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The fruitless Gene Is Required for the Proper Formation of Axonal Tracts in the Embryonic Central Nervous System of Drosophila
Ho-Juhn Song1,a, Jean-Christophe Billeter2,b, Enrique Reynaud3,c, Troy Carlob, Eric P. Spana4,d, Norbert Perrimond, Stephen F. Goodwin2,b, Bruce S. Bakerc, and Barbara J. Tayloraa Department of Zoology, Oregon State University, Corvallis, Oregon 97331-2914,
b Department of Biology, Brandeis University, Waltham, Massachusetts 02454-9110,
c Department of Biological Sciences, Stanford University, Stanford, California 94305-5020
d Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115
Corresponding author: Barbara J. Taylor, 3029 Cordley Hall, Oregon State University, Corvallis, OR 97331-2914., taylorb{at}bcc.orst.edu (E-mail)
Communicating editor: T. SCHÜPBACH
 | ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The fruitless (fru) gene in Drosophila melanogaster is a multifunctional gene that has sex-specific functions in the regulation of male sexual behavior and sex-nonspecific functions affecting adult viability and external morphology. While much attention has focused on fru's sex-specific roles, less is known about its sex-nonspecific functions. We have examined fru's sex-nonspecific role in embryonic neural development. fru transcripts from sex-nonspecific promoters are expressed beginning at the earliest stages of neurogenesis, and Fru proteins are present in both neurons and glia. In embryos that lack most or all fru function, FasII- and BP102-positive axons have defasciculation defects and grow along abnormal pathways in the CNS. These defects in axonal projections in fru mutants were rescued by the expression of specific UAS-fru transgenes under the control of a pan-neuronal scabrous-GAL4 driver. Our results suggest that one of fru's sex-nonspecific roles is to regulate the pathfinding ability of axons in the embryonic CNS.
THE fruitless (fru) gene has a prominent role in developing the potential for male sexual behavior as well as adult viability and normal external morphology (
The male-specific behavioral functions of fru depend on transcripts produced from the P1 promoter that are sex-specifically spliced (
The products of the P2, P3, and P4 fru promoters differ from those of the P1 promoter in their spatial patterns of expression, their protein coding sequences, and their functions (
To gain a better understanding of the role of fru's P2, P3, and P4 transcripts in development, we determined the expression pattern of fru RNAs and proteins in the embryonic CNS. In this article, we show that fru RNAs and proteins are produced in a dynamic pattern during embryogenesis and are widely expressed in the developing embryonic CNS. Neuronal precursors as well as neurons and glial cells express Fru proteins. The embryonic CNS is an important model system for defining how specific genes govern neuronal identity and the process of axonal pathfinding necessary for the formation of proper neuronal connections (reviewed in
Analyses of fru mutants, in which specific subsets of transcripts are disrupted or in which no fru transcripts are made, revealed that Fasciclin II (FasII)-positive axons had abnormal trajectories, as did axons producing other longitudinal and commissural tracts in the CNS. Our findings suggest that fru transcripts are needed in the embryonic CNS for the formation of the wild-type pattern of axonal tracts. The defects in the FasII-positive and BP102-positive axonal tracts found in fru mutants were rescued by the expression of specific fru isoforms under the control of the pan-neuronal sca-GAL4 driver, which has a pattern of expression similar to that of Fru proteins. In contrast, these mutant phenotypes were not rescued by fru transgene expression driven by the elav-GAL4 driver, which is expressed exclusively in postmitotic neurons. The neurons that pioneer the FasII fascicles express markers appropriate to their wild-type identity in fru mutants. Our analysis suggests that fru's primary function during neurogenesis is the regulation of fasciculation and defasciculation processes involved in the growth of neuronal axons along their wild-type pathway through the CNS.
| MATERIALS AND METHODS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Fly stocks and crosses:
Canton-S flies were used as the wild-type genotype. The following fru mutant alleles were used: fru3; fru4; frusat; In(3R)fru1 (fru1); Df(3R)ChaM5 (ChaM5); Df(3R)fruw24 (fruw24); In(3R) fruw27 (fruw27); Df(3R)frusat15 (frusat15); Df(3R)fru4-40 (fru4-40); In(3R)fruw12 (fruw12); Df(3R)P14 (P14) (
A deficiency, Df(3L)XDI98 (65A02-65E1), was used to examine the phenotypes associated with the non-fru inversion breakpoint, at 65C-D, of the fruw12 chromosome. fruw12/Df(3L) XDI98 mutants were fully viable, fertile, and morphologically normal adults (n = 10 male; n = 10 female). Deficiency heterozygous males did not show courtship chaining behaviors, a typical fru mutant phenotype (n = 20, 10 males each group). Due to the similarity of phenotypes, a spread mutant allele (sprd05284;
Generation and molecular characterization of the fruAJ96u3 deficiency:
The AJ96w+ P element (
Lethal phase, phenotypic, fertility, and behavioral analysis of fruAJ96u3 mutant animals:
To characterize the fruAJ96u3 mutation, trans-heterozygotes between fruAJ96u3 and other fru alleles were examined. In those crosses in which adult fru trans-heterozygotes did not survive, the pupal stage at which the fru mutant combination died was determined from aged collections of white prepupae and by staging based on
For sterility tests, virgin males were collected a few hours after eclosion and housed as groups of 8–10 males in food vials. After 4 days, individual males were mated with 2 or 3 Canton-S virgin females and the vials were examined after 7 days for the presence of larvae and/or pupae. To determine whether males would form courtship chains (
Embryonic immunohistochemistry:
Timed embryo collections were staged by morphological criteria (
For detection of primary antibodies, we used secondary antibodies, which were directly conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA), with alkaline phosphatase (AP; Jackson ImmunoResearch), or with the fluorochromes Alexa-594, Alexa-488, or Alexa-395 (Molecular Probes, Eugene, OR). For anti-Repo labeling, a biotinylated secondary antibody was used followed by incubation with the ABC reagent (Vector Laboratories, Burlingame, CA). For some double-label experiments, colorimetric visualization of diaminobenzidine (Sigma) was nickel enhanced (
Labeled whole-mount and filleted embryos were viewed and photographed with a Sony DKC-5000 digital camera under differential interference contrast optics, using an Olympus Vanox-TX microscope. For certain double- or triple-labeling experiments, fluorescently labeled embryos were viewed on a TCS-Leica confocal microscope. Composite images were assembled in Adobe Photoshop 5.0.
In situ hybridization of embryos:
Single-strand antisense and sense riboprobes for in situ hybridization were prepared as previously described (
The protocols for fixation and in situ hybridization were according to
Generation of transformants:
Six different UAS-fru constructs were made from fru cDNAs subcloned in pBluescript KS (
The pUAST vectors also contained a mini-white reporter gene and the final transgene constructs were introduced into the Df(1)yw parental strain by germline transformation as described in 
2-3 (100 µg/ml). The chromosomal location of 3–10 transformant lines was determined. If a transformant line contained two insertions on different chromosomes, the transgenes were segregated and treated as independent lines. Each UAS-fru construct was crossed into a fruW12 or frusat15 mutant background and balanced over TM3, Sb, and P[ftz-lacZ]. At least two independent lines for each construct were tested and analyzed.
To express these various UAS-fru constructs in the CNS, lines containing scabrous-GAL4 (sca-GAL4) and embryonic lethal abnormal vision-GAL4 C155 (elav-GAL4;
Generation of Fru antibodies:
For the glutathione S-transferase (GST) fusion constructs, coding regions for the fru BTB domain and alternative ZnF A and C (Fig 1) were generated by PCR. Oligonucleotide pairs containing sites for in-frame directional cloning in the pGEX-4T-1 vector (Pharmacia, Piscataway, NJ) were designed for BTB and each unique ZnF sequence. DNA was amplified from fru cDNA clones (primer sequences for BTB domain are BTB-1-For, GGG GGA ATT CAT GGA CCA GCA ATT CTG CTT 3'; BTB-115-Rev, GGG GGC TCG AGC TAG TTG TTA TCT GTG AGA 3'; those for A form ZnF domain are A-For, 5' CCG GAA TTC CAG CAG CGC CCG CCA CC 3'; A-Rev, 5' GCC GCT CGA GCG GGA TGG GCT GCA CTT GGG C; and those for C form ZnF domain are C-For, 5' CCG GAA TTC CGC GTC AAG TGT TTT AAC ATT AAG C 3'; C-Rev, 5' CCG CTC GAG GTT TGC TTG ATT CTT GGT TAC TTA G 3'), digested with EcoRI and XhoI enzymes, and cloned into the polylinker of the pGEX-4T-1 vector. Individual recombinant clones were validated by sequencing.
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Fusion proteins were purified according to
Statistics:
The frequency of defective embryos in various fru mutant and wild-type embryos was analyzed statistically by one-way ANOVA (SAS program version 6.12; SAS Institute) and post hoc analyzed by Tukey HSD comparisons or by two-sample t-test (SAS program version 6.12).
| RESULTS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Isolation and characterization of a new fru null mutation:
The AJ96w+ P element, which labels a pair of midline neurons in each segment of the embryonic CNS, was mapped molecularly to 
3 kb downstream of the 3' end of the fru locus (Fig 1; MATERIALS AND METHODS;
We mobilized this P element and generated a deficiency line, Df(3R)AJ96U3 (fruAJ96u3). This deletion extends from sequences within the AJ96w+ P element to sequences between the P3 and P4 promoters, thus deleting all fru common coding sequences (Fig 1; see MATERIALS AND METHODS). As the smallest available deficiency of the fru locus, it appears to remove fru and only one other putative open reading frame located between the 3' end of the fru locus and the AJ96w+ P element (29,144–27,188 bp, accession no. AE003722;
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Temporal and spatial distribution of fru mRNA during embryogenesis:
To determine the spatial and temporal distribution of embyonic fru transcripts, we performed in situ hybridizations with antisense-BTB and antisense-Com riboprobes, which detect most or all fru transcripts (
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The fru locus encodes a complex set of transcripts (Fig 1;
An additional complexity in fru transcripts reflects alternative splicing at the 3' end, which generates transcripts containing one of three different pairs of Zn-finger domains (Fig 1;
To confirm that we were detecting authentic fru transcripts, fru mutant embryos, fruw24/fruw24 and frusat15/frusat15, in which the coding regions of the fru gene are deleted, were labeled with the antisense-BTB, antisense-Com, and antisense-A 3' end riboprobes. At the beginning of embryogenesis, we detected transcripts in these null mutant embryos only up to stage 5; later stage embryos were not labeled (Fig 2C and Fig F). By contrast, fru transcripts in wild-type embryos are present up to stage 16 (Table 2A). From these results, we infer that fru transcripts in very early mutant and wild-type embryos are maternally derived, but that the transcripts found only in older wild-type embryos are zygotically generated. The presence of maternal and zygotic fru transcripts was consistent with the results of Fru protein immunohistochemistry (see below).
The temporal and spatial expression of Fru protein during embryogenesis:
To determine whether the fru transcripts present during embryogenesis are translated, embryos were labeled with antibodies directed against the common coding regions of Fru proteins (anti-FruCOM and anti-FruBTB') and against the class A 3' end (anti-FruA') and the class C 3' end (anti-FruC'; see MATERIALS AND METHODS). We found that the localization of Fru proteins during embryogenesis is largely consistent with the spatial and temporal RNA distribution described above (Table 2A). Before neuroblast delamination, mesectodermal cells (st 9), but not neuroectodermal cells, were labeled with both anti-FruCOM and anti-FruBTB' antibodies (Fig 2E; Table 2B). All delaminating neuroblasts and their progeny appear to express Fru proteins (st 9–10; Fig 2J). Ganglion mother cells (GMCs) located in the anterior regions of each segment label more strongly for Fru proteins than do those in the posterior regions (st 10; Fig 2K). At later stages (st 11–16), many cells in the CNS were strongly labeled (Fig 2L).
To determine whether Fru proteins were expressed in neurons and/or glia of stage 13–16 embryos, we carried out double- and triple-labeling experiments with anti-Elav antibodies to identify Fru-positive neurons and with anti-Repo antibodies to identify Fru-positive lateral glia (Fig 3A and Fig B). From a detailed comparison of double- (n = 5) and triple-labeled (n = 3) CNSs, it appeared that all Elav-positive cells in the CNS coexpress Fru proteins, even though there were variable levels of Fru protein expression in individual cells (Fig 3C). Likewise, all Repo-positive cells in the CNS were also Fru positive (n = 5, Fig 3C). In these experiments, some midline cells were only Fru positive (arrows, Fig 3C and Fig F). By their location, these cells are likely to be midline glia, which do not express Repo protein (
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We used antibodies specific to either the A or the C ZnF carboxy termini found in Fru isoforms to determine whether cells have Fru proteins with only one or both of these isoforms (see MATERIALS AND METHODS). The pattern of cells labeled with anti-FruA' is very similar to the spatial and temporal pattern of cells detected with anti-FruCOM and anti-FruBTB' (Table 2B). In contrast, in young embryos (st 1–11) the anti-FruC' labeling pattern is similar to the pattern of cells stained with anti-FruCOM and anti-FruBTB', whereas at later stages (st 12–16), fewer cells were labeled with anti-FruC' than with anti-FruCOM and anti-FruBTB'. By double- and triple-labeling experiments in stage 16 embryos, all Elav-positive neurons, Repo-positive lateral glia, and putative midline glial cells were FruA positive (n = 4, Fig 3, D–F). However, all neurons, but only some Repo-positive glia, were FruC' positive (n = 5, Fig 3, G–I). These results suggest that all neurons and some lateral glia contain Fru isoforms having at least two different carboxy termini, but that some lateral glia and midline cells contain Fru proteins that may have only one type of carboxy terminus. Currently, no antibody specific to the B carboxy terminus is available.
In contrast to the findings from the in situ hybridization experiments, Fru proteins were detected in neurons and glial cells in the PNS by anti-FruBTB', anti-FruCOM, anti-FruA', and anti- FruC' immunohistochemistry (Fig 3, J–L). In double- and triple-labeling experiments, some Fru-positive cells, labeled with anti-FruBTB' or anti-FruCOM antibodies, were Elav positive and by location were judged to be mechanosensory and chordotonal organ neurons as well as the more internal multidendritic neurons. Another population of Fru-positive cells were Repo-positive and thus were identified as peripheral glial cells (Fig 3J; Table 2B). However, double- and triple-labeling experiments using anti-FruA' and anti-FruC' antibodies showed that only a subset of the Fru-positive sensory neurons and peripheral glia had Fru proteins with the A and C carboxy termini, from which we infer that some of these cells have Fru proteins with the B carboxy terminus (Fig 3K and Fig L; Table 2B).
Fru proteins are also expressed in other embryonic tissues. The posterior epidermis (st 13–16) and most body wall muscles (st 14–16, Table 2B) were Fru positive by anti- FruBTB' and anti-FruCOM labeling. These tissues were labeled with anti-FruC' but not with anti-FruA' antibodies (Table 2B). These findings suggest that Fru protein isoforms with specific ZnF domains have a tissue-specific pattern in non-CNS tissues. The presence of Fru proteins in cell types that were not labeled in the in situ hybridization experiments, such as the PNS or muscle, may be due to differences in sensitivity of the molecular probes used and/or the relative levels of fru transcripts and proteins in these different cell types.
To be certain that authentic Fru proteins were being labeled in wild-type embryos, fru mutant embryos, fruw24/fruw24 and frusat15/frusat15, which are deleted for the fru gene, were labeled with anti-FruBTB', anti-FruCOM, anti-FruA', and anti-FruC' antibody (Table 2B). At early embryonic stages (st 1–5), the pattern of Fru protein expression in null mutant embryos was comparable to that in wild-type embryos. At later stages (st 6–16), no Fru proteins were detected in null mutant embryos (Fig 2F), even though there was still robust labeling in wild-type embryos. These results indicate that the earliest transcripts present, which we infer to be maternal in origin, are translated in very early stage embryos and that Fru proteins in stage 6–16 embryos are largely or exclusively derived from zygotic fru transcripts.
Neurons in the embryonic CNS and PNS are labeled in fru P-element lines:
To determine whether P elements inserted within or near the fru locus behave as enhancer traps of the embryonic pattern of fru expression, we examined ß-galactosidase activity in fru3, fru4, and frusat mutant and AJ96w+ embryos (Fig 1;
In fru P-element mutant and control sibling embryos, cells in the CNS and PNS were labeled by anti-ß-galactosidase. Similar temporal and spatial labeling patterns were observed in embryos from the fru3 and fru4 lines. In embryos from these lines, anti-ß-galactosidase expression was detected in the mesectoderm (st 8), delaminating neuroblasts (st 9; Fig 4A and Fig B), and a variety of smaller cells, which appear by their location to be GMCs (st 10), neurons (st 11), and lateral glia and midline cells (st 12; Fig 4C). ß-Galactosidase labeling persisted in these CNS cells until stage 16. In the embryonic PNS, sensory organ precursors (st 10) followed by the external and chordotonal sensory neurons (st 12–16) were ß-galactosidase positive (Fig 4C). In addition, tracheal cells (st 11) and epidermal cells in the posterior part of each hemisegment (st 13–16) were labeled (Fig 4C). By contrast, in embryos from the frusat line, no ß-galactosidase-positive cells were found in the embryonic CNS, and only the chordotonal neurons in the PNS were labeled (data not shown). In summary, these results indicate that ß-galactosidase expression from the P elements inserted in fru3 and fru4 lines largely replicates fru's pattern in the embryonic CNS and PNS.
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We also examined the expression pattern of the enhancer trap line AJ96w+. Previous studies have shown that the MP2 neuroblast and its progeny, the dorsal and ventral midline precursor neurons (dMP2 and vMP2), express ß-galactosidase in the AJ96w+ line (
fru function is required for the formation of axonal tracts within the CNS:
Given the findings that the fru gene is widely expressed in the embryonic CNS, we expected that fru mutant embryos would have defects in CNS development and used two antibodies, anti-FasII and mAb BP102, to assay axonal projections within the CNS. FasII is a neural adhesion molecule expressed on the cell surface of axons forming specific longitudinal fascicles or tracts running throughout the entire ventral nerve cord and into the brain (Fig 5A;
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FasII-positive tracts are abnormal in fru mutants lacking all or most fru transcripts. Between 12 and 25% of fru null mutant embryos (e.g., frusat15/fruAJ96u3) had abnormal FasII-positive tracts (Fig 5B and Fig C; Table 3D). By comparison, <3% of wild-type embryos had any segments with disrupted FasII tracts (Table 3A). In all fru null mutant CNSs, FasII-positive axons no longer formed distinct tracts in one or more adjacent hemisegment, suggesting that these axons had defasciculated from other axons within the tracts. In some cases, axons that had defasciculated crossed and joined an adjacent fascicle or approached and crossed the midline (Fig 5C). In other cases, the left and right medial tracts appeared to merge along the midline.
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To determine whether many or most axonal tracts were disrupted in fru mutants, we labeled the longitudinal connectives and commissures with the BP102 antibody (Fig 5D;
To demonstrate that these FasII and BP102 axonal defects depended on the loss of fru function, we used the fruw12 allele, which has a chromosomal break within the fru locus, in combination with fru deletion mutations (Fig 1; Table 3C). We found that 15–23% of these mutant embryos had defects in their FasII and BP102 tracts; the frequency and the severity of the defects were similar to those found in fru null embryos (Fig 5B and Fig C). The fruw12 allele is caused by a chromosomal inversion, and to rule out the possibility that the non-fru inversion breakpoint contributes to the FasII mutant phenotype, we examined fruw12/Df(3L)XD198 mutants and found that the pattern of FasII fascicles was wild type (n = 10; data not shown; see MATERIALS AND METHODS). These results show that it is the loss of fru function that causes the axonal defects in FasII and BP102 tracts (Table 3C and Table 3D). The chromosomal break in the fruw12 allele in the fru locus separates the P1, P2, and P3 promoters from the fru coding region but leaves the P4 fru transcription unit intact (
To further define which fru transcripts are required for the formation of FasII and BP102 tracts, we examined fru mutants in which P1 transcripts are affected or where P1 and P2 transcripts are eliminated (e.g., fru4-40/frusat15; Table 3B;
fru function is required for wild-type orientation of pioneering axonal projections:
If the axonal pathfinding defects in fru mutants are due to the loss of fru function in neuronal precursors or in neurons themselves, then there are two likely explanations for the altered axonal trajectories in these mutants. One explanation is that neurons have not adopted, or only partially adopted, their wild-type identity in fru mutants and thus their axons fasciculate with different axonal partners as they grow in the CNS. An alternative explanation is that neurons in fru mutants adopt their wild-type identity but are unable to carry out their normal program of axonal pathfinding and differentiation. To distinguish between these two possibilities, we used neuronal and axonal markers to identify the earliest developmental abnormalities in fru mutants.
To assess whether fru played a role in neuroblast delamination and identity, we first labeled developing neuroblasts with antibodies to the Hunchback (Hb) protein, which labels all delaminating neuroblasts by early stage 9 in wild-type embryos (Fig 6A, n = 10). Fewer neuroblasts were labeled with anti-Hb antibody in early stage 9 fruw12/frusat15 embryos than in wild type, but neuroblasts in all three rows did become Hb positive in late stage 9 embryos (Fig 6B and Fig C; n = 10 both stages). Thus, the final pattern of Hb expression was wild type in these fru mutant embryos but there was a slight temporal delay in either neuroblast delamination itself or the onset of Hb expression in delaminating neuroblasts.
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We next examined the development of neurons in fru mutants that pioneer the FasII fascicles to better assess whether there were changes in neuronal identity or early defects in axonal differentiation. In the wild-type CNS, pCC, vMP2, dMP2, and MP1 axons initiate the formation of the medial and intermediate FasII tracts within each segment (
We therefore examined the development of aCC, pCC, vMP2, and dMP2 neurons in frusat15/fruAJ96u3 and fruw12/fruAJ96u3 embryos. Along the midline, the MP2 precursor expresses the Odd-skipped protein and upon division the dMP2 daughter cell maintains Odd-skipped expression while the vMP2 daughter downregulates Odd-skipped expression (
To assess the early outgrowth of axons from these neurons as they begin to differentiate their axons, we labeled neurons with markers to assess axonal outgrowth. The mab22C10 antibody recognizes the futsch protein, which labels the vMP2 and dMP2 axons and is needed for the normal outgrowth of axonal processes (Fig 6D;
fru function is not required for lateral and midline glial cell survival:
The axonal phenotypes in fru mutant embryos might also result from the loss of midline or lateral glial cells, both of which have been shown to be necessary for the formation of normal axonal tracts and to express Fru proteins (Fig 3C, Fig F, and Fig I;
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The expression of specific UAS-fru transgenes rescues mutant defects in the CNS of fru mutant embryos:
The phenotypic analysis of fru mutant embryos along with fru's temporal and spatial expression pattern suggests that the fru gene functions during axonal outgrowth. To positively demonstrate fru's role in the CNS, we rescued fru mutant defects by the expression of UAS-fru transgenes controlled by a pan-neuronal driver, scabrous (sca)-GAL4 (
The expression of UAS-fruA and UAS-fruC transgenes under the control of a sca-GAL4 driver was sufficient to rescue the defects in FasII and BP102 axonal tracts in fru mutant embryos (Fig 8A, Fig C, Fig D, and Fig F; Table 4). Embryos from two lines with independent UAS-fruA and UAS-fruC insertions in the fruw12/frusat15 mutant background were examined (Fig 8A and Fig C; Table 4). In both lines of sca-GAL4/UAS-fruA; fruw12/frusat15 embryos, the wild-type BP102 pattern was also restored (Fig 8D; Table 4). However, the BP102 pattern was rescued in embryos from only one of the UAS-fruC; sca-GAL4; fruw12/frusat15 lines (Fig 8F); in the other line, 33% of fru mutant embryos had an abnormal BP102 pattern (Table 4).
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In contrast to the results obtained from expressing the UAS-fruA and UAS-fruC transgenes, the FasII and BP102 mutant phenotypes were more severe in the CNSs of sca-GAL4/UAS-fruB; fruw12/frusat15 embryos than in the CNSs of the corresponding fru mutants (Table 3C and Table 4). The FasII labeling in the CNS of these embryos was diffuse, indicative of highly defasciculated axonal projections, and axons frequently crossed between fascicles or across the midline (Fig 8B; Table 4). Not surprisingly, the BP102 pattern in both lines of sca-GAL4/UAS-fruB; fruw12/frusat15 embryos was also severely disrupted (Fig 8E; Table 4). Sibling embryos, sca-GAL4/UAS-fruB; fru-/fru+, also had had defective FasII and BP102 axonal tracts (95% embryos, n = 20, figure not shown). Taken together, these results indicate that the sca-GAL4-driven expression of UAS-fruB may be exerting a dominant negative effect on the development of these FasII and BP102 axonal phenotypes. In addition, the defects in mab22C10-positive axons in the dMP2 and vMP2 pioneer neurons in UAS-fruA, UAS-fruB, or UAS-fruC fruw12/frusat15 mutants were not rescued (n = 50 hemisegments of fru mutant embryos expressing each construct; data not shown).
To determine whether Fru function in postmitotic neurons might be sufficient to restore wild-type FasII tracts in fru mutants, we used an elav-GAL4 driver to express the UAS-fru transgenes (
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The male-specific transcripts of fru derived from the P1 promoter encode an additional N-terminal extension to the common coding sequences of most fru transcripts but are not expressed in the embryo (Table 2A;
| DISCUSSION |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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fru transcripts and proteins are expressed during embryogenesis:
This is the first report showing that fru transcripts and proteins are expressed during embryogenesis (but see
The presence of multiple isoforms and tissue-specific patterns of expression are common characteristics of the BTB/POZ-ZnF transcription factor family. For example, the tramtrack (ttk) gene encodes two isoforms, Ttkp69 and Ttkp88, that are expressed in the CNS and PNS. The Ttkp69 protein is expressed in CNS glial cells and Ttkp88 is expressed in the peripheral nervous system (
Compared to these BTB/POZ-ZnF genes, the fru gene has additional complexity, including multiple promoters as well as alternative 5' and 3' end splicing. This transcript complexity means that tissue- and stage-specific gene expression may be regulated by the choice of the promoter as well as alternative splicing. In the best-understood example of fru transcript regulation, P1 fru transcripts are expressed in the CNS from late larval through adult stages and show sex-specific splicing at the 5' end of the primary transcript and sex-nonspecific alternative splicing of the 3' ends (
For the other fru transcripts derived from the P2, P3, and P4 promoters, little is known of their regulation. Our results suggest that in the CNS most or all neurons and glia express transcripts derived from both the P3 and P4 promoters. The finding that transcripts having any one of the three different 3' ZnF domains are also widely expressed suggests that neurons and glia likely coexpress multiple Fru isoforms with different ZnF domains. If both P3 and P4 transcripts are spliced to use the full range of alternative 3' ends, there may be as many as six different fru transcripts within individual neurons or glia. These alternative 3' ends encode peptides that differ in size as well as in the position and sequence of the ZnF domain (
Our finding of a good correspondence between the cell types that were ß-galactosidase positive in the fru3 and fru4 P-element lines and the pattern of Fru antibody labeling suggests that enhancers sufficient to control fru's embryonic expression pattern are located in nearby regions. The similarity in the pattern of label in the fru3 and fru4 lines indicates that these embryonic enhancers are likely to be distributed in the 40-kb region between the relevant P-element insertion sites located between the P2 and P3 promoters (
The formation of longitudinal and commissural tracts in the embryonic CNS depends on fru function:
Our results show that the fru gene has sex-nonspecific functions in the development of the embryonic axonal scaffold. fru mutants that lack most or all fru transcript classes formed longitudinal and commissural axonal tracts in which axons did not coalesce into fascicles, fasciculated with inappropriate partners, or were unable to maintain proper fasciculation. Consistent with the in situ hybridization experiments, fru mutants, in which P1 or P1 and P2 transcripts were disrupted, formed wild-type FasII and BP102 tracts. By contrast, in fru mutants where P1, P2, and P3 transcripts were disrupted, but P4 transcripts were present, the defects in FasII and BP102 axonal tracts were as severe as the defects in embryos completely lacking fru function. Consideration of the axonal phenotypes in these different fru mutant genotypes suggests that P3 fru transcripts are sufficient for the formation of wild-type FasII and BP102 tracts. Even though this explanation is the simplest that accounts for our data, we are unable to assess the effects of the loss of transcripts from only the P2, P3, or P4 promoter. Thus, we are unable to rule out the possibility that elimination of other fru transcripts or combinations of transcripts might also result in defective axonal pathfinding.
The embryonic phenotypes of complete loss of fru mutants are mild with only a fraction of mutant embryos showing defects in their axonal tracts. This relatively benign phenotype suggests that the activity of other genes may be able to compensate for the loss of fru function. Mutants in other genes, such as fasII, Dlar, and other receptor protein tyrosine phosphatases that encode fasciculation and guidance molecules, also exhibit weak phenotypes in single mutants but show much stronger phenotypes in double mutants or when heterozgyous with mutations that reduce the function of genes that operate in the same developmental pathway (
fru expression in the embryonic CNS rescues axonal pathfinding defects in fru mutants:
The widespread expression of the fru gene in neurons and lateral and midline glia suggests that its function may be required in each of these three cell types and that it influences a variety of cellular processes necessary for the formation of a wild-type axonal scaffold. Each of these three cell types has been shown to function during the creation of the wild-type axonal scaffold (
By contrast, the UAS-fruB transgene did not rescue the mutant axonal phenotypes when expressed with the same pan-neuronal sca-GAL4 driver, but instead led to an increase in the severity and frequency of abnormal FasII and BP102 tracts. The simplest explanation for these results is that the FruB isoform, unlike the FruA and FruC isoforms, is not able to functionally replace other Fru isoforms. The increased severity of the phenotypes in these embryos suggests that the FruB isoform may interfere, perhaps as a dominant negative factor, with the function of the FruA and/or FruC isoforms or with other proteins involved in axonal pathfinding. Alternately, sca-GAL4-driven FruB expression may not replicate the wild-type temporal or spatial pattern or expression level of FruB proteins and these differences in expression may be responsible for the exacerbated axonal phenotypes. Furthermore, our results also show that the misexpression of all of the male-specific Fru isoforms leads to an increased severity in the FasII and BP102 mutant phenotypes of fru mutant and control sibling embryos. Since embryos do not normally produce these FruM proteins, the ectopic expression of these FruM proteins appears to interfere with the function of one or more of the other Fru isoforms or with the activity of proteins needed for wild-type axonal pathfinding.
In contrast, the expression of fru transgenes in postmitotic neurons by the elav-GAL4 driver failed to rescue fru mutant axonal phenotypes. This failure to rescue suggests that the fru gene may function at earlier stages in neuronal development, such as in neuroblasts or in GMCs, in order for the axons to make the right axonal pathfinding decisions or that fru function in other cell types, such as glial cells, is also required for wild-type axonal pathfinding. This explanation is supported by the finding of genetic interactions between fru and other genes involved in generating or responding to repulsive signaling of axons crossing the midline (H.-J. SONG and B. J. TAYLOR, unpublished observations). Somewhat surprising was the finding that fru mutant embryos developed a more severe axonal defasciculation when fru transgenes were expressed only in neurons. The exacerbation of the fru mutant phenotype may indicate that the level of fru transcripts in neurons is important and that levels of Fru proteins different from those found in wild-type neurons might result in disruptions of normal axonal pathfinding or the interaction of these neurons with fru- glial cells.
fru's role in the embryonic CNS appears to be largely in the regulation of axonal outgrowth, not in the initial acquisition of neuronal identity:
The FasII and BP102 axonal pathfinding defects we found in fru mutant CNSs might result from the failure of neurons to adopt their proper cell fate or their ability to differentiate according to their fate. Since P3 and P4 fru transcripts are strongly expressed during NB delamination and early neurogenesis, the time at which neuronal fate decisions are being made, it was possible that fru's main function would be in fate determination. We found that four neurons, dMP2, vMP2, aCC, and pCC, responsible for pioneering the medial and intermediate FasII tracts expressed the appropriate identity markers, Odd-skipped and FasII (
In fru mutants, the earliest defects we observed in FasII pioneering neurons were in the orientation and outgrowth of their initial axonal projections. In some neurons, axonogenesis appeared to be delayed, whereas in other neurons the initial axonal process was oriented abnormally and/or did not appear to be fasciculating properly with other axons. If these pioneering axons are unable to form normal fascicles or are unable to coalesce into discrete fascicles, then other later developing neurons may also be expected to have difficulties in fasciculating along their normal pathways. These results suggest that the loss of fru function may very well affect the expression of the specific receptor systems on axons that are necessary to recognize their fasciculation partners (for review, see
Glial cells have been implicated as important regulators of axonal pathfinding by neurons. Glial cells in the CNS can be grouped into two major categories, midline and the lateral glia, according to their position and gene expression profiles in wild-type embryos. Four segmental midline glial cells, closely associated with the developing commissures, are characterized by the expression of the epidermal growth factor receptor, argos, and pointedP2 (
Glial cells of both subtypes are required for the formation of the axonal scaffold of the ventral nerve cord (
From our findings, we conclude that the fru gene functions in the process of axonal pathfinding by neurons in the embryonic CNS. The earliest neuronal defect that we observed was in the initial outgrowth of axons, which suggests that the fru gene plays an important role in neurons during axonogenesis. Since fru is expressed in neuronal progenitors as well as in neurons and glia, fru may also have a role in cell fate acquisition or maintenance in these cell types.
On the possible function of sex-nonspecific fru transcripts in the adult CNS:
The male-specific Fru proteins derived from the male P1 transcripts are involved in the control of male-specific behaviors (
| FOOTNOTES |
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1 Present address: Functional Genomics Department, Novartis Pharmaceuticals, Summit, NJ 07901. 
2 Present address: Division of Molecular Genetics, University of Glasgow, Glasgow G11 6NU, United Kingdom.
3 Present address: Departamento de Genética del Desarrollo, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca Morelos 62250, Mexico.
4 Present address: Syngenta Biotechnology Inc., Research Triangle Park, NC 27709-2257.
| ACKNOWLEDGMENTS |
|---|
We thank N. Patel, A. Travers, and J. Skeath for generously supplying us with antibodies and J. C. Hall for critical advice and review. We also thank G. Carney, M. Foss, K. Latham, C. Rivin, T. Dreher, J. Giebultowicz, S. Tornquist, and anonymous reviewers for helpful comments on this work and manuscript. This work was supported by National Institutes of Health grant NS-33352 to B.S.B., J.C.H., and B.J.T. Current financial support to S.F.G., from the Wellcome Trust and Division of Molecular Genetics, University of Glasgow, is gratefully acknowledged. J.-C.B. is supported by a University of Glasgow postgraduate scholarship and an Overseas Research Studentship award.
Manuscript received January 14, 2002; Accepted for publication September 19, 2002.
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