BIOLOGICAL OVERVIEW

Lola is a transcription factor that regulates axon guidance. One spliced variant of Lola, that contains a DNA binding domain, is made in glial cells, the accessory cells of neurons. This molecular form probably regulates the production of glial factors, which in turn regulate guidance. Another alternatively spliced variant lacking a DNA binding domain, is produced in neurons. In this case the Lola variant regulates neural molecules responsible for guidance (Giniger, 1994).

Lola is required for pathfinding and targeting of the SNb motor nerve in Drosophila. With complete penetrance and very high expressivity, the motor axons that project through the SNb peripheral nerve in lola mutants fail to form connections to their cognate muscles. Most commonly, SNb appear to 'stall' somewhere between the point at which they would normally separate from the intersegmental nerve and the muscle 6/3 junction. In other hemisegments, SNB axons project through the muscle field but fail to branch into the muscle clefts where synapses should be formed. Careful analysis of muscle phenotypes show that muscle identities are not specified by lola, suggesting that the observed phenotypes are directly due to defective axon guidance. lola is shown to be a dose-dependent regulator of SNb development: by varying the expression of one lola isoform, the extent of interaction of SNb motor axons with their target muscles can be progressively titrated, from no interaction at all, through wild-type patterning, to apparent hyperinnervation. In embryos that overexpress lola in muscle, an apparent 'overgrowth' of Fasciclin 2-immunoreactive is observed where SNb axons contact the VLMs. Some of this material is in the clefts between adjacent muscles, but more is spread out of the clefts and over the surfaces of the muscles. Moreover, in many cases, broad branches extend over the muscles quite separate from the normal projections in the clefts. The Fas 2 immunoreactivity is likely to reflect expanded domains of synaptogenesis. The phenotypes observed from altered expression of Lola suggest that this protein may help orchestrate the coordinated expression of the genes required for faithful SNb development. In addition to its control of SNb morphogenesis, lola also regulates growth of CNS longitudinal axons between successive neuromeres, and growth of ISN axons along lateral peritracheal cells. It may be that some cell surface proteins act in multiple axon growth and guidance decisions, and act in multiple axon growth and guidance decisions, and that lola regulates some of the same genes in all three developmental contexts (Madden, 1999).

Alternative splicing of the lola gene creates a family of 19 transcription factors. All lola isoforms share a common dimerization domain, but 17 have their own unique DNA-binding domains. Seven of these 17 isoforms are present in the distantly-related Dipteran Anopheles gambiae, suggesting that the properties of specific isoforms are likely to be critical to lola function. Analysis of the expression patterns of individual splice variants and of the phenotypes of mutants lacking single isoforms supports this idea and establishes that the alternative forms of lola are responsible for different functions of this gene. Thus, in this system, the alternative splicing of a key transcription factor helps to explain how a small genome encodes all the information that is necessary to specify the enormous diversity of axonal trajectories (Goeke, 2003).

Two isoforms have been reported, and a third isoform is identified in the Berkeley Drosophila Genome Database. All three forms share four exons that encode a common N-terminal region, including the BTB dimerization domain. Two of these isoforms, T and F, splice to unique fifth exons, in each case encoding a pair of zinc fingers. The other isoform, A, is produced if translation continues past the common region, into the adjacent intron, bypassing the final splice. Another lola splice variant was discovered by isolating and sequencing additional lola cDNAs. This splice variant (N) follows the same pattern seen in T and F: the four common exons are spliced to a unique 3' sequence that contains a pair of zinc fingers (Goeke, 2003).

The diversity of Lola protein forms was surveyed by immunoblotting total embryo extract with antibody directed against the Lola common region, and at least 11 apparent Lola protein products were discovered. The pattern of Lola proteins evolves over the course of embryonic development, hinting that some of the different forms of Lola might have different functions (Goeke, 2003).

Since three of the known forms of Lola include zinc finger DNA-binding domains, computational searches of the lola region of the D. melanogaster genome sequence were performed, looking for additional potential zinc fingers. A variety of search motifs were used, including motifs based on a consensus of all zinc fingers or on the consensus of zinc fingers found in previously published BTB-containing proteins. Applying these searches to the sequence of the BAC that encompasses lola revealed 32 potential zinc finger sequences in the genomic DNA corresponding to the lola gene. Each of the putative fingers had strong statistical support (P < 10^-10) in at least one search and predicted protein sequences consistent with the zinc finger structure. Each of these putative fingers is embedded within a larger open reading frame. The distribution of the apparent fingers was consistent with the idea that they define 17 distinct molecular species, 15 bearing pairs of zinc fingers and two bearing single fingers (Goeke, 2003).

The expression of all 17 predicted lola 3' sequences was confirmed by performing RT-PCR on mRNA isolated from embryonic, larval and adult animals, using a forward primer from the common region and reverse primers from the putative finger containing exons. Expression of different combinations of lola isoforms was detected at different developmental stages. All isoforms except for isoform O are expressed in embryos; larvae express all isoforms, and adult males express all except isoforms C and R. Adult virgin females express all isoforms except O (Goeke, 2003).

By sequencing the RT-PCR products, it was found, in all cases, that a single finger or pair of fingers is spliced back to the common region. Eleven lola splice variants have the lola common region spliced directly to a unique final exon containing a pair of zinc fingers, whereas two isoforms have a single zinc finger. Four isoforms have the 3' portion split by an additional intron. No evidence was found for the splicing of multiple finger pair exons to make an isoform with more than two fingers. An additional isoform (M), identified from the D. melanogaster expressed sequence tag (EST) database, splices to 3' exons that do not contain zinc fingers. Remarkably, M and the finger-containing isoform N share a small interstitial exon before diverging by splices to different final exons. All isoforms include the BTB dimerization domain at their N terminus (Goeke, 2003).

The functional significance of lola gene structure was investigated by analyzing its conservation in the mosquito, Anopheles gambiae, which diverged from Drosophila ~250 million years ago. The A. gambiae genome was searched by BLAST searches with the sequence of the BTB domain and all 17 zinc-finger moieties from lola in D. melanogaster. Eight of the seventeen D. melanogaster zinc finger domains clearly had single best matches in the A. gambiae genome, as identified by high percent identity and similarity. These eight matches identified seven unique zinc finger units in A. gambiae that all mapped to a small region on a single genomic scaffold (GenBank accession AAAB008900.1, gi 19611997). Two of the D. melanogaster zinc finger sequences, K and T (which are 92% identical), identified the same zinc finger unit in mosquito. The same genomic region was also selected by BLAST searches with the lola BTB domain. These seven putative finger moieties, together with the BTB domain, identify a likely lola ortholog in A. gambiae. Several details of lola genomic organization were conserved between Anopheles and Drosophila, including the presence of an intron within the sequence encoding the first finger of isoform I (A. gambiae finger sequence 4), the existence of a single-finger isoform (isoform E and A. gambiae finger sequence 2), as well as conservation of the overall order of orthologous zinc fingers along their respective chromosomes. Analysis of the lola locus in A. gambiae also identified two lola-related finger sequences that did not have obvious closest relatives among D. melanogaster lola finger exons (Goeke, 2003).

Antisense RNA probes were generated from the lola RT-PCR products and they were used to assay the expression patterns of the embryonic lola isoforms. The probes excluded the finger sequences themselves to reduce the danger of cross-hybridization; no other portions of the 3' exons showed significant sequence identity in pairwise comparisons. The 19 probes revealed a wide variety of expression patterns for different lola isoforms. Many probes are expressed in broad, overlapping patterns, including expression in whole germ layers or even throughout the embryo. In contrast, some probes show strong enrichment in small sets of cells, such as gonad, invaginating tracheal pits, imaginal discs, a dorsal cell layer of the CNS or isolated cells in the neuroectoderm (Goeke, 2003).

Whereas most lola alleles, such as lola^ORC46 and lola^ORE76, knock out all known lola functions in parallel, several alleles disrupt specific subsets of lola-dependent guidance decisions. These have been termed 'decision-selective alleles'. For example, lola^ORC50 disrupts several CNS functions of lola but not the development of the ISN_b peripheral nerve, whereas lola^ORC4, lola^ORE50, and lola^ORE119 largely block development of ISN_b but have only mild effects on CNS patterning. The defects in decision-selective alleles do not follow any simple allelic series of overall phenotypic strength, but are more readily consistent with the hypothesis that these alleles specifically disrupt separate activities of lola (Goeke, 2003).

Sequencing the terminal lola splice variant exons in flies bearing decision-selective alleles identified molecular lesions in single lola splice variants in three cases: lola^ORE119, lola^ORC4 and lola^ORE50. These have been termed 'single-isoform mutants.' In lola^ORC4 and lola^ORE50, mutations have been identified in the same isoform (K). In lola^ORC4, a (C-->T) mutation introduced a stop codon at amino acid 771 (out of 970 residues in the predicted protein), and consistent with this, Western analysis of extract from lola^ORC4 homozygous embryos using anti-Lola antibodies verified that one of the largest Lola isoforms was absent, but a new, smaller protein species appeared. No change was observed in the level of any other Lola protein species in this experiment. In lola^ORE50, a 4-bp deletion throws the same isoform out of frame at residue 776. In both cases, the mutation is predicted to delete the zinc fingers from the protein, and is therefore expected to be null for this isoform. Consistent with sequencing data, the ISN_b phenotypes of lola^ORC4 and lola^ORE50 homozygous embryos were found to be quantitatively indistinguishable, and were also identical to the phenotype of heteroallelic embryos that are lola^ORC4/ORE50. Since the ISN_b motor nerve fails to innervate its target muscles in lola^ORC4 and lola^ORE50, and isoform K is expressed preferentially in the CNS, the simplest hypothesis is that this isoform acts cell-autonomously in motor neurons, though a contribution cannot be ruled out from low-level epithelial staining of probe K that was observe in some experiments (Goeke, 2003).

In lola^ORE119, a single base pair change in isoform L leads to a single amino acid substitution: Pro712 was changed to a leucine in the sequence between the two fingers. Nine of the 15 zinc finger pairs contain prolines at the analogous position, and moreover, a proline at this position is common in proteins containing multiple zinc fingers. Mutation of the corresponding prolines in TFIIIA and ADR1 reduces DNA binding affinity by 95%. Together, these facts suggest that the mutation in isoform L would likely knock out its function. lola^ORE119 disrupts ISN_b innervation of target muscles, as do lola^ORC4 and lola^ORE50, but in this case probably reflecting the expression of isoform L in mesoderm. The basis of the modest CNS defects in lola^ORE119 are not yet clear; perhaps they arise from a function of isoform L in the mesoderm-derived CNS midline cells. Alternatively, low-level CNS expression is observed of isoform L and it is possible that this contributes to one or both of the phenotypes of lola^ORE119. As expected, Western analysis did not detect a change in the level of any Lola protein species in embryos homozygous for the lola^ORE119 missense mutation. Surprisingly, lola^ORC4 (a mutation inactivating isoform K) did not fully complement the isoform L mutation, lola^ORE119 (Goeke, 2003).

The observation that three single-isoform mutants produce decision-selective mutant phenotypes provides direct evidence that different lola isoforms make unique contributions to specific lola functions. Mutations that inactivate isoform K (lola^ORC4 and lola^ORE50) largely block muscle innervation by ISN_b motoneurons, whereas a mutation inactivating isoform L (lola^ORE119) disrupts both muscle innervation and, to a lesser extent, CNS development. Isoforms K and L differ in sequence, with 27 differences among 55 amino acids in the zinc fingers. The expression patterns of isoforms K and L also differ, with K expressed mainly in the CNS (but with a low level of epithelial expression) and L expressed mainly in mesoderm (but with a low level of CNS expression). It will be interesting to determine how much of the functional difference between isoforms K and L arises from the difference in their expression patterns, and how much arises from the difference in protein sequence (Goeke, 2003).

The data show that some lola isoforms have unique functions. Other experiments demonstrate that some lola functions require combinations of different isoforms. The phenotypes of mutations in isoforms K and L establish that each makes unique contributions to motoneuron pathfinding. However, it is striking that both are required for the same process: muscle innervation by the ISN_b motor nerve. Evidently, innervation of these muscles requires cooperation between at least these two lola isoforms. The need for functional cooperation between these isoforms is driven home by the observation that embryos that are doubly heterozygous for mutations in isoforms K and L display highly penetrant defects in ISN_b development (lola^ORC4/ORE119), even though each mutation by itself is recessive. Trans-heterozygous mutant phenotypes have often been observed for genes whose products function together closely. Since it is not possible to rule out some coexpression of K and L, it may be that their synthetic phenotype arises from the requirement for a K/L heterodimer. Alternately, it may be that these two isoforms regulate the expression of, respectively, a receptor and its ligand that function together in ISN_b development, much as lola co-regulates robo and slit in the CNS. The trans-heterozygous interaction of K and L mutations in this case could reflect their concerted effect on the expression of these target genes (Goeke, 2003).

The data above demonstrate that the diversity of lola isoforms contributes to the complex pattern of lola functions. Yet more diversity could potentially arise from heterodimerization. Taking into account overlap in expression patterns, if the Lola isoforms dimerize with each other, then association of different isoforms could produce over 100 distinct combinations of zinc finger DNA-binding domains. Moreover, the BTB domain is highly conserved among BTB proteins, and there are indications of heterodimerization between lola and other BTB-containing proteins. In addition, multiple splice variants have been identified for some of these potential lola partners. If lola dimerization partners show as much alternate splicing as does lola, the potential diversity of Lola-containing species would increase geometrically (Goeke, 2003).

With the sequencing of various complete genomes, a common problem has emerged: understanding how functional diversity is generated from a compact genome. The role of alternative splicing in increasing the diversity of cell surface proteins is well established. These data extend this principle to gene regulatory proteins, and it is suggested that alternative splicing may be used by transcription factors as extensively as it is by cell surface proteins as a means to increase the overall functional diversity of the genome (Goeke, 2003).

GENE STRUCTURE

lola codes for three RNA transcripts of 3.8, 4.7 and 4.9 kb sharing a common core sequence with different 5' and 3' ends. Both 3.8 and 4.7 kb transcripts have a 399 base pair 5' UTR whose sequences differ in the first 178 bases. The 3.8 kb transcript has a 180 base pair 5' UTR. The 4.7 kb transcript has a 750 base pair 5' UTR. The proteins are identical up to the 454th amino acid. The long form continues from there, while the short form has 13 amino acids that differ from the long form.

PROTEIN STRUCTURE

Amino Acids - 467 for the 3.8 kb RNA and 894 for the 4.7 kb RNA

Structural Domains and Evolutionary Homologs

The long transcript has a Cys2-His2 type of double zinc finger. The short transcript has no zinc finger. Both transcripts have a BTB domain (Giniger, 1994).

A novel zinc finger protein, ZID (standing for zinc finger protein with interaction domain) was isolated from humans. ZID has four zinc finger domains and a BTB domain, also know ans a POZ (standing for poxvirus and zinc finger) domain. At its amino terminus, ZID contains the conserved POZ or BTB motif present in a large family of proteins that include otherwise unrelated zinc fingers, such as Drosophila Abrupt, Bric-a-brac, Broad complex, Fruitless, Longitudinals lacking, Pipsqueak, Tramtrack, and Trithorax-like (GAGA). The POZ domains of ZID, TTK and TRL act to inhibit the interaction of their associated finger regions with DNA. This inhibitory effect is not dependent on interactions with other proteins and does not appear dependent on specific interactions between the POZ domain and the zinc finger region. The POZ domain acts as a specific protein-protein interaction domain: The POZ domains of ZID and TTK can interact with themselves but not with each other, or POZ domains from ZF5, or the viral protein SalF17R. However, the POZ domain of TRL can interact efficiently with the POZ domain of TTK. In transfection experiments, the ZID POZ domain inhibits DNA binding in NIH-3T3 cells and appears to localize the protein to discrete regions of the nucleus (Bardwell, 1994).

Transcriptional Regulation

The wide-ranging defects in dendrites and axons indicate that sequoia functions to regulate axonal and dendritic morphogenesis in most neurons. Alternatively, it is conceivable that sequoia regulates the expression of genes generally required for neuronal differentiation. To gain mechanistic insight into sequoia function, the transcript profiles in wild-type and sequoia mutant embryos were compared based on microarray analyses of over 3,000 genes or ESTs, corresponding to about 25% of the Drosophila genome. The vast majority of these genes show comparable expression levels, including genes for cytoskeletal elements, genes that specify neuronal cell fates, and genes generally required for neurite outgrowth such as cdc42. Interestingly, a small fraction of the genes/ESTs analyzed showed clearly distinct expression ratios in sequoia mutants. Of these, 93 (3.1%) different transcripts were reduced by at least one-third of the wild-type level, and 34 (1.1%) different transcripts were increased by at least 75% of the wild-type level. A number of genes that appear to be regulated by sequoia, directly or indirectly, correspond to genes implicated in the control of axon morphogenesis rather than neuronal fate. These include known genes such as connectin, frazzled, roundabout 2, and longitudinals lacking, in addition to novel molecules with homology to axon guidance molecules including slit/kekkon-1 and neuropilin-2. It is noteworthy that two of the genes showing increased transcript ratios, roundabout 2 and CG1435, a novel calcium binding protein, were both also identified in a gain-of-function screen affecting motor axon guidance and synaptogenesis. In addition to genes that have clearly been implicated in axon development based on previous studies or sequence similarity, microarray data reveal that other genes potentially regulated by sequoia include peptidases, lipases, and transporters, as well as novel zinc finger proteins. It should be noted that transcripts that are broadly expressed and increased or decreased in sequoia mutants may actually be altered to a greater extent within neurons, because sequoia likely functions cell autonomously and is only expressed in the nervous system (Brenman, 2001).

Targets of Activity

By transfection experiments, a 72-bp enhancer sequence has been identified within the Drosophila copia retrotransposon, which is involved in the control of the transcription level of this mobile element in cells in culture. Gel shift assays with nuclear extracts from Drosophila hydei-derived DH-33 cells demonstrate specific interactions of at least two nuclear factors with this enhancer sequence. Using this sequence as a probe for the screening of an expression cDNA library constructed from DH-33 cells RNA, a cDNA clone encoding a 110-kDa protein was isolated with features common to those of known transcription factors: these include a two-zinc-finger motif at the C terminus; three glutamine-rich domains in the presumptive activation domain of the protein, and an N-terminal domain that shares homology with the Bric-a-brac, Tramtrack, and Broad-Complex BTB boxes. The precise DNA recognition sequence for this transcription factor has been determined by both gel shift assays and footprinting experiments with a recombinant protein made in bacteria. The functionality of the cloned element has been demonstrated upon transcriptional activation of copia reporter genes, as well as of a minimal promoter coupled with the identified target DNA sequence, in cotransfection assays in cells in culture with an expression vector for the cloned factor. Southern blot and nucleotide sequence analyses reveal a related gene in Drosophila melanogaster (the lola gene) previously identified by a genetic approach as involved in axon growth and guidance. Transfection assays in cells in culture with lola gene expression vectors and in situ hybridization experiments with lola gene mutants finally provided evidence that the copia retrotransposon is regulated by this neurogenic gene in D. melanogaster, with a repressor effect in the central nervous systems of the embryos (Cavarec, 1997).

The pattern and level of expression of axon guidance proteins must be choreographed with exquisite precision for the nervous system to develop its proper connectivity. Previous work has shown that the transcription factor Lola is required for central nervous system (CNS) axons of Drosophila to extend longitudinally. Lola is simultaneously required to repel these same longitudinal axons away from the midline, and it acts, in part, by augmenting the expression of both the midline repellant, Slit, and its axonal receptor, Robo. Lola is thus the examplar of a class of axon guidance molecules that control axon patterning by coordinating the regulation of multiple, independent guidance genes, ensuring that they are co-expressed at the correct time, place and relative level (Crowner, 2002).

The reduction of Robo expression seen in lola mutants is relatively modest (~40%). It is known, however, that a 50% diminution in Robo is sufficient by itself to cause some inappropriate midline crossing, and this effect is strongly enhanced by a simultaneous 50% reduction in Slit. Loss of lola causes a greater reduction than this in Slit levels. Thus, it is plausible that the change in Slit and Robo levels could account for much of the midline phenotype observed in embryos that bear strong lola mutations. But why are weaker lola alleles like lola^1A4 able to cause extra midline crossing when their effect on target gene expression is presumably proportionately less? It is likely that regulation of Slit and Robo expression is only one part of the control of midline crossing by lola, and that a significant contribution to the phenotype is made by changes in the expression of other, interacting guidance genes that are also controlled by lola. For example, aspects of the lola midline phenotype resemble details of the axon pattern observed upon mutation of genes encoding receptor tyrosine phosphatases, suggesting that these are good candidates for potential lola effectors. Moreover, it is known that the Notch-dependent mechanism that promotes the alternative (longitudinal) trajectory of CNS axons also requires lola. The multiplicity of genes contributing to the midline/longitudinal axon growth decision underscores the need for a gene, like lola, to coordinate the expression of all these cooperating guidance factors. It is suggested that it is the combination of many quantitative effects, each individually modest, which together produce the profound effects of lola on axon patterning (Crowner, 2002).

Many questions remain from these studies. (1) Though Lola itself is a transcriptional regulator, it is not known whether robo and slit are direct Lola targets or whether Lola initiates a longer chain of events leading only indirectly to robo and slit. For example, Lola could regulate other genes that themselves control the stability of robo or slit RNA or protein, or the splicing or translation efficiency of these genes. Analysis of this issue will require unambiguous identification of the exact lola isoforms required for expression of robo and slit, and characterization of their DNA-binding specificities in combination with their appropriate dimerization partner(s). (2) Only the accumulation of Robo and Slit protein has been characterized in lola mutants, and not transcript levels. The inherent variability of whole-mount RNA in situ hybridization has prevented sufficiently precise quantification of robo and slit RNA levels for this purpose. Nonetheless, the observation that ectopic expression of lola 4.7 leads to ectopic expression of slit RNA strongly argues that lola is upstream of slit transcription, though it remains possible that Robo and Slit expression are also subject to lola-dependent regulation at some post-transcriptional level (Crowner, 2002).

lola does not just regulate midline crossing, but also controls extension of some peripheral motor axons and orientation of lateral chordotonal neurons in the embryo, as well as pathfinding of some axons of the adult wing. In each case, it apparently establishes a precise balance of guidance factors. How can one transcription factor exert such subtle control over such a diverse array of developmental events? This remains to be determined, but we have recently found that lola encodes a large number of protein isoforms. At least in some cases, lola isoforms with different predicted DNA binding specificities are expressed in different tissue specific patterns, potentially allowing the regulation of distinct cohorts of downstream target genes. Moreover, it is known that a single, direct Lola target gene can be activated by lola in one tissue and repressed in another . Both of these properties are likely to contribute to the ability of Lola to modulate gene expression programs in distinct ways in different cells (Crowner, 2002).

The problem of ensuring appropriate relative levels of multiple guidance genes is not unique to the midline crossing versus longitudinal growth decision in the fly CNS, but rather is an inherent feature of all axon guidance decisions. It is therefore imagined that lola is not unique in its property of co-regulating multiple, interacting guidance genes, but rather is the exemplar of a class of transcriptional regulators that will be found to be widespread in distribution and critical in importance in the regulation of axon patterning (Crowner, 2002).

Alternative trans-splicing of constant and variable exons of lola

longitudinals lacking (lola) is a complex Drosophila gene encoding at least 20 protein isoforms, each bearing the same N-terminal constant region linked to a different C-terminal variable region. Different isoforms specify different aspects of axon growth and guidance. lola mRNAs are generated by alternative trans-splicing of exons sequentially encoded by the same DNA strand. Chromosomal pairing facilitates interallelic trans-splicing, allowing complementation between mutations in the constant and those in the variable exons. At least one variable exon is transcribed from its own promoter and is then trans-spliced to the constant exons transcribed separately (Horiuchi, 2003).

To dissect the genetic properties of the lola locus, interallelic complementation tests were performed among five ethylmethane sulfonate (EMS)-induced mutant alleles, lola^ORE120, lola^ORC46, lola^ORC4, lola^ORE50, and lola^ORE119, whose molecular lesions are already identified. Exons C5-8 constitute the constant region for all isoforms, and V10-11, V21-22, V23, and V32 correspond to the variable region of isoforms B, K, L, and T, respectively. All are homozygous lethal with severe defects in embryonic axon guidance. Flies die that are heterozygous for alleles mutated in the same exon unit (lola^ORE120/lola^ORC46 for C5-8 and lola^ORE50/lola^ORC4 for V21-22). In contrast, animals heterozygous for mutations in exons V21-22 and V23 (lola^ORE50/lola^ORE119 and lola^ORC4/lola^ORE119) show nearly normal viability. The interallelic complementation between alleles mutated in different variable exons is reasonable, since a functional version of each isoform is supplied by one of the homologous chromosomes. Surprisingly, however, combinations of mutations in the constant exons with those in the variable exons (lola^ORC46/lola^ORE50, lola^ORE120/lola^ORE119, and so on) also result in viable flies (Horiuchi, 2003).

To explain these unexpected results, three models were postulated: (1) Functional mRNAs are produced through interallelic trans-splicing or any other mechanisms recombining two pre-mRNAs; (2) a wild-type locus is generated through recombination or gene conversion, or (3) functional complementation occurs between the two mutant proteins. Model 3 is rather unlikely, because lola^ORC46 contains a nonsense mutation within a constant exon, so that its mRNA would be degraded quickly via nonsense-mediated mRNA decay; even if it could survive, the translated protein lacks the entire variable region. In models 1 and 2, wild-type mRNA variants should be present in the hybrid animals. To examine this possibility, the sequences were determined of C5-8/V21-22 and C5-8/V23 cDNAs amplified by reverse transcriptase PCR (RT-PCR) from total RNA fractions of flies of the genotype lola^ORE120/lola^ORC4 and lola^ORC46/lola^ORE119, respectively. Indeed, C5-8/V21-22 and C5-8/V23 cDNA clones were found with no molecular lesions. In model 2, DNA recombination or gene conversion at the lola locus should occur at an extraordinarily high frequency at a very early stage of development. Whether it happened in the germ-line cells, which start dividing at a very early stage, was examined. lola^ORE120/lola^ORC4 and lola^ORC46/lola^ORE119 flies were crossed to Df(2R)stan2, a deficiency uncovering the entire lola region. None of the hemizygous offspring were viable, suggesting that DNA recombination or gene conversion at the lola locus is unlikely. Therefore, model 1 seemed the most likely mechanism to explain the interallelic complementation between mutations in the constant and those in the variable regions and the presence of chimeric mRNAs. In all sequenced clones including those described below, joining of exons occurs invariably at consensus splicing sites, suggesting that recombinant mRNAs are generated through trans-splicing, rather than any other mechanism (Horiuchi, 2003).

It is important to note that the variable exons (V23 and V32) assort independently of the constant exons; they are trans-spliced to the constant exons derived from either homologous chromosome at nearly equal frequency. This suggests that the splicing must occur when the pre-mRNAs from the two homologous chromosomes are in close proximity. In Drosophila and other dipteran insects, genes on homologous chromosomes are thought to be physically proximal to each other through pairing. Transvection and trans-sensing effects are severely affected when homologous chromosome pairing is disrupted by chromosomal rearrangements. To examine the influence of chromosomal rearrangements on the frequency of interallelic trans-splicing in the lola locus, SNP analyses of F1 hybrids was performed with a CKG30 balancer chromosome with multiple inversions, and those with T(2;3)X3, a translocation involving the lola locus. The frequency of cDNA clones with interallelic hybrid haplotypes was significantly reduced by CKG30 and by T(2;3)X3. These results clearly indicate that interallelic trans-splicing occurs frequently when the two chromosomal loci are positioned such that they can pair effectively (Horiuchi, 2003).

In summary, therefore, alternative trans-splicing at the constant/variable junction generates a significant portion of the mRNA from the lola locus. For some isoforms, nearly half of the mature RNAs were chimeras between pre-mRNAs derived from the homologous chromosomes, indicating that the mRNAs were generated through trans-splicing exclusively. Finally, in one case, a variable exon was found being expressed from a dedicated promoter in the preceding intron. lola provides the first demonstration of abundant mRNAs generated by alternative trans-splicing of exons sequentially encoded by the same DNA strand. The frequent interallelic trans-splicing events support the pairing of homologous chromosomes in Drosophila. In contrast, mammalian hybrid alpha/gamma protocadherin mRNAs, which are most likely generated by trans-splicing, can be spliced only intrachromosomally. This difference may be due to a lack of chromosomal pairing during transcription in mammalian cells, and/or a high competence of Drosophila pre-mRNAs to be trans-spliced (Horiuchi, 2003).

There are important implications regarding the mechanism of trans-splicing. Because the efficiency of interchromosomal trans-splicing was dramatically reduced by chromosomal rearrangements, it is inferred that trans-splicing occurs locally either cotranscriptionally or posttranscriptionally. Cotranscriptional trans-splicing is consistent with a recent view of gene expression in which all steps of mRNA processing take place cotranscriptionally. It is also supported by reported examples of trans-splicing events detected in mammalian cells in which trans-splicing occurred between pre-mRNAs derived from the same genes or closely linked ones. Trans-splicing of mod(mdg4) pre-mRNAs occurs between those derived from the original locus and a transgene inserted in a different chromosome. However, this may not represent the natural situation, because a high level of transgene expression was induced by the GAL4-UAS system, and RT-PCR not controlled for template switching was used to detect trans-spliced mRNAs. Therefore, it may be reasonable to assume that natural trans-splicing occurs locally, most likely cotranscriptionally, as is the case for cis-splicing. An additional requirement for trans-splicing would be a mechanism allowing integration of pre-mRNAs encoding variable exons into the spliceosome operating in the constant region. The outron sequences (transcribed region preceding the variable exons) may contain cis-elements required for this process (Horiuchi, 2003).

In addition, variable regions may consist of multiple independent transcription units whose pre-mRNAs are each trans-spliced to the constant exons. With internal promoter(s) for variable exons and cotranscriptional alternative trans-splicing, the regulation of lola isoform expression could be much simplified. Unlike alternative cis-splicing, simultaneous expression of multiple isoforms can be achieved without complex regulation of splicing and polyadenylation sites of a single mRNA. Multiple pre-mRNAs containing different variable exons could be expressed in the same cells depending on their own promoter activities, which would then be trans-spliced to the constant exons, possibly in a concentration-dependent manner. Furthermore, the internal promoter(s) for variable exons may be tissue-specific, and thus it could also simplify the mechanism of tissue-specific expression of particular variants as well (Horiuchi, 2003).

lola shares many molecular features with mod(mdg4), and it is therefore possible that mod(mdg4) isoforms sequentially encoded in the same DNA strand could also be generated through trans-splicing. Furthermore, there are other fly genes encoding BTB-Zn finger proteins, such as fruitless and Br-C, some of which show complex patterns of intragenic complementation reminiscent of what is seen with lola. These genes produce multiple splice variants, often with a complex pattern of expression. Trans-splicing may be a general means to generate splice variants in this family of genes (Horiuchi, 2003).

DEVELOPMENTAL BIOLOGY

Embryonic

All three RNA transcripts of lola are present maternally, although the 4.9 kb RNA disappears rapidly after fertilization. The 3.8 kb RNA continues to be present throughout embryogenesis, whereas the 4.7 kb transcript decays at about 9 hours, at which time the 4.9 kb RNA reappears. The 4.7 kb isoform preferentially appears in mesodermal and mesectodermal cells after germ band extension. The mesectodermal cells are probably glial precursors. Label associated with the short form concentrates in neural tissue between stage 11 and 13, concommitant with the reappearance of the 4.9 kb form.

RNA detectable with the long probe appears in clusters of peripheral cells that are probably precursors of imaginal discs and abdominal histoblasts and tracheal histoblasts and in a small sector of the brain, possibly the optic lobe anlagen (Giniger, 1994).

Effects of mutation or deletion

lola was isolated from a screening for disrupted axons of 550 homozygotic lethal insertions of a transposon. Disrupting genes with exogenous DNA is a convenient method to search for and manipulate new genes. Mutation of lola leads to disrupted axon tracts in the central nervous system, and displacement of chordotonal organs (Giniger, 1994).

REFERENCES

Bardwell, V. J. and Treisman, R. (1994). The POZ domain: a conserved protein-protein interaction motif. Genes Dev. 8: 1664-1677

Brenman, J. E., Gao, F.-B., Jan, L. Y. and Jan, Y. N. (2001). Sequoia, a Tramtrack-related zinc finger protein, functions as a pan-neural regulator for dendrite and axon morphogenesis in Drosophila. Dev. Cell 1: 667-677. Medline abstract: 11709187

Cavarec, L., et al. (1997). Molecular cloning and characterization of a transcription factor for the copia retrotransposon with homology to the BTB-containing lola neurogenic factor. Mol. Cell. Biol. 17(1): 482-94. Medline abstract: 97127405

Crowner, D., Madden, K., Goeke, S. and Giniger, E. (2002). Lola regulates midline crossing of CNS axons in Drosophila. Development 129: 1317-1325. Medline abstract: 11880341

Giniger, E., Tietje, K., Jan, L.Y. and Jan, Y.N. (1994). lola encodes a putative transcription factor required for axon growth and guidance in Drosophila. Development 120: 1385-1398 Medline abstract

Goeke, S., et al. (2003). Alternative splicing of lola generates 19 transcription factors controlling axon guidance in Drosophila. Nature Neurosci. 6: 917-924. Medline abstract: 12897787

Horiuchi, T., Giniger, E. and Aigaki1, T. (2003). Alternative trans-splicing of constant and variable exons of a Drosophila axon guidance gene, lola. Genes Dev. 17: 2496-2501. Medline abstract: 14522953

Madden. K., Crowner, D. and Giniger, E. (1999). lola has the properties of a master regulator of axon-target interaction for SNb motor axons of Drosophila. Dev. Biol. 213(2): 301-13. Medline abstract: 99410338

date revised: 20 February 2004

Please e-mail comments/corrections to brodyt@codon.nih.gov