated. Our study thus provides the first in vivo evidence to support an essential and specific role for de novo synthesis of guanine nucleotides in axon guidance.

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THE establishment of neuronal connections in the developing embryo involves the differentiation of neuron precursor cells, the directed growth of axons from developing neurons, and the selection of specific target cells by the growing axons. The growth and targeting of an axon is directed by its growth cone, an expanded structure located at the leading edge of the growing axon. The growth cone expresses cell-surface receptors that recognize and integrate the guidance signals present along the path of axonal projection (TESSIER-LAVIGNE and GOODMAN 1996; GUAN and RAO 2003). Subsequently, the activation of the growth-cone receptors triggers a cascade of signaling events that ultimately converts guidance signals into directed motility (TESSIER-LAVIGNE and GOODMAN 1996; GUAN and RAO 2003).

Accumulated evidence points to the importance of purine nucleotides in mediating growth-cone signaling during development. For instance, GTP is required for the activation of Rho-family small GTPases, which are key players in mediating the reorganization of cytoskeletal structures in the growth cone (LUO 2000; DICKSON 2001). Pharmacological studies demonstrate that changes in the levels of cAMP and cGMP activities can switch the response of a growth cone to an extracellular cue (SONG et al. 1997, 1998; NISHIYAMA et al. 2003). For instance, the ratio of cAMP to cGMP activities has been shown to determine if a Xenopus spinal neuron growth cone makes an attractive or a repulsive response to netrin-1 in vitro (NISHIYAMA et al. 2003). A higher ratio of cAMP/cGMP signaling favors attraction, while a lower ratio favors repulsion. Genetic studies in Drosophila also demonstrate that cAMP/PKA signaling antagonizes the Semaphorin-induced repulsive response via Nervy, a PKA anchoring protein, which binds directly to the cytoplasmic domain of the Semaphorin receptor PlexA (TERMAN and KOLODKIN 2004).

The cellular level of guanine nucleotides in animals can be maintained through the de novo and salvage pathways. The de novo synthesis of guanine nucleotides involves a number of reactions that assemble the guanine ring from precursors, including amino acids, carbon dioxide, and tetrahydrofolate derivatives (ZALKIN and DIXON 1992). In contrast, the salvage pathway synthesizes guanine nucleotides by utilizing the available free guanine bases generated by the degradation of nucleic acids and nucleotides (ZOLLNER 1982). Energetically, the salvage pathway appears to be much less costly than de novo synthesis and is generally believed to be the principal pathway for the supply of guanine nucleotides in animals. While the de novo synthesis of guanine nucleotides has been shown to play a role in the proliferation of leukocytes (EUGUI et al. 1991; DAYTON et al. 1992; HAUSER and STERZEL 1999; GU et al. 2000, 2003), it is unknown if de novo synthesis is essential for any other biological processes when the food supply is sufficient. In this study, we show that the de novo synthesis of guanine nucleotides is specifically required for the pathfinding of photoreceptor (R cell) axons in the developing Drosophila visual system.

In the Drosophila adult visual system, R cells in the retina project axons directly into the optic lobe of the brain (MEINERTZHAGEN and HANSON 1993). The formation of the R-cell-to-optic-lobe connection pattern begins at the third-instar larval stage when differentiating R cells in the eye-imaginal disc send out axons through the optic stalk into appropriate topographic locations in the developing optic lobe (CLANDININ and ZIPURSKY 2002; TAYLER and GARRITY 2003). Different subclasses of R cells (i.e., R1–R6, R7, and R8) within each ommatidium establish connections with different target layers in the optic lobe: R1–R6 axons terminate within the superficial lamina layer, while R7 and R8 axons project through the lamina and innervate two distinct sublayers (i.e., M6 and M3, respectively) in the medulla.

In a search for genes that are required for establishing R-cell connectivity, we found that the bur gene is specifically required in R cells for the guidance of R-cell axons in the developing optic lobe. bur encodes an evolutionarily conserved GMP synthetase that catalyzes the final reaction of the de novo GMP synthesis. These findings support an essential requirement for the GMP de novo synthesis in axon guidance during neural development.

Genetics:

Bur¹, l(2)k07130 (bur^P), UAS–RacN17, UAS–RacL89, ras¹¹, ras^G0002, ras^G0436, and Df(2L)TW161 were obtained from the Bloomington Drosophila Stock Center. ES2-2^e10 (bur^e10) was provided by Gerald Rubin (NEUFELD et al. 1998). To generate single bur mutant R-cell axons, hsFLP, UAS–mCD8::GFP, elav–GAL4 (C155); bur^P, FRT40A/Bc flies were crossed with Tub–GAL80, FRT40A flies. The progeny were heat shocked at 37° for 1 hr at larval stage to induce mitotic recombination. To express the UAS–bur under control of the eye-specific driver GMR–GAL4 in bur mutants, GMR–GAL4, bur^e10/Bc flies were crossed to bur^e10/Bc; UAS–bur flies. The R-cell projection pattern in GMR–GAL4, bur^e10/bur^e10; UAS–bur/+ was compared to that in GMR–GAL4, bur^e10/bur^e10 larvae. To express the UAS–bur under control of the neuronal-specific driver elav–GAL4 in bur mutants, elav–GAL4; bur^e10/Bc flies were crossed to bur^e10/Bc; UAS–bur flies. The R-cell projection pattern in elav–GAL4/+; bur^e10/bur^e10; UAS–bur/+ was compared to that in bur^e10/bur^e10; UAS–bur larvae. Similar genetic schemes were used to examine the rescue of the phenotype in bur^P mutants by eye- or neuronal-specific expression of bur and also used to examine if UAS–mcm10 or UAS–Ret rescues the R-cell guidance phenotype in bur mutants. Guanine-depleted fly food was prepared similarly as described previously (JOHNSTONE et al. 1985).

Molecular biology:

The 3.3-kb fragment from the bur RE18382 EST clone containing the full-length bur cDNA was subcloned into the NotI and XbaI sites of the pUAST vector. The UAS–mcm10 construct was generated by subcloning the 2.9-kb XhoI–NotI fragment from the EST clone LD15957 (Research Genetics, Huntsville, AL) containing the full-length mcm10 cDNA into the pUAST vector. To generate the UAS–Ret construct, the 3.06-kb XhoI–NotI and 1.87-kb XhoI partial ret cDNA fragments were ligated together and subsequently subcloned into the NotI and XhoI sites of the pUAST vector. The resulting UAS constructs were introduced into flies to generate transgenic lines by using standard methods.

Histology and immunohistochemistry:

Eye–brain complexes from third-instar larvae were dissected and stained as described (RUAN et al. 1999). Primary antibodies were used at the following dilutions: mAb 24B10 [1:200 dilution, Developmental Studies Hybridoma Bank (DSHB)], Bar (1:200 dilution), Boss (1:2000 dilution), Elav (1:10 dilution), Repo (1:10 dilution, DSHB), and ß-galactosidase (1:1000 dilution). The secondary antibodies (i.e., HRP-, Texas-red-, and FITC-conjugated goat anti-rabbit or anti-mouse secondary antibodies) (Jackson Immunochemicals, West Grove, PA) were used at 1:200 dilution. Epifluorescent images of Repo and ß-galactosidase double staining were captured using a high-resolution fluorescence imaging system (Canberra Packard) and analyzed by two-dimensional deconvolution using MetaMorph imaging software (Universal Imaging, Brandywine, PA).

The severity of the R-cell hyperfasciculation phenotype was quantified by counting the number of separate and distinct R-cell axon bundles that were located between lamina and medulla. In bur mutants, this number decreased dramatically due to the hyperfasciculation of axon bundles.

Identification and characterization of the bur gene:

In a search for genes required for R-cell axon guidance in the fly visual system, we found that larvae homozygous for each of two bur mutant alleles l(2)k07130 (bur^P) and ES2-2^e10 (bur^e10) displayed severe R-cell axon guidance defects in the optic lobe. bur^P is a P-insertion mutation, while bur^e10 is an EMS-induced mutation isolated originally as an enhancer of the rough-eye phenotype caused by misexpression of sina (NEUFELD et al. 1998). The detailed phenotypic analysis is described below.

Both bur^P and bur^e10 are allelic to bur¹ located at the cytological region 39B1–2, which has been shown previously to be auxotrophic for guanosine (JOHNSTONE et al. 1985), suggesting that the bur gene encodes for an enzyme required for the de novo GMP synthesis. Consistently, the BDGP/Celera Drosophila Genome Project predicts a gene encoding a GMP synthetase at 39B1–2, whose homologs in other species catalyze the amination of XMP to GMP at the final step of the de novo GMP synthesis. We performed plasmid rescue to determine the P-element insertion site in bur^P and found that the P element is inserted into the first exon of this GMP synthetase gene (Figure 1A), 35 bp downstream of the putative transcriptional start site. To determine the mutation site in bur^e10, we amplified the predicted coding sequence of the bur gene in bur^e10 by polymerase chain reaction (PCR) and subsequently sequenced the PCR fragments. A nonsense mutation was identified in the bur coding sequence, which leads to the premature termination of protein translation at the amino-terminal amino acid residue 61 in bur^e10. Thus, bur^e10 appears to be a genetically null allele, while phenotypic analysis indicates that bur^P is a hypomorphic allele (see below).

View larger version (45K): In this window In a new window Download PPT slide   FIGURE 1.— Molecular characterization of the bur gene. (A) Predicted genomic organization of the bur and two nearby genes mcm 10 and Ret at 39B1–2 by the BDGP/Celera Drosophila genome project. The exon–intron boundaries in the bur gene were determined by comparing the sequence of the full-length EST cDNA clone RE18382 to the genomic sequence. The P-element insertion site for burP is located within exon 1, 35 bp downstream of the putative bur transcriptional start site. In bure10, a C-to-T nonsense mutation in the third exon changes the codon for R61 into a stop codon. Open boxes, noncoding regions; solid boxes, coding regions. (B) Alignment of the Bur protein sequence with GMP synthetases in human (HIRST et al. 1994) and Escherichia coli (TIEDEMAN et al. 1985). Identical residues are boxed.

 
To further characterize the bur gene, we obtained an EST cDNA clone (i.e., RE18382) from Research Genetics. The cDNA clone was completely sequenced and compared to the bur genomic sequence available from the BDGP/Celera Drosophila Genome Project. We found that the bur gene consists of 10 exons and 9 introns (Figure 1A). Conceptual translation of the cDNA sequence reveals an open reading frame of 683 amino acids (Figure 1B). To further determine if bur is indeed the corresponding gene for the guidance phenotype in bur mutants, we subcloned the bur cDNA clone and the full-length cDNAs from the two nearby genes mcm10 and Ret into the pUAST vector to generate transgenic lines for rescue experiments. The mcm10 and Ret genes are located 244 and 2697 bp upstream of the putative bur transcriptional initiation site, respectively. The mcm10 gene encodes a protein that binds to the members of the prereplicative complex and is required for chromosome condensation (CHRISTENSEN and TYE 2003), while the Ret gene encodes an evolutionarily conserved receptor tyrosine kinase (HUEN et al. 2000; HAHN and BISHOP 2001). We found that the expression of the bur transgene, but not of mcm10 or Ret, in homozygous bur^P (data not shown) or bur^e10 mutants (see below), almost completely rescued the phenotype, confirming that the lesion in the bur gene is responsible for the R-cell guidance phenotype in bur mutants.

The bur gene encodes a GMP synthetase:

The Bur protein is highly homologous to the members of the evolutionarily conserved GMP synthetases in other species. The amino acid identity between Bur and the human GMP synthetase over the entire polypeptide is 56.8% (Figure 1B), and the percentage of additional conserved amino acid changes between the two proteins is 17%. The similarity between Bur and the Escherichia coli GMP synthetase GuaA is also highly significant with 31.2% amino acid identity and 16% conserved changes. Like the human GMP synthetase, the Bur polypeptide contains three distinct domains, including the amino-terminal glutamine amidotransferase class-I (GATase-1) domain (aa 20–230), a central region (aa 230–290) containing an ATP-binding 3-PP-loop consensus sequence, and the highly conserved carboxyl-terminal domain (aa 429–683) (Figure 1B). The GATase-1 domain in GMP synthetases is responsible for binding glutamine and catalyzing its hydrolysis. The cysteine 95 residue in this domain, conserved in all known glutamine amidotransferases, binds to glutamine and forms a glutamyl -thioester intermediate during the catalytic reaction. The PP-loop motif is found in many ATP pyrophosphatases (BORK and KOONIN 1994) and may play a role in the hydrolysis of ATP into AMP and inorganic pyrophosphate, which generates the energy to drive the amination reaction. To generate GMP by amination of XMP, the remaining carboxyl-terminal sequence may bind XMP and subsequently add to XMP the amino group resulting from glutamine hydrolysis (ZALKIN et al. 1985).

bur mutations caused severe R-cell axon pathfinding defects:

To determine the effect of bur mutations on R-cell projections, R-cell axons in homozygous bur mutants were stained using the monoclonal antibody 24B10 that recognizes Chaoptin, an R-cell-specific cell adhesion molecule (VAN VACTOR et al. 1988). In wild type (Figure 2A), each differentiating R-cell cluster or ommatidium sends out a single axon bundle consisting of eight axons toward the most posterior end of the eye-imaginal disc, where it converges with axon bundles from other ommatidia and subsequently enters the optic stalk. After exiting the optic stalk, R-cell axons project evenly over the superficial lamina layer. R1–R6 axons terminate within the lamina, and their growth cones expand significantly in size and establish a smooth termination layer. R7 and R8 axons project through the lamina into appropriate topographic locations in the medulla, where their growth cones also expand and display a characteristic "Y-like" morphology.

View larger version (65K): In this window In a new window Download PPT slide   FIGURE 2.— Mutations in the bur gene disrupted R-cell axon pathfinding. (A–C) R1–R8 axons in third-instar larvae were stained with mAb 24B10. In wild type (A), R-cell axons elaborate smooth retinotopic arrays in the lamina (la) and medulla (me). The expanded R1–R6 growth cones form a continuous layer in the lamina. R7 and R8 axons establish a smooth topographic array in the medulla, where individual expanded "Y-shaped" growth cones can be readily identified. (B) In a burP homozygote, thicker bundles (arrow) were frequently observed. Growth cones displayed irregular morphology. The array of R7 and R8 growth cones in the medulla was disorganized. (C) In a bure10 homozygote, the phenotype was much more severe. Axon bundles were much thicker than that in the burP mutant (B). Most R7 and R8 growth cones failed to expand and could not be identified individually. Crossing over of axons (arrow) was frequently observed. (D–F) Wild-type (D) or bur mutant axons (E and F) were labeled with the MARCM method (LEE and LUO 1999). (D) Labeled wild-type axons projected into appropriate topographic locations. Their growth cones expanded significantly when reaching the target region (arrow). (E) Labeled burP mutant axons in a mosaic larva did not expand their growth cones (arrow). (F) A labeled burP mutant axon turned abnormally along the dorsal–ventral axis and projected into an incorrect topographic location in medulla (arrow). Bar, 20 µm.

 
In homozygous bur^P mutant larvae (Figure 2B), R-cell axons migrated properly toward the posterior end of the eye disc and then entered the optic stalk normally. The axon tract within the optic stalk was morphologically indistinguishable from that in wild type. After R-cell axons exited the optic stalk, however, they failed to maintain an appropriate neighbor relationship. Many R-cell axons formed abnormally thicker bundles in the lamina and medulla. The retinotopic array of R7 and R8 growth cones within the medulla appeared much less organized than that in wild type. Crossing over of neighboring axon bundles was frequently observed in the medulla. R7 and R8 growth cones were less expanded and displayed irregular morphology. The phenotype in homozygous bur^e10 mutant larvae (Figure 2C) was much more severe than that in bur^P (Figure 2B). In all bur^e10 mutant individuals examined (n > 60 hemispheres), most if not all R-cell axons were present within abnormally large bundles, causing the appearance of hyper- and hypoinnervated regions (Figure 2C). The R-cell terminal field in bur mutants was much smaller than that in wild type. R7 and R8 growth cones in the medulla failed to expand, and most of them could not be identified individually. These phenotypes were not enhanced when bur^e10 was placed over the deficiency Df(2L)TW161 (100%, n > 10 hemispheres) (data not shown), consistent with the fact that bur^e10 is a null allele (see above).

To determine if the R-cell projection phenotype in bur mutants reflects a cell-autonomous role for bur in R-cell axons, we used the mosaic analysis with a repressible cell marker (MARCM) system (LEE and LUO 1999) to examine the projection pattern of positively labeled homozygous bur mutant axons in otherwise heterozygous or wild-type larvae. In controls (Figure 2D), the vast majority of labeled wild-type R-cell axons project into appropriate topographic locations (40 of 41 labeled axons examined), where their growth cones expand significantly in size (36 of 41 labeled axons examined, Figure 2D). By contrast, many labeled bur mutant axons did not expand upon reaching the target region (89% penetrance, n = 38, Figure 2E), and some displayed an abnormal topographic projection pattern (16% penetrance, n = 38, Figure 2F). These results suggest a cell-autonomous role for bur in R-cell axonal projections.

bur mutations did not affect lamina-specific termination of R2–R5 axons:

To determine if the above guidance defects affect the binary lamina vs. medulla target selection of R-cell axons, we used the marker ro--lacZ to examine the targeting of R2–R5, a subset of R1–R6 axons, in third-instar bur^e10 mutant larvae. Surprisingly, although R-cell axons displayed severe pathfinding defects in the optic lobe (see above), we found that the vast majority of R2–R5 axons in bur^e10 mutants (Figure 3B), like that in wild type (Figure 3A), terminated correctly within the lamina.

View larger version (125K): In this window In a new window Download PPT slide   FIGURE 3.— bur is not required for lamina-specific termination of R2–R5 axons. (A and B) R2–R5 axons in wild type (A) and bure10 mutant (B) were labeled with the larval R2–R5 marker ro--lacZ. In wild type (A), the vast majority of ro--lacZ-labeled R2–R5 axons terminated within the lamina. Only a few (i.e., 2–5) R2–R5 axons mistarget into the medulla. In a bure10 homozygote (B), the average number of mistargeted R2–R5 axons was 6 (n = 22 hemispheres), which was not significantly different from that in wild type (A). (C and D) R-cell axons (red) and laminal glial cells (green) in wild-type (C) and bure10 (D) third-instar larvae were double stained with anti-ß-galactosidase antibody and anti-Repo antibodies, respectively. Both wild type and bure10 mutants carry a glass–lacZ transgene in which the expression of lacZ is under control of the eye-specific glass promoter (MISMER and RUBIN 1987), which allows the visualization of all R-cell axons with anti-ß-galactosidase staining. Anti-Repo recognizes a glial-specific nuclear protein. In wild type (C), glial cells (green) migrate from progenitor regions into the lamina where they are organized into two layers, the epithelial (eg) and marginal glia (mg), presenting a stop signal for the termination of R1–R6 growth cones (red) at the lamina plexus (lp). (D) In a bure10 homozygote, although glial cells (green) migrated correctly into the lamina, they appeared less organized. Bar, 20 µm.

 
Previous studies demonstrate complex interactions between R-cell axons and laminal glial cells: R-cell axons induce the differentiation and migration of laminal glial cells (PEREZ and STELLER 1996; SUH et al. 2002; DEARBORN and KUNES 2004), and conversely laminal glial cells present a stop signal for terminating R1–R6 axons within the lamina (POECK et al. 2001). To determine the potential effect of bur mutations on the development of laminal glial cells, we stained the third-instar optic lobe using a monoclonal antibody that recognizes the glial-specific nuclear protein Repo. In wild type (Figure 3C), differentiating glial cells migrate into the lamina in response to an unknown signal from R-cell axons (SUH et al. 2002), forming two layers of glial cells (i.e., epithelial and marginal glia), which in turn present a stop signal for terminating R1–R6 growth cones in the lamina (POECK et al. 2001). In bur^e10 mutants, the differentiation and migration of laminal glial cells appeared largely normal (Figure 3D). That R2–R5 axons still terminated correctly in bur mutants (Figure 3B) indicates that these laminal glial cells were still able to present correct targeting information for R1–R6 axons in the lamina. The density of glial cells in the lamina, however, was increased significantly, which was likely due to the reduction in the R-cell terminal field.

In summary, although loss of bur caused severe defects in R-cell fasciculation, retinotopy, and growth-cone morphology, it did not affect layer-specific termination of R2–R5 axons.

R-cell differentiation and patterning in the developing eye disc occurred normally in bur mutants:

The bur null allele bur^e10 was originally isolated as a strong enhancer of the sina-misexpression-induced rough-eye phenotype (NEUFELD et al. 1998). However, analysis of the overall patterning of mutant ommatidia in adult bur^e10 mosaic animals did not reveal any defect in either the organization of ommatidia or the number and positioning of R-cell cell bodies within each ommatidium (NEUFELD et al. 1998). These data argue against the possibility that the severe pathfinding phenotype in bur mutants was secondary to the defects in R-cell differentiation or ommatidial organization. To confirm this, we examined the development of the eye disc in bur mutants at the third-instar larval stage, using cell-type-specific markers including Chaoptin (R1–R8 cell bodies) (Figure 4, A and E), Elav (R1–R8 nuclei) (Figure 4, B and F), Bar (R1 and R6 nuclei) (Figure 4, C and G), and Boss (R8 cell) (Figure 4, D and H). No defect in R-cell differentiation or in the organization of R-cell clusters was detected in homozygous bur^e10 mutant eye-imaginal discs. Thus, R-cell guidance defects in bur mutants were unlikely to be due to abnormal R-cell differentiation or irregular spacing of ommatidial clusters in the developing eye.

View larger version (157K): In this window In a new window Download PPT slide   FIGURE 4.— R-cell differentiation and patterning remained normal in bur mutants. (A and E) All R-cell bodies in the third-instar wild-type (A) and bure10 mutant (E) eye disc were visualized with mAb 24B10 staining. In wild type (A), ommatidial clusters of R-cell bodies are highly organized. (E) In a homozygous bure10 mutant eye disc, R-cell clusters maintained an appropriate neighbor relationship (n > 30 discs). (B and F) Differentiating R1–R8 nuclei in the third-instar wild-type (B) and bure10 mutant (F) eye disc were stained with anti-Elav antibody, which recognizes the panneuronal nuclear protein Elav. The organization of R-cell clusters in the bure10 mutant (F) (n = 44 discs) was indistinguishable from that in wild type (B). (C and G) R1 and R6 nuclei in the third-instar wild-type (C) and bure10 mutant (G) eye disc were stained with anti-Bar antibody. No obvious defect was observed in the bure10 mutant (n = 16 discs). (D and H) R8 cell bodies in the third-instar wild-type (D) and bure10 mutant (H) eye disc were stained with anti-Boss antibody. In a bure10 homozygote (H), like that in wild type (D), each ommatidium contains only a single R8 cell (n = 14 discs). Bar, 20 µm.

 

The axon guidance phenotype in bur mutants could be rescued by cell-type-specific expression of a bur transgene:

Genetic mosaic analysis suggests a cell-autonomous role for bur in R-cell axon guidance (Figure 2, E and F). To further confirm that R-cell axonal projection defects in bur mutants were indeed caused by loss of bur in R cells, we performed cell-type-specific rescue experiments by expressing a UAS–bur transgene in homozygous bur mutants under control of the neuronal-specific elav–GAL4 or eye-specific GMR–GAL4 driver. We found that both neuronal-specific and eye-specific expression of the UAS–bur transgene almost completely rescued the guidance defects in bur mutants (compare Figure 5, B and F, to 5A; Table 1). As R cells are the only neuronal cell types in the developing eye, these results, taken together with that from genetic mosaic analysis (Figure 2, E and F), indicate that bur is specifically required in R cells for the proper guidance of R-cell axons in the optic lobe.

View larger version (109K): In this window In a new window Download PPT slide   FIGURE 5.— Rescue of the R-cell pathfinding phenotype by expressing the bur transgene in R cells. Third-instar eye–brain complexes were stained with mAb 24B10 to visualize R1–R8 axons. In a bure10 homozygote (A), R-cell axons displayed hyperfasciculation, aberrant topographic order, and growth-cone morphology. Genotype: bure10, GMR–GAL4/bure10. In a bure10 homozygote carrying a UAS–bur transgene under control of the eye-specific GMR–GAL4 driver (B), the R-cell projection pattern was indistinguishable from that in wild type (see Figure 2A). Note the appearance of expanded "Y-like" growth cones in the medulla. Genotype: bure10, GMR–GAL4/bure10; UAS–bur/+. (C and D) Enlarged views of the boxed regions in A and B, respectively. (E) In a bure10 homozygote expressing a UAS–mcm 10 transgene or a UAS–Ret transgene (data not shown), R-cell axons still displayed severe pathfinding defects. Genotype: bure10, GMR–GAL4/bure10; UAS–mcm 10/+. (F) In a bure10 homozygote carrying a UAS–bur transgene under control of the neuronal-specific elav–GAL4 driver, R-cell axons displayed a wild-type-like innervation pattern in the optic lobe. Genotype: elav–GAL4/+; bure10/bure10; UAS–bur/+. (G and H) Enlarged views of the boxed regions in E and F, respectively. Bars, 20 µm in A, B, E, and F; 5 µm in C, D, G, and H.

 

View this table: In this window In a new window   TABLE 1 Transgenic rescue of R-cell axonal hyperfasciculation phenotype in bur mutants

 
Although many bur^e10 mutants can reach the third-instar larval stage, all of them die at pupation, indicating that bur is also required in other tissues for viability. We examined the ability of the bur transgene to rescue the pupal lethality. Three independent UAS–bur transgenes were used under control of the neuronal-specific elav–GAL4 driver in the rescue experiments. We found that all three UAS–bur transgenes could substantially rescue the pupal lethality. The neuronal-specific expression of one UAS–bur transgene allowed 50% of bur^e10 homozygous mutants to reach the adult stage, while 26 and 34% survival rates were obtained by expressing another two UAS–bur transgenes. As the compound eye is not essential for viability, the rescue of the bur pupal lethality by expressing the UAS–bur transgene in postmitotic neuronal cells raises the interesting possibility that bur is also required in neurons in other tissues for the wiring of the neural network.

Raspberry is also required for R-cell axon pathfinding:

The bur phenotype may reflect a role for the de novo GMP synthesis in R-cell axon guidance. Alternatively, bur may function in other processes to regulate R-cell axonal projections. To distinguish among these possibilities, we examined if other enzymes involved in the de novo GMP synthesis are also required for R-cell axon guidance. Mutant alleles for the raspberry (ras) gene (SLEE and BOWNES 1995), encoding for inosine monophosphate dehydrogenase (IMPDH) that catalyzes the first step of the IMP–XMP–GMP conversion during de novo GMP synthesis, were analyzed for potential defects in R-cell projections. Indeed, we found that loss of ras caused an R-cell axon guidance phenotype identical to that in bur mutants using three different ras alleles (compare Figure 6, C and D, to 6B). These results indicate an essential role for de novo GMP synthesis in R-cell axon guidance.

View larger version (143K): In this window In a new window Download PPT slide   FIGURE 6.— Mutations in the ras gene caused a bur-like phenotype in R-cell axon guidance. Third-instar eye–brain complexes were stained with mAb 24B10. (A) Wild type. (B) bure10 homozygote. (C) In a rasG0002 hemizygote, R-cell axons displayed a severe axon guidance phenotype (24 of 26 hemispheres examined) that was very similar to that in bure10. (D) A temperature-sensitive ras11 homozygote (100% penetrance, n = 92 hemispheres) grown at restrictive temperature (i.e., 29°). Bar, 20 µm.

 

De novo GMP synthesis is sufficient for maintaining the GMP level required for R-cell axon guidance:

The above results establish an essential role for de novo GMP synthesis in maintaining the necessary level of GMP for R-cell axon guidance. To further determine the contribution of the GMP salvage synthesis pathway, we examined the effect of depleting guanine from fly food on R-cell projections. Wild-type fly embryos could still survive and develop into adults when fed with guanine-depleted food. In contrast, when bur^P or bur^e10 homozygous mutants were fed with guanine-depleted food, none of them could survive to the third-instar larval stage (data not shown), confirming the role of Bur in de novo GMP synthesis. Examination of the R-cell projection pattern in wild-type larvae fed with guanine-depleted food did not reveal any obvious defect (Figure 7B), suggesting that the level of GMP maintained by de novo GMP synthesis is sufficient for R-cell axon guidance.

View larger version (80K): In this window In a new window Download PPT slide   FIGURE 7.— Depleting guanine from the fly food did not affect R-cell axon guidance. Third-instar eye–brain complexes were stained with mAb 24B10. (A) R-cell projection pattern in a wild-type larva grown on normal food. (B) In a wild-type larva grown on guanine-depleted food, the R-cell projection pattern remained normal (n = 50 hemispheres). Bar, 20 µm.

 

bur interacts with Rac genetically:

After synthesis, GMP can be converted into cGMP and GTP, and both are important signaling molecules. Interestingly, Rac, the Rho family small GTPase whose activation requires GTP, has been shown previously to be required for the proper fasciculation of R-cell axons in the Drosophila visual system (HAKEDA-SUZUKI et al. 2002). To address the possibility that de novo synthesis of GMP is required for maintaining a high level of GTP that allows the proper function of Rac in R-cell axons, we examined the potential genetic interaction between bur and Rac during R-cell axon guidance. When the Rac1 dominant-negative transgene RacN17 was expressed in R cells, we observed a weak axonal hyperfasciculation phenotype (Figure 8A). This phenotype was enhanced when the dosage of bur was reduced by half (Figure 8B). Similar enhancement was observed when the dosage of bur was reduced in larvae expressing another Rac1 dominant-negative transgene RacL89 (compare Figure 8D to 8C). These results are consistent with a requirement for de novo GMP synthesis in the upregulation of Rac signaling in R-cell axons.

View larger version (148K): In this window In a new window Download PPT slide   FIGURE 8.— Reducing the dosage of bur enhanced the Rac phenotype. Third-instar eye–brain complexes were stained with mAb 24B10. (A) R-cell projection pattern in a wild-type larva expressing the dominant-negative RacN17 transgene under control of the eye-specific GMR–GAL4 driver. The average number (25 ± 5, n = 12) of clearly separated axonal bundles between lamina and medulla was decreased compared to that in wild type (31 ± 6) due to axonal hyperfasciculation. Genotype: GMR–GAL4/+;UAS–RacN17/+;. (B) In a RacN17-expressing larva in which the dosage of bur was reduced by 50%, the phenotype became more severe. Compared to that in A (25 ± 5, n = 12), the average number (21 ± 4, n = 22) of axonal bundles was decreased (P < 0.05). Genotype: GMR–GAL4, bure10/+;UAS–RacN17/+. (C) In a larva expressing the dominant-negative RacL89 transgene, the R-cell projection pattern was severely disrupted. Genotype: GMR–GAL4/UAS–RacL89. (D) In a RacL89-expressing larva in which the dosage of bur was reduced by 50%, the R-cell axonal hyperfasciculation phenotype was enhanced. Compared to that in C (9 ± 5, n = 9), the average number (5 ± 2, n = 9) of axonal bundles was significantly decreased (P < 0.05). Genotype: GMR–GAL4, bure10/UAS–RacL89. Bar, 20 µm.

 

In this study, we show that the bur gene is required for R-cell axon guidance in the fly visual system. In bur mutants, R-cell axons displayed hyperfasciculation, disrupted topographic innervation, and abnormal growth-cone morphology. Phenotypic analysis using a collection of cell-type-specific markers shows that the pathfinding phenotype was not caused by abnormal R-cell differentiation or retinal patterning. Genetic mosaic analysis and cell-type-specific rescue experiments demonstrate that bur is required in R cells. bur encodes a GMP synthetase that catalyzes the final reaction of the de novo GMP synthesis, the amination of XMP to GMP. Like bur, mutations in the ras gene encoding for IMPDH catalyzing the conversion of IMP to XMP in de novo GMP synthesis, also disrupted R-cell axon guidance. Bur displayed dosage-sensitive genetic interaction with the GTP-binding protein Rac. We propose that the de novo synthesis of GMP is essential for maintaining a high level of guanine nucleotides, which are required for growth-cone signaling during axon pathfinding.

bur has been shown previously to interact with sina (NEUFELD et al. 1998), which encodes for a transcriptional factor required for specifying cell fate determination in the fly eye (CARTHEW and RUBIN 1990). Reducing the dosage of bur enhanced the sina-overexpression eye phenotype (NEUFELD et al. 1998). This interaction, however, does not appear to reflect a relevant in vivo interaction between bur and sina for two reasons. First, unlike some other enhancers (e.g., Sin3A^es2-3) of the sina-overexpression eye phenotype identified from the same screen, bur did not display a dosage-sensitive genetic interaction with a hypomorphic sina loss-of-function allele (NEUFELD et al. 1998). And second, unlike loss of sina, loss of bur did not affect R-cell fate determination (Figure 4) (NEUFELD et al. 1998). These results argue strongly against the suggestion that Bur is a component of the Sina signaling pathway in R cells.

In mammals, de novo GMP synthesis has been shown to be required for cell proliferation during immunoresponse. Inhibitors of IMPDH, which catalyzes the conversion of IMP into XMP during de novo GMP synthesis, could block lymphocyte proliferation leading to immunosuppression (SOLLINGER et al. 1992; EUGUI and ALLISON 1993). Consistently, it has been shown that the proliferation of lymphocytes could be significantly suppressed when the genes encoding for IMPDH were disrupted in mice (GU et al. 2000, 2003). Surprisingly, no defect in cell proliferation was observed in bur and ras mutants. The size of the mutant disc and the number of differentiating R cells were indistinguishable from that in wild type. Importantly, the expression of bur in postmitotic neurons could completely rescue the R-cell guidance phenotype and also largely rescue the pupal lethality, arguing strongly against a role for Bur in cell proliferation. One likely explanation is that in the absence of Bur, the salvage GMP synthesis pathway maintains a certain level of GMP, which may be sufficient for cell proliferation during development. This level of GMP generated by the salvage pathway, however, is not sufficient for certain signaling events necessary for specific growth-cone guidance decisions.

How does the de novo synthesis of guanine nucleotides play a role in R-cell growth-cone guidance? We speculate that the de novo GMP synthesis is required for generating a certain level of GTP necessary for R-cell growth-cone signaling. The GTP-binding protein Rac, a member of the Rho-family small GTPases, has been shown to be required for R-cell axon guidance in Drosophila (HAKEDA-SUZUKI et al. 2002). Like loss of bur, loss of Rac or loss of its activator Trio also caused defects in R-cell fasciculation and growth-cone expansion (NEWSOME et al. 2000; HAKEDA-SUZUKI et al. 2002). Our results showing that bur genetically interacts with Rac during R-cell axon guidance (Figure 8) are consistent with the notion that de novo GMP synthesis is required for upregulating Rac activity. Alternatively or additionally, the de novo GMP synthesis may also be required for maintaining a certain level of cGMP in R-cell growth cones for guidance decisions. Pharmacological studies demonstrate that an increase in cGMP activity is necessary for the repulsive response of Xenopus spinal cord neuron growth cones to netrin-1 (NISHIYAMA et al. 2003). It is possible that a similar cGMP-dependent repulsive interaction exists between R-cell axons, which is necessary for maintaining an appropriate neighbor relationship between R-cell axons. If so, loss of bur could cause a decrease in the level of cGMP, which disrupts the repulsive interaction between neighboring axons, resulting in the hyperfasciculation phenotype. Future studies are necessary to determine if cGMP signaling also plays a role in R-cell axon guidance.

The human genome carries a single GMP synthetase gene (HIRST et al. 1994), which exhibits extensive homology to the fly Bur (Figure 1B). It is highly possible that the de novo GMP synthesis mediated by this enzyme is also required for the wiring of the neural network during the development of the human brain. In this context, it is notable that mutations in the gene encoding for adenylosuccinate lyase (ADSL), which is required for the de novo synthesis of both AMP and GMP (ZALKIN and DIXON 1992), cause severe mental retardation and autism (JAEKEN and VAN DEN BERGHE 1984). How the ADSL deficiency causes mental retardation and autism is still unclear. Given the specific guidance phenotype in the fly bur mutants, it will be of interest to determine if these neurological disorders in ADSL-deficiency patients are caused by malformation of neuronal connections during embryonic development. It will also be interesting to determine if any other human neurological diseases are caused by mutations in the GMP synthetase gene or genes encoding for other enzymes of the de novo GMP synthesis pathway.

We thank Don van Meyel for critical reading of the manuscript, the members of the Rao lab for helpful discussions, the Berkeley Drosophila Genome Project and Bloomington Stock Center for fly stocks, Developmental Studies Hybridoma Bank at University of Iowa for mAb 24B10 and anti-Repo antibodies, David Huen for Ret cDNAs, and Gerald Rubin for bur^e10. This work was supported by an operating grant (MOP-14688) awarded to Yong Rao from the Canadian Institutes of Health Research.

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