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 lola generates 19 transcription factors controlling axon guidance in Drosophila

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).

A genome-wide analysis reveals that the Drosophila transcription factor Lola promotes axon growth in part by suppressing expression of the actin nucleation factor Spire

The phylogenetically conserved transcription factor Lola is essential for many aspects of axon growth and guidance, synapse formation and neural circuit development in Drosophila. To date it has been difficult, however, to obtain an overall view of Lola functions and mechanisms. Expression microarrays were used to identify the lola-dependent transcriptome in the Drosophila embryo. lola was found to regulate the expression of a large selection of genes that are known to affect each of several lola-dependent developmental processes. Among other loci, it was found that lola is a negative regulator of spire, an actin nucleation factor that has been studied for its essential role in oogenesis. spire was shown to be expressed in the nervous system and is required for a known lola-dependent axon guidance decision, growth of ISNb motor axons. It was further shown that reducing spire gene dosage suppresses this aspect of the lola phenotype, verifying that derepression of spire is an important contributor to the axon stalling phenotype of embryonic motor axons in lola mutants. These data shed new light on the molecular mechanisms of many lola-dependent processes, and also identify several developmental processes not previously linked to lola that are apt to be regulated by this transcription factor. These data further demonstrate that excessive expression of the actin nucleation factor Spire is as deleterious for axon growth in vivo as is the loss of Spire, thus highlighting the need for a balance in the elementary steps of actin dynamics to achieve effective neuronal morphogenesis (Gates, 2011).

The transcription factor Lola is required for a variety of axon growth and guidance events in the developing fly embryo. Expression microarray analysis of lola mutant embryos now reveals that, rather than producing large changes in the levels of a restricted number of major-effect downstream targets, Lola appears to exert its influence via the cumulative effects of small, quantitative changes in a broad spectrum of downstream loci. One key Lola target is spire, which encodes an actin nucleation factor that has been studied intensively for its role in regulating cytoskeletal structure in the developing fly oocyte. spire, like lola, is required for development of ISNb motor axons, its level goes up in lola mutants, and reduction of spire dosage suppresses, but does not eliminate, the ISNb mutant phenotype of lola (Gates, 2011).

Previous analysis of candidate genes implicated in various lola-dependent axon guidance processes identified several whose expression was subtly affected by lola, but none that were dramatically altered. This and other observations led to the proposition that Lola might execute its effects by fine-tuning the expression levels of genes that contribute quantitatively to various guidance decisions, and not simply by turning these genes ON versus OFF. As an unbiased test of this hypothesis, expression microarrays were used to perform a genome-wide comparison of the embryonic transcriptome of wild-type and lola zygotic mutant embryos. RNA isolated from animals 10 to 12 hours after egg laying was analyzed, at a time when a large number of lola-dependent axons are extending. By this analysis, the expression of no single-copy Drosophila gene was altered more than four-fold by lola, and few were altered more than 2.5-fold. It is possible that this is an underestimate due to the compression of expression ratios in microarray experiments, but qRT-PCR results were largely consistent with the array data. It is also possible that expression of some genes may have been altered by a greater factor in just a small subset of expressing cells, but it is noted that most lola isoforms are themselves expressed very broadly, making this possibility less likely. Finally, some genes can be affected oppositely by different lola isoforms, or in different tissues, so it may be that a small net change in expression of a lola target gene hides larger but counteracting changes in different cells. Nonetheless, it remains that a genome-wide analysis failed to identify any single major-effect lola target that would account for the lola axonal phenotypes. It is also true that there is a substantial maternal contribution of Lola to the embryo, and this may limit the measured effect of the mutation on downstream targets. It is noted, however, that it is the zygotic mutant phenotype of lola that this study seeks to explain, and it is therefore the quantitative effect of that zygotic mutant that is the relevant measurement for investigating the phenotype (Gates, 2011).

Microarray analysis has been widely used to identify genes associated with, or responsible for, many developmental and physiological processes. Typical analyses of expression microarray data emphasize genes whose level is strongly altered by the biological manipulation, often setting numerical cutoffs for change in expression level, together with statistical criteria, to identify true positives. In these experiments, it was necessary to eschew the use of a quantitative cutoff in fold change; for example, a commonly used criterion of a two-fold minimum change would have excluded from analysis all but 26 single-copy genes in the genome. Rather, the nature of the biological process studied, and the nature of lola, required that the biological and technical variance be minimized to achieve exceptionally tight statistics. In the end, qRT-PCR validation of expression changes from 1.2-fold (genderblind) to 2.5-fold (spire) provided support for 50% of the putative downstream effects of lola. It is noteed that this is likely to be an underestimate of the reliability of the array results since these small fold differences were at or beyond the usual sensitivity of RT-PCR itself, and it is as likely that RT-PCR was reporting false negatives as the microarrays were reporting false positives. Validity of the results was also supported more globally by independent expression profiling of another lola allele. Thus, these data underscore the efficacy of microarray analysis for detecting reliably even quite small changes in expression level. Known genes whose expression was altered in the lola mutant shed light on many lola-dependent processes. Previous experiments had led to the notion that lola likely coregulates a suite of interacting genes that are important for particular axon guidance decisions, and indeed, it was found that expression of a number of well-characterized guidance receptors is altered in lola. frazzled, which was identified as a downstream target of lola, is on its own known to be required for three lola-dependent axonal processes: ISNb development in the periphery, and both longitudinal and commissural axon extension in the CNS. Among other factors downstream of lola are midline fasciclin (longitudinal and commissural axons), fasciclin 3 and capricious (ISNb) and neural lazarillo (thought to be involved in both longitudinal and commissural axon guidance). Also identified were genes for a number of ligands, receptors and receptor-modifying proteins not previously associated with lola-dependent processes, such as sugarless, dallylike, wnt4a and PVF-1. It now becomes interesting to investigate the potential role of these genes in axon patterning, and in migration and orientation of sensory neurons. Aside from cell surface and extracellular proteins, expression of genes encoding a number of intracellular signaling proteins was found to be altered, including prospero (which in hypomorphic alleles produces a phenotype very similar to that of lola), as well as moesin, Rac2, and a calmodulin-dependent protein kinase (CAKI). An unexpected cluster of downstream effects comprised genes for proteins modulating microtubule structure and function, including katanin, stathmin, NudC and KLP-59C. lola also interacts genetically with the axon patterning function and other activities of the receptor Notch, and a cluster of affected genes was found that modulate Notch action, including sca, Nak, Dap-160 and O-fut1. In addition to these known genes, Gene Ontology analysis identifies a large number of lola-dependent loci that have not yet been characterized in the fly, but whose annotations cluster with lola-dependent genes of known function. This provides a substantial list of excellent candidates for additional contributors to lola-dependent processes. Unfortunately, the large number of Lola isoforms, and their heteromeric combinations, makes it impossible to extract Lola binding site consensus sequences from these candidates using standard computational approaches. Extensive molecular experiments will be necessary in the future to identify response elements for individual heteromeric forms of Lola (Gates, 2011).

lola has several characterized functions outside of axon patterning. For example, it affects cell fates in the eye, and indeed, there is a substantial group of eye patterning genes included in the list of lola-affected loci (sickle, charlatan, asense, rap, roughex, Lobe, target of eyeless and fat facets). Additionally, consistent with the role of lola in controlling programmed cell death during oogenesis, grim, scylla, charybde, bunched and Nedd2-like caspase were found among the downstream effects. It should be noted that since the microarray analysis was performed only with mid-stage embryos, it cannot be distinguished whether the effects of lola on these postembryonic processes are mediated by the same downstream targets that were see affected during embryogenesis. Seeing that these genes can be modulated by lola at one stage of the lifecycle, however, makes them more attractive candidates for analysis at other stages. Finally, in addition to genes affecting known lola-dependent processes, the set of genes altered in lola mutants identifies clusters associated with new processes that would be worth investigating for a role of lola. These include aging, oxidative stress, hormonal regulation of development, tracheal development and maintenance, cell polarity and olfactory learning, among others (Gates, 2011).

One of the most robust putative downstream effects identified for lola was downregulation of the actin nucleation factor Spire. This was immediately striking since spire is known to be a critical regulator of the oocyte cytoskeleton during Drosophila oogenesis. spire is required for both anteroposterior and dorsoventral patterning of the developing oocyte. By modulating actin structure, Spire restrains bundling of oocyte microtubules, thereby blocking cytoplasmic streaming in the oocyte until critical anteroposterior and dorso-ventral polarity cues become stably bound to cortical anchoring sites or initiate irreversible signaling cascades. At the biochemical level, Spire nucleates actin filaments by bringing together actin monomers to assemble a filament nucleus, and it may then transfer this nucleus to the associated formin, Cappuccino, which stimulates filament growth. While the developmental function of spire has been studied most thoroughly in the oocyte, strong mutations in this gene are largely lethal, with only small numbers of escapers surviving to adulthood, and this suggested the existence of as yet uncharacterized zygotic functions of spire. Moreover, a mouse ortholog of spire is expressed in the developing and adult brain. This study found that spire is required for a well-characterized lola-dependent neuronal process, extension of the ISNb motonerve. ISNb was an ideal candidate for the sort of function that had previously been hypothesized for lola, since it is known to depend on the summed, quantitative effects of a large collection of regulators. Therefore ISNb was exploited to examine more carefully the potential interaction of lola and spire, and it was found that genetic reduction of spire suppressed the ISNb mutant phenotype of lola, consistent with the upregulation of spire in a lola mutant making a significant contribution to ISNb axon stalling in lola. By itself, expression analysis cannot distinguish whether spire is a direct target of Lola or whether the upregulation of spire message is a downstream consequence of other changes set in motion by lola. Further biochemical studies of the DNA binding properties of Lolaisoforms will be necessary to assess this. Finally, lola has other phenotypes that are not suppressed by reduction of spire. These may reflect, for example, roles of lola-dependent guidance molecules that are themselves spire -independent, or the action of Spire-independent aspects of growth cone signaling (Gates, 2011).

Efforts to mimic the lola ISNb phenotype by overexpression of spire were not successful. There are several possible reasons for this. First, there are thought to be at least eight Spire protein isoforms, based on cDNA and expressed sequence tag data, and it may be that a particular combination of isoforms, or a specific ratio of expression levels of different isoforms, is necessary to give the ISNb stalling phenotype. Alternatively, it may be that this phenotype is produced only when spire upregulation occurs in the context of some other downstream effect(s) of lola. Additional experiments will be necessary to discriminate among these models (Gates, 2011).

Superficially, it seems remarkable that complete loss of spire causes stalling of ISNb axons, yet the upregulation of spire that occurs in a lola mutant also contributes to ISNb stalling. Evidently, excessive nucleation of actin filaments from spire overexpression is as detrimental to growth cone motility as is the failure of actin nucleation from absence of the protein. Similar non-linearity has been observed in the effects of a number of signaling and cytoskeletal regulatory proteins in other axon guidance paradigms, and it appears to be a common feature of the relationship of signaling to morphogenesis. Thus, for example, even though Abl tyrosine kinase pathway signaling appears to be essential for most axon growth, extension of longitudinal pioneer axons of the fly CNS requires suppression of Abl signaling to achieve the proper balance in the steps of actin dynamics. Similarly, for the Rac GTPases, expression of dominant negative and dominant constitutive forms of the protein are equally effective for inhibiting axon motility, but in one case from excessive stabilization of actin filaments and in the other from insufficient stabilization. Spire now provides another example of this generalization, and underscores the need for signaling networks to evoke a balance in the steps in actin dynamics, thus optimizing throughput through the mechanical cycle of growth cone motility (Gates, 2011).

lola mutants have profound effects on axon patterning, even though systematic molecular analysis reveals only subtle modulation of downstream target gene expression. This observation highlights the exquisite sensitivity of motility and guidance to the balance among cell signaling networks, and thus also to the gene expression mechanisms that set the boundaries of that balance (Gates, 2011).

This study has used a genome-wide analysis to identify the suite of genes whose expression is altered in embryos lacking the Drosophila transcription factor Lola. Gene Ontology analysis sheds light on the regulation of several characterized lola functions, including axon guidance, synapse formation, eye development and oogenesis, by revealing the loladependence of genes known to be involved in those processes, and also by identifying a large number of previously uncharacterized lola-dependent genes that are likely to contribute to these processes. Additionally, these results identify novel processes that are likely to be regulated by lola. Regarding axon patterning, this analysis reveals that Lola suppresses expression of the actin nucleation factor Spire, and this is crucial for its ability to promote growth of motor axons in vivo. These data underscore the critical importance of ensuring the correct levels of actin regulatory proteins in a cell to promote motility effectively (Gates, 2011).

A comprehensive temporal patterning gene network in Drosophila medulla neuroblasts revealed by single-cell RNA sequencing

During development, neural progenitors are temporally patterned to sequentially generate a variety of neural types. In Drosophila neural progenitors called neuroblasts, temporal patterning is regulated by cascades of Temporal Transcription Factors (TTFs). However, known TTFs were mostly identified through candidate approaches and may not be complete. In addition, many fundamental questions remain concerning the TTF cascade initiation, progression, and termination. This work used single-cell RNA sequencing of Drosophila medulla neuroblasts of all ages to identify a list of previously unknown TTFs, and experimentally characterize their roles in temporal patterning and neuronal specification. This study reveals a comprehensive temporal gene network that patterns medulla neuroblasts from start to end. Furthermore, the speed of the cascade progression is regulated by Lola transcription factors expressed in all medulla neuroblasts. This comprehensive study of the medulla neuroblast temporal cascade illustrates mechanisms that may be conserved in the temporal patterning of neural progenitors (Zhu, 2022).

scRNA-Seq analysis revealed the temporal progression of transcriptional profiles as medulla NBs age at single-cell resolution. Candidates were discovered of critical temporal patterning regulators including eight previously unknown TTFs, as well as TFs such as Nerfin-1 and Lola, that are also involved in the temporal patterning process. Further experimental validation of previously unknown TTFs and other crucial regulators confirmed the accuracy of the high-resolution data, supporting that scRNA-seq is a powerful tool to study the highly dynamic temporal patterning process. This analysis and further experimental investigation revealed a comprehensive temporal cascade in Drosophila medulla NBs: Hth+SoxN+dmrt99B->Opa->Ey+Erm->Ey+Opa->Slp+Scro->D->BarH1&2->Tll, Gcm (see A schematic model summarizing the medulla TTF cascade and its regulation.), and also illustrated several principles that are likely conserved during the temporal patterning of neural progenitor (Zhu, 2022).

The expression of Scro is indicated only by its transcriptional pattern. The expression of Hth, SoxN, and Dmrt99B all start in the neuroepithelium (NE), Erm has a stripe in the transition zone from NE to NB, whereas other TTFs are initiated in NBs. Different isoform compositions of Lola are indicated by different colors in the Model. The number of NE and NB cells does not indicate the actual number of cell cycles they go through. Extensive cross-regulations were identified between these TTFs, which generally follow the rule that a TTF is required to activate the next TTFs and repress the previous TTF, but with a few important exceptions. This TTF cascade controls the sequential generation of different neural types by regulating the expression of neuronal transcription factors, and examples of neural types were also indicated (Zhu, 2022).

First, this study identified early temporal factors that initiate the medulla neuroblast TTF cascade. Before this study, Hth was proposed to be the only TTF at play during the earliest temporal stage. Hth is expressed in the neuroepithelium and the youngest NBs. It is necessary for the generation of Bsh neurons, but is required neither for the NE to NB transition nor for the further temporal cascade progression. Loss of Ey also does not affect the termination of Hth. These data suggested missing links between Hth and the later TTF cascade. Here,several previously unknown TTFs were identified that linked the whole cascade together. Two of those TTFs that start their expression in the NE, SoxN, and Dmrt99B, are also required for the first temporal fate (Bsh neurons), and Dmrt99B is required for the timely activation of Opa in the youngest NBs. Opa is then required to activate Ey and repress Hth. Interestingly, the three TTFs inherited from NE maintain their expression for different durations in NBs, as Hth is repressed by Opa and Erm, SoxN is repressed by Ey, whereas Dmrt99B expression extends until the Slp stage. Whether this differential downregulation is significant for temporal patterning is currently unknown. However, it is worth noting that the expression of mammalian orthologs of Dmrt99B, Dmrt3, and Dmrta1, also starts in symmetrically dividing early cortical progenitors (NE), and decreases gradually in asymmetrical dividing cortical progenitors due to the direct suppression by FoxG1, the mammalian ortholog of Slp1/2 Given the essential role of Dmrt99B in initiating temporal patterning in medulla neuroblast, it will be interesting to investigate whether its mammalian orthologs play conserved roles in the temporal patterning of cortical progenitors (Zhu, 2022).

Second, it was shown that a broad temporal stage can be divided into sub-temporal stages by combinations of TTFs, which determine the progeny fates. This is well-illustrated in the Ey stage. The first stripe of Opa is necessary to initiate the expression of Erm and Ey, which are then required to repress Opa in a negative feedback loop, generating a gap in Opa expression. Furthermore, the data suggest that Ey may first enhance the activation of Erm at the gap, but then possibly a higher level of Ey is required to repress Erm, either directly or indirectly. After Erm is turned off, Opa is turned back on. At the same time, Slp has been gradually activated by Ey and Scro, and when it reaches a certain level, it will repress Opa and Ey to end the Ey stage. Thus, cross-regulations among TTFs divide the Ey stage into (at least) two (sub-)temporal stages determined by the co-expression of Ey and Erm, or Ey and Opa. It was shown that different neural types are generated in these two sub-temporal stages, and the first set of neurons require both Ey and Erm, whereas the second set of neurons require both Ey and Opa. It is interesting to note, the mammalian ortholog of Erm, Fezf2, is also expressed in cortical progenitors and plays important roles in cortical neuron specification (Zhu, 2022).

Third, this study demonstrated that a TTF that is required for the switch to gliogenesis at the final stage is also required for the cell-cycle exit and termination of the medulla TTF cascade. Previously it was thought that Tll stage NBs switch to gliogenesis and then exit the cell cycle, but whether Tll indeed plays a role in these processes has not been studied. In this study the scRNA-Seq data suggested another final temporal stage marked by the expression of Gcm and Dap. Further, it was shown that BarH1 and BarH2 are required to activate both Tll and Gcm, but Tll is activated first, and when Gcm is activated, Gcm represses Tll. Gcm but not Tll was shown to be required for the NBs to switch to gliogenesis and exit the cell cycle. Gcm is well-known for its role in gliogenesis, but this study shows that it is also required and sufficient to activate Dap expression in NBs, possibly through which to promote cell-cycle exit and end the temporal progression. In vertebrate retina, scRNA-seq analysis of retinal progenitor cells identified NFI factors as required for both late-born cell fates including Muller glia and for exiting the cell cycle. As neural progenitors often switch to produce glia at the end of the lineage, it is possibly a general mechanism that factors required for the switch to gliogenesis are also required for the mitotic exit to end the temporal progression (Zhu, 2022).

Another factor that is likely involved in the final stage is Nerfin-1. The expression of Nerfin-1 is observable mostly in maturing neurons, and is required to prevent neurons from de-differentiation. However, this TF responsible for maintaining the differentiation status of neurons, is turned on in the final-stage NBs, where it may function to promote gliogenesis and help terminate the temporal cascade on time. The fast exit of the cell cycle at the final stage is likely accomplished because self-renewal repressors that usually function in GMCs and neurons, such as Prospero and Nerfin-1, gather and cooperate in the oldest NBs. Whether Nerfin-1 can be characterized as a TTF is a remaining question. Since Nerfin-1 expression in both the oldest NBs and the newly born glia is very transient, and cell cycle exit is coupled with glia generation in the oldest NBs, it is not easy to distinguish when exactly Nerfin-1 functions to contribute to the termination of the final temporal stage. The mechanism behind Nerfin-1's requirement at the final stage may be different from the mechanism used in neurons preventing their de-differentiation. One evidence is that while a previous study showed that double knockdown of Nerfin-1 and Su(H) could reduce most ectopic NBs generated by single knockdown of Nerfin-1, suggesting that Nerfin-1 represses Notch signaling in neurons to prevent their de-differentiation, there are always several ectopic NBs remaining located at the medial edge inside the double knockdown clones86. The location of those ectopic NBs indicates that they are likely the oldest NBs unable to exit the cell cycle. Therefore, Nerfin-1 may function through a different mechanism in the final-stage NBs, which is not dependent on the downregulation of Notch signaling. Finally, this study showed that Nerfin-1 is not required for Gcm expression, and it remains to be determined whether Gcm regulates Nerfin-1 expression in this process (Zhu, 2022).

Fourth, complex cross-regulations were observed among TTFs that form temporal gene networks. The model for the cross-regulations between medulla TTFs was that each TTF activates the next TTF and inhibits the previous TTF from the Ey stage to the end of the cascade, exhibiting a simple combination of feedforward activation and feedback repression. However, based on the experimental evidence this study produced as well as inferred from the scRNA-seq data that the cross-regulations among TTFs are more complex. One TTF is not necessarily repressed by the very next TTF, or activated by the exactly previous TTF. Hth is repressed by Opa and Erm. SoxN is repressed by Ey, while Dmrt99B is likely to be repressed by Slp or later TTFs. Tll is activated just before Gcm, however, Tll is not required for Gcm's activation. The complexity of their cross-regulation is a way to increase the number of combinations of TTFs in aging NBs, thereby increasing the number of possible neuron fates determined along with the temporal progression. However, the overall trend that early TTFs activate late TTFs, and late TTFs repress early TTFs remains valid (Zhu, 2022).

Finally, this study demonstrated that the speed of the TTF cascade progression is regulated by Lola factors expressed in all NBs. Lola proteins belong to a BTB/POZ family of proteins which have been shown to be involved in chromatin remodeling and organization. Certain isoforms of Lola are expressed in all NBs, e.g., Lola-F is activated one cell cycle earlier than Opa. This study showed that Lola proteins function as a speed modulator of the temporal cascade progression. It represses the expression of Hth, facilitates the activation of Opa and the following TTFs to different extents, thereby guaranteeing a quick transition from the NE TTF network to the NB TTF network. Interestingly, the vertebrate ortholog of lola, Zbtb20, was also found to modulate the sequential generation of different neural types in cortical progenitors. Loss of Zbtb20 causes the temporal transitions to be delayed further and further, very similar to the loss of lola phenotype in this system. Thus, it is possible that lola/Zbtb20 play conserved roles in the temporal patterning of neural progenitors (Zhu, 2022).

In summary, the entire life of a medulla neuroblast from the beginning to the end was revealed in this study. This comprehensive study of the medulla neuroblast temporal cascade illustrated mechanisms that may be conserved in the temporal patterning of neural progenitors. The single-cell RNA-sequencing data provide a plethora of information that allows further exploration of the mechanisms of temporal patterning (Zhu, 2022).

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 it has recently been 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).

SWI/SNF regulates the alternative processing of a specific subset of pre-mRNAs in Drosophila melanogaster

The SWI/SNF chromatin remodeling factors have the ability to remodel nucleosomes and play essential roles in key developmental processes. SWI/SNF complexes contain one subunit with ATPase activity, which in Drosophila is called Brahma (Brm). The regulatory activities of SWI/SNF have been attributed to its influence on chromatin structure and transcription regulation, but recent observations have revealed that the levels of Brm affect the relative abundances of transcripts that are formed by alternative splicing and/or polyadenylation of the same pre-mRNA. This study investigated whether the function of Brm in pre-mRNA processing in Drosophila is mediated by Brm alone or by the SWI/SNF complex. The effects of depleting individual SWI/SNF subunits on pre-mRNA processing was examined throughout the genome, and a subset of transcripts was identified that are affected by depletion of the SWI/SNF core subunits Brm, Snr1 or Mor. The fact that depletion of different subunits targets a subset of common transcripts suggests that the SWI/SNF complex is responsible for the effects observed on pre-mRNA processing when knocking down Brm. Brm was also depleted in larvae, and it was shown that the levels of SWI/SNF affect the pre-mRNA processing outcome in vivo. This study has shown that SWI/SNF can modulate alternative pre-mRNA processing, not only in cultured cells but also in vivo. The effect is restricted to and specific for a subset of transcripts. These results provide novel insights into the mechanisms by which SWI/SNF regulates transcript diversity and proteomic diversity in higher eukaryotes (Waldholm, 2011).

Previous studies have shown that Brm influences the alternative processing of a subset of pre-mRNAs in human and insect cell lines (Batsche, 2006; Ito, 2008; Tyagi, 2009) but the mechanisms responsible for such regulation are not known. In these studies, a functional link was found between the levels of Brm and the splicing outcome was established after experimental alteration of the Brm levels in cultured cells, either by over-expression or by RNAi-mediated depletion. These studies focused on the Brm subunit. This study has now extended the previous studies and asked whether depletion of other SWI/SNF subunits also results in alterations of pre-mRNA processing. In addition to Brm, this study disrupted Mor, Snr1, PB, Bap170 and Osa in Drosophila S2 cells. Also, microarray data was mined, looking for genes whose relative abundances between alternative transcripts are changed by the RNAi treatment in a different manner than the abundances in mock-treated samples. The number of genes affected was low, but many of the detected events could be validated. This small collection of validated genes is valuable for future mechanistic studies (Waldholm, 2011).

The reasons for the low number of genes affected could be partly technical. First, the data used was obtained from expression arrays that do not cover all the splicing variants of the transcriptome of Drosophila. Second, the variances in the datasets were relatively high and, for this reason, attempts were made to avoid false positives by establishing stringent criteria and discarding genes that did not show consistent results in the replicates. In spite of these limitations, a total of 45 genes were identified for which the pre-mRNA processing levels changed after depletion of SWI/SNF subunits. Depletion of different SWI/SNF subunits affected different genes with a statistically significant overlap, in particular for the core subunits of the SWI/SNF complex. Indeed, a group of ten genes were identified that, according to the microarray data, were affected by depletion of at least two different core subunits. In summary, these results show that depletion of other core subunits apart from Brm influences pre-mRNA processing. This conclusion agrees with observations in human cells, where Brm modulates the splicing of the TERT transcripts in concert with the mRNA-binding protein p54(nrb) (Ito, 2008). In the same study, it was shown that p54(nrb) and core subunits of the SWI/SNF complex interact physically (Waldholm, 2011).

This study has analyzed the decay of the transcripts affected by depletion of SWI/SNF subunits and differential stability can be ruled out as a major cause for the differences observed in the relative abundances of alternative transcripts. Using ChIP, it was also shown that Brm, Snr1 and Mor are asociated with the genes affected. Altogether, these observations support the conclusion that the mechanism by which SWI/SNF affects pre-mRNA processing is direct and cotranscriptional (Waldholm, 2011).

Alternative splicing and polyadenylation are major sources of transcript diversity and proteomic diversity in higher eukaryotes. Complex regulatory networks determine the premRNA processing outcome and play critical roles in differentiation and development. Key elements in such regulatory networks are the splicing and polyadenylation factors that influence the choice of alternative processing sites by binding to cis-acting elements, either enhancers or silencers, in the pre-mRNAs. Recent research has revealed that, in addition to the RNA sequence itself, the chromatin environment and the transcription machinery contribute to the recruitment of regulatory factors to their target transcripts during transcription. Genome-wide studies have shown that certain histone modifications are non-randomly distributed in exons and introns, and that nucleosomes are enriched in exonic sequences. The functional significance of these observations is not fully understood, but there are examples of adaptor proteins that bind both to splicing factors and to specific histone modifications, and such adaptors may play important roles in the targeting of regulatory factors to the pre-mRNA. Another determinant of the splicing outcome is the elongation rate of RNAP II. A reduction of the RNAP II elongation rate at specific positions along the gene can facilitate the assembly of the splicing machinery at weak splice sites and promote the inclusion of proximal exons. hBrm regulates the alternative splicing of the CD44 pre-mRNA in human cells by decreasing the elongation rate of RNAP II and inducing the accumulation of the enzyme at specific positions in the gene. In the case of the CD44 gene, Brm favors the usage of proximal processing sites. This study has shown that in Drosophila the SWI/SNF complex regulates the processing of a subset of pre-mRNAs through somehow different mechanisms. In some of the cases that this study has analyzed, depletion of SWI/SNF promotes the use of a proximal splice site (for instance, the up-regulation of the lola-RA transcript), which cannot be explained by the same mechanisms that act on the human CD44 gene. Several alternative mechanisms can be envisioned. In one scenario, SWI/SNF either decreases or increases the transcription rate, depending on the genomic context and on the presence of specific regulators. Alternatively, SWI/SNF could act by a mechanism that is independent from the transcription kinetics. It was previously shown that a fraction of SWI/SNF is associated with nascent transcripts, while other studies have shown that Brm and specific mRNA-binding proteins interact. It is tempting to speculate that SWI/SNF plays a more direct role in pre-mRNA processing, possibly by modulating the recruitment and/or assembly of splicing or polyadenylation factors (Waldholm, 2011).

Previous research on the role of Brm in pre-mRNA processing was carried out in cultured cells. This study has now depleted Brm in larvae and detected changes in pre-mRNA processing in vivo. Depletion of Brm had no significant effect on two of the four genes analyzed, CG3884 and mod(mdg4). Depletion of Brm in vivo affected lola and Gpdh, in contrast, in a similar manner to its effect in S2 cells. It is important to point out that this study analyzed RNA extracted from total larvae, not from individual organs, which might have occluded tissue-specific effects. Indeed, the gene expression data in FlyAtlas (http://www.flyatlas.org/) shows that the expressions of the analyzed genes vary among organs and throughout development. Analyzing total larvae gives an average of the effects in the entire organism, which might not reflect the physiological regulation of the target genes in any specific tissue. However, the fact that Brm depletion affects the processing of the lola and Gpdh transcripts in larvae shows that the reported effect of SWI/SNF on pre-mRNA processing is not an artefact that occurs only in cultured cells (Waldholm, 2011).

The lola and Gpdh genes are structurally very different. Gpdh is a relatively short gene with three alternative mRNAs that encode nearly identical proteins. The alternative processing of the Gpdh pre-mRNA determines the sequence of the 3' UTRs, which can have a profound impact on the stability of the transcripts, their regulation by microRNAs and their translational properties. The lola gene, in contrast, is very long with at least 26 different transcripts that code for a plethora of protein isoforms characterized by different types of DNA-binding motifs. Therefore, in vivo regulation of lola by SWI/SNF affects the abundances of protein isoforms with different biological activities (Waldholm, 2011).

This study has shown that SWI/SNF can modulate alternative pre-mRNA processing, not only in cultured cells but also in vivo. The effect is restricted to and specific for a subset of transcripts, both in S2 cells and in larvae. The results provide novel insights into the mechanisms by which SWI/SNF regulates transcript diversity and proteomic diversity in higher eukaryotes (Waldholm, 2011).

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).

Epigenetic silencers Lola and Pipsqueak collaborate with Notch to promote malignant tumours by Rb silencing

Cancer is both a genetic and an epigenetic disease. Inactivation of tumour-suppressor genes by epigenetic changes is frequently observed in human cancers, particularly as a result of the modifications of histones and DNA methylation. It is therefore important to understand how these damaging changes might come about. By studying tumorigenesis in the Drosophila eye, two Polycomb group epigenetic silencers, Pipsqueak and Lola, have been identified that participate in this process. When coupled with overexpression of Delta, deregulation of the expression of Pipsqueak and Lola induces the formation of metastatic tumours. This phenotype depends on the histone-modifying enzymes Rpd3 (a histone deacetylase), Su(var)3-9 and E(z), as well as on the chromodomain protein Polycomb. Expression of the gene Retinoblastoma-family protein (Rbf ) is downregulated in these tumours and, indeed, this downregulation is associated with DNA hypermethylation. Together, these results establish a mechanism that links the Notch-Delta pathway, epigenetic silencing pathways and cell-cycle control in the process of tumorigenesis (Ferres-Marco, 2006).

Correct organ formation depends on the balanced activation of conserved developmental signalling pathways (such as the Wnt, Hedgehog and Notch pathways). If insufficient signals are received, organ growth may be deficient. By contrast, excess signalling leads to an overproduction of progenitor cells and a propensity to develop tumours. When such hyperproliferation is associated with the capacity of cells to invade surrounding tissue and metastasis to distant organs, cancer develops. Indeed, activation of the Wnt, Hedgehog and Notch pathways is a common clinical occurrence in cancers. Curiously, activation of any of these pathways in animal models seems to be insufficient for cancer to develop, indicating that synergism with other genes is required for these pathways to produce cancer (Ferres-Marco, 2006).

Cellular memory or the epigenetic inheritance of transcription patterns has also been implicated in the control of cell proliferation during development, as well as in stem-cell renewal and cancer. Proteins of the Polycomb group (PcG) are part of the memory machinery and maintain transcriptional repression patterns. The upregulation of several PcG proteins has been associated with invasive cancers. Thus, increased amounts of EZH2, the human homologue of the Drosophila histone methyltransferase E(z), is associated with poorer prognoses of breast and prostate cancers (Ferres-Marco, 2006).

Another histone methyltransferase implicated both in gene silencing and in cancer is SUV39H1, a homologue of Drosophila Su(var)3-9. SUV39H1 and Su(var)3-9 methylate histone H3 on lysine 9 (H3K9me), and this epigenetic tag is characteristic of heterochromatin and DNA sequences that are constitutively methylated in normal cells. DNA methylation is another mechanism involved in cellular memory that actively contributes to cancer. Indeed, numerous tumour-suppressor genes, including the retinoblastoma gene RB, are silenced in cancer cells by DNA hypermethylation. Inactivation of the RB tumour-suppressor pathway is considered an important step towards malignancy; thus, it is important to understand how these damaging epigenetic changes are initiated in cells that become precursors of cancer. Moreover, it is equally important to determine the connection between these processes and the developmental pathways controlling proliferation (Ferres-Marco, 2006).

Forward genetic screening in suitable animals is a powerful tool with which to identify tumour-inducing genes and to reveal changes that precede neoplastic events in vivo. The developing eye of Drosophila melanogaster is a good model for such studies because it is a simple and genetically well-defined organ. The growth of the eye depends on Notch activation in the dorsal-ventral organizer by its ligands Delta (human counterparts, DLL-1, -3, -4) and Serrate (human counterparts, JAGGED-1, -2). This study used the 'large eye' phenotype, produced by overexpression of Delta, as a tool to screen for mutations that interact with the Notch pathway and convert tissue overgrowths into tumours. One mutation, eyeful, was isolated that combined with Delta induces metastatic tumours. eyeful forces the transcription of two hitherto unsuspected growth and epigenetic genes, lola and pipsqueak (psq). The identification of eyeful has been a starting point from which to unravel crosstalk between the Notch and epigenetic pathways in growth control and tumorigenesis. The fact that many epigenetic factors are involved in cancer suggests that these processes may be more generally involved in tumorigenesis than at first it might seem (Ferres-Marco, 2006).

To identify genes that interact with the Notch pathway and that influence growth and tumorigenesis, the Gene Search (GS) system was used to screen for genes that provoked tumours when coexpressed with Delta in the proliferating Drosophila eye. The ey-Gal4 line was used for both eye-specific and ubiquitous induction, resulting in the transactivation of UAS-linked genes throughout the proliferating eye discs. It was through such a screen that the GS88A8 line was isolated. Generalized overexpression of Delta by ey-Gal4 (hereafter termed ey-Gal4 > Dl) produces mild eye overgrowth. In most of the flies in which the GS88A8 line was coexpressed with Delta, tumours developed in the eyes. Moreover, in ~30% of the mutant flies, secondary eye growths were observed throughout the body, and in some flies the whole body filled up with eye tissue. These secondary eye growths had ragged borders, indicating invasion of the mutant tissue into the surrounding normal tissue. As a result, the GS88A8 line was named 'eyeful' (Ferres-Marco, 2006).

A developmental analysis of the tumours was undertaken. To facilitate analyses, a triple mutant strain was generated carrying the eyeful, UAS-Dl and ey-Gal4 transgenes all on the same chromosome (ey-Gal4 > eyeful > Dl. In this strain, mutant eye discs showed massive uncontrolled overgrowth (some discs were more than five times their normal size). In most discs, the epithelial cells had lost their apical-basal polarity, and some had a disrupted basement membrane and grew without differentiating (Ferres-Marco, 2006).

These results were extended to the wing disc. (1) dpp-Gal4 was used to direct coexpression of eyeful and Delta along the anterior-posterior boundary of the wing (perpendicular to the endogenous Delta domain along the dorsal-ventral boundary. In a normal wing disc, the dpp-Gal4 driver typically establishes a stripe of green fluorescent protein (GFP) expression with a sharp border at the boundary. Whereas wild-type (or single eyeful) cells expressing GFP conformed with this pattern, some of the eyeful and Delta cells were found outside this stripe, indicating that the mutant cells can disseminate and invade adjacent regions of the disc. (2) The MS1096-Gal4 line was used to direct expression in the dorsal wing disc compartment. Under these conditions, the wing tissue grew massively and aggressively, and the mutant tissue failed to differentiate. These observations suggest that, when coupled with Delta overexpression, an excess of the gene products flanking the eyeful insertion site induces the formation of tumours capable of metastasising (Ferres-Marco, 2006).

The genomic DNA flanking the eyeful P-element was isolated and sequenced. eyeful is inserted in an intron of the gene longitudinals lacking (lola), which is known to be a chief regulator of axon guidance. lola encodes 25 messenger RNAs that are produced by alternative splicing and that generate 19 different transcription factors. All of the different isoforms share four exons that encode a common amino terminus, which contains a BTB or POZ domain. In addition, all but one of these transcription factors are spliced to unique exons encoding one or a pair of zinc-finger motifs (Ferres-Marco, 2006).

The GS P-elements allow Gal4-dependent inducible expression of sequences flanking the insertion site in both directions. The nearest gene in the opposite direction to transcription of lola is the psq gene. This gene encodes nine variants produced by alternative splicing and alternative promoter use, generating four different proteins. Three of the psq isoforms contain a BTB or POZ domain in the N terminus, and a histidine- and glutamine-rich region downstream of this domain. Two of the BTB-containing isoforms and the isoform that lacks this domain contain four tandem copies of an evolutionarily conserved DNA-binding motif, the Psq helix-turn-helix (HTH) motif (Ferres-Marco, 2006).

psq was initially identified for its 'grandchildless' and posterior group defects and was subsequently shown to have a role in retinal cell fate determination. Psq is essential for sequence-specific targeting of a PcG complex that contains histone deacetylase (HDAC) activity. Psq binds to the GAGA sequence, which is present in many Hox genes and in hundreds of other chromosomal sites (Ferres-Marco, 2006).

Both polymerase chain reaction with reverse transcription (RT-PCR) and in situ hybridization experiments confirmed that transcription of lola and psq was influenced by eyeful in response to Gal4 activation (Ferres-Marco, 2006).

To determine whether lola and/or psq was responsible for the tumour phenotype, 11 enhancer promoter (EP) P-elements inserted into the lola and psq region were tested. In contrast to the GS lines, the EP lines allows Gal4-dependent inducible expression of sequences flanking only one end of the P-element. It was found that none of the EP lines induced tumours; thus, it was reasoned that the deregulation of both genes might be required to produce the tumours (Ferres-Marco, 2006).

The complexity of lola and psq loci, which together produce 23 proteins, hampers identification of the transcripts responsible for the eyeful phenotype by gain-of-expression mutants (that is, by expressing individual or combinations of isoforms). Therefore, this issue was resolved by isolating point mutations that reverted the phenotype caused by deregulated expression of lola and psq. In this analysis, the chemical mutagen ethyl-methane sulphonate (EMS) was used to induce preferentially single nucleotide changes (Ferres-Marco, 2006).

The parental eyeful GS line was viable in trans with deficiencies that removed both lola and psq. In contrast, a set of 14 EMS-induced mutations on the eyeful chromosome failed to complement these deficiencies and were found to be alleles of psq or lola. The EMS-induced mutations that best recovered a normal eye size were sequenced. Each individual mutation had a single base change or a small deletion that considerably altered the predicted Psq or Lola proteins (Ferres-Marco, 2006).

All psq^- mutations induced on the eyeful chromosome prevented eyeful from producing eye tumours and metastases. Three alleles affected the BTB domain (psq^rev2, psq^rev7 and psq^rev9), and three other alleles contained either a premature stop codon that would produce truncated proteins lacking the Psq HTH repeats (psq^rev4 and psq^rev14) or a missense mutation that would change a conserved amino acid in the third Psq HTH repeat (psq^rev12). All lola^- mutations induced on the eyeful chromosome, including the presumptive null allele (lola^rev6), reduced eye tumour size but still permitted sporadic secondary growth (Ferres-Marco, 2006).

These data show an unequal contribution of Psq and Lola in this process, whereby Psq is the most important factor in the tumorigenic phenotype. The BTB subfamily of transcriptional repressors includes the human oncogenes BCL6 and PLZF. In these oncogenes, the BTB domain is crucial for oncogenesis through the recruitment of PcG and HDAC complexes. It is therefore speculated that deregulated Psq and Lola could lead to tumorigenesis by epigenetic processes and that Drosophila counterparts of HDACs and PcG proteins might be involved in the progression of these tumours. Indeed, genetic evidence was found that both Lola and Psq function as epigenetic silencers in vivo (Ferres-Marco, 2006).

Attempts were made to determine the specific epigenetic mechanisms through which deregulation of Psq and Lola might induce tumorigenesis in conjunction with Delta overexpression. Methylation of histone on lysine is a central modification in both epigenetic gene control and in large-scale chromatin structural organization. For example, trimethylation of histone H3 on K4 (H3K4me3) is associated with the active transcription of genes and open chromatin structure. By contrast, histone hypoacetylation and H3K9 and H3K27 methylation are characteristic of heterochromatin state and gene silencing. To determine whether any changes in these epigenetic markers might coincide with the induction of tumorigenesis, eye discs were immunolabelled with antibodies against specific histone H3 modifications. Because dorsal eye disc cells are refractory to Delta, the dorsal region of the discs provided an internal control for these studies (Ferres-Marco, 2006).

With the exception of some scattered cells, a prominent loss or strong reduction of H3K4me3 was observed in the ventral region of the mutant discs. Notably, although the loss of Notch in clones does not affect this epigenetic tag, overexpression of Delta caused a significant reduction in staining for H3K4me3. The H3K4me3 depletion was already apparent in discs showing moderate hyperplasia and thus preceded neoplasm formation. Changes in other epigenetic tags (such H3K9me3 and H3K27me2) could not be reproducibly resolved; perhaps more sensitive methods or antibodies might facilitate detection of such changes (Ferres-Marco, 2006).

H3K4 methylation is thought to be permissive for maintaining and propagating activated chromatin and is thought to neutralize repressor tags by precluding binding of the HDAC complex and impairing SUV39H1-mediated H3K9 methylation. Thus, H3K4me3 depletion may contribute to tumour formation by permitting aberrant chromatin silencing. It was found that a 50% reduction in dosage of the HDAC gene Rpd3 or of Su(var)3-9 decreased the tumour phenotype dominantly. In contrast, reducing the activity of the H3K4 histone methyltransferase genes Trx (known as ALL1 or MLL in humans) or Ash1, which would be expected to deplete the H3K4me3 tag further, did not visibly enhance the tumours (Ferres-Marco, 2006).

E(z) when complexed with the Extra sex combs (Esc) protein becomes a histone methyltransferase. The E(z)-Esc complex and its mammalian counterpart Ezh2-Eed show specificity for H3K27 but may also target H3K9. The complex also contains the HDAC Rpd3, and this association with Rpd3 is conserved in mammals. H3K27 methylation facilitates binding of the chromodomain protein Pc (HPC in humans), which then creates a repressive chromatin state that is a stable silencer of genes (Ferres-Marco, 2006).

Although loss of E(z) does not cause proliferation defects within discs, halving the E(z) gene dosage dominantly suppressed tumorigenesis, indicating that histone methylation by the E(z)-Esc complex is also a prerequisite for the excessive proliferation of these tumours. Accordingly, Esc^- or Pc^- mutations also notably reduced the tumours (Ferres-Marco, 2006).

Together, these findings suggest that the development of these tumours involves, at least in part, changes in the structure of chromatin brought about by covalent modifications of histones. These changes probably switch the target genes from the active H3K4me3 state to a deacetylated H3K9 and H3K27 methylation silent state (Ferres-Marco, 2006).

From this above data, it is considered that the tumours might form as a result of aberrant gene silencing. If so, then the expression of genes involved in cell-cycle control is likely to be altered in the mutant cells. The transcription of 12 tumour-related genes in the mutant and wild-type discs was compared. Transcription of the gene Rbf, a fly homologue of the RB/Rb family of genes, was strongly downregulated in this assay (and even in ey-Gal4 > Dl flies). A second Rb gene, Rbf2, remained unchanged in the different genetic backgrounds, highlighting the specificity of Rbf silencing (Ferres-Marco, 2006).

It was found that Rbf depletion seems to be intricately associated with tumorigenesis: (1) reducing Rbf gene dosage by 50% enhanced tumour growth; (2) re-establishing Rbf expression in the eye (using an UAS-Rbf transgene) consistently prevented eye tumours and occurrence of secondary growths (Ferres-Marco, 2006).

Inactivation of RB1 in retinoblastoma, a form of eye cancer in children, can occur through DNA hypermethylation of the promoter. Unlike in mammals, however, there is little cytosine methylation of the genome in Drosophila during developmental stages, and its potential role during tumorigenesis is unknown. DNA methylation seems to depend on one DNA methyltransferase, Dnmt2, that preferentially methylates cytosine at CpT or CpA sites. The fly genome also encodes one methyl-CpG DNA-binding MBD2/3 protein. Because there are no known Dnmt2 loss-of-function mutations, the role of this gene in tumorigenesis could not be tested (Ferres-Marco, 2006).

Nevertheless, whether the CpG islands that were observed in the Rbf gene were potential targets for repression by DNA methylation was tested by two methods. (1) Methylation-sensitive restriction enzymes analysis was used; this showed that the regions around the promoter and transcription start site of the Rbf gene are susceptible to methylation. This approach showed aberrant DNA hypermethylation of Rbf in eyeful and Delta eye discs and mild hypermethylation in Delta discs; however, at best only very mild methylation was detected in discs from wild-type flies or from flies with the control psq gene (Ferres-Marco, 2006).

(2) Direct bisulphite sequencing of genomic DNA was carried out from mutant discs. This approach confirmed the notable increase in methylated DNA in eyeful and Delta discs when compared with wild-type discs (and a moderate increase in methylated DNA in the Delta discs). Hypermethylation of the Rbf promoter was not simply the result of de novo transcription of Dnmt2 (ey-Gal4 > Dnmt2), indicating that activation of the Notch pathway is a crucial step in this de novo hypermethylation of Rbf (Ferres-Marco, 2006).

This study has used Drosophila genetics to search for genes that collaborate with the Notch pathway during tumorigenesis in vivo. Psq and Lola were identified as decisive factors to foment tumour growth and invasion when coactivated with the Notch pathway. These proteins are presumptive transcription repressors that contain a BTB domain and sequence-specific DNA-binding motifs and behave as epigenetic silencers in vivo (Ferres-Marco, 2006).

In addition, crosstalk between the Notch pathway and different epigenetic regulators was identified. It is likely that alterations in this crosstalk provoke the aberrant epigenetic repression (and perhaps also derepression) of genes that contribute to cellular transformation. The Rbf gene was identified as one target for this epigenetic regulation and Rbf depletion was shown to directly contribute to the tumours (Ferres-Marco, 2006).

It is proposed that the sequence of events that leads to these tumours commences with hyperactivation of the Notch pathway, which initiates gene repression. Subsequently, or at the same time as Notch, Psq-Lola could bind to the silenced genes and enforce silencing by recruitment of HDAC or PcG repressors. Given the conservation of the Psq-like HTH domains in Psq and of BTB domains, it seems likely that other transcriptional repressors containing such domains strongly influence the tumour-inducing capacities of HDACs and PcG repressors in human cancers (Ferres-Marco, 2006).

Finally, the collaboration between PcG-mediated cellular memory and the Notch pathway may have implications in other processes controlled by Notch, including the second mitotic wave in the Drosophila eye, and the organization of eye and wing growth. In these processes, the memory mechanism could ensure that cells keep a record of the Notch signals received at an earlier stage or when the progenitor cells were closer to the Delta source. In this way, they might remain proliferative without having to receive continuous instructions from Notch. Likewise, such a situation could be conceived for tumorigenesis. The oncogenic signals could opportunistically take advantage of the memory mechanism to fix and to maintain their instructions of continuous proliferation in progenitor or stem cells, thereby fostering tumour growth and metastasis (Ferres-Marco, 2006).

Lola regulates cell fate by antagonizing Notch induction in the Drosophila eye

Lola is a transcription repressor that regulates axon guidance in the developing embryonic nervous system of Drosophila. Lola regulates two binary cell fate decisions guided by Notch inductive signaling in the developing eye: the R3-R4 and the R7-cone cell fate choices. Lola is required cell-autonomously in R3 for its specification, and Lola transforms R4 into R3 if overexpressed. Lola also promotes R7 fate at the expense of cone cell fate. Lola antagonizes Notch-dependent gene expression and Notch-dependent fate transformation. Expression analysis shows that Lola is constitutively present in all photoreceptors and cone cells. It is proposed that when a precursor cell receives a weak Notch inductive signal, it is not sufficiently strong to overcome the constitutive repression of target gene transcription by Lola. A precursor that receives a strong Notch inductive signal expresses target genes despite a constitutive repression by Lola. The predicted consequence of this mechanism is to sharpen a cell’s responsiveness to Notch signaling by creating a threshold (Zheng, 2008).

Lola regulates two cell-fate decisions in the eye: the R3 vs. R4 decision and the R7 vs. cone cell decision. Lola promotes R3 fate at the expense of the R4 fate, and it promotes R7 fate at the expense of the cone cell fate. Lola appears to influence these binary fate decisions by acting on responsiveness to Dl-N signaling. Lola regulates N-dependent gene expression, and Lola is able to suppress the effects of constitutively-active N on cell fate decisions. This latter result argues that Lola either acts downstream of N in the N signaling pathway or acts in a pathway that is convergent downstream with the N signaling pathway (Zheng, 2008).

Lola antagonizes 'strong N' signaling, which is evident by overexpressed Lola antagonizing strong N-dependent gene expression in R4 and transforming it into an R3-type. This is interpreted to mean that Lola normally makes the R3 precursor more resistant against induction into a R4-type, an interpretation that is consistent with mosaic analysis indicating that Lola is required in the R3 cell. Within the R3 precursor cell, Lola might repress transcription of genes that are normally activated by strong N signaling, such as the E(spl)m∂ gene. Doing so, Lola then would ensure the precursor would not inappropriately adopt an R4 cell fate. However, this notion raises a paradox: since Lola is expressed somewhat equivalently in all photoreceptors including R4, why does it not repress genes such as E(spl)m∂ in the R4 cell? One explanation is that it represses these genes in the R4 precursor but this repression is not strong enough to block their expression when induced by strong N signaling. Two lines of evidence support this explanation. Overexpression of Lola in the R4 precursor is sufficient to block expression of the E(spl)m∂ gene. This argues that Lola activity is a limiting component in N-dependent gene expression. Second, analysis of mutant lola clones reveals that R4 cells in mosaic ommatidia are more likely to be mutant for the lola gene. This result implies that Lola antagonizes N-activated programming of R4 fate in the R4 cell itself (Zheng, 2008).

Based on these observations, a model is proposed in which Lola acts as a transcription repressor of certain N-dependent genes that program the R4 cell fate. It acts on these genes in all cells as they are induced by EGFR to differentiate. In cells that receive no Dl-N signal or a weak Dl-N signal, repression is sufficiently strong to block N-dependent gene expression. However, in cells such as the R4 precursor that receive a strong Dl-N signal, repression is not sufficient to block gene expression, and the cells follow an R4 fate. Thus, Lola functions to make cells respond with a sharper threshold to Dl-N signaling. Since Lola functions in this manner only in cells that have already been induced by EGFR signaling, Lola seems to aid in crosstalk between the two signal transduction pathways. Moreover, the lola gene is repressed by Dl-N signaling; loss of N activity results in ectopic lola expression in many precursor cells. This maybe ensures that only cells induced to differentiate will respond with a sharp threshold to later Dl-N signals they might receive (Zheng, 2008).

Lola protein has sequence-specific transcriptional repressor activity and it genetically interacts with histone remodeling proteins. Thus, Lola could antagonize N signaling by virtue of it specifically binding and repressing transcription of genes that are activated by N signaling. Alternatively, Lola might render genes less responsive to N by specifically interacting with effectors of N signaling. In this regard, strong genetic interactions have been observed between Lola and Hairless, a co-repressor of Su(H) in Drosophila (Muller, 2005). It is not yet possible to distinguish between these two models of interaction (Zheng, 2008).

The Dl-N pathway is also required to specify both R7 and cone cell fates; strong Dl-N signaling induces cone fate and weaker Dl-N signaling induces R7 fate. Precursors with high levels of constitutively-active Notch become cone cells. This transformation is suppressed by Lola overexpression and leads to formation of more R7 cells. Conversely, loss of Lola leads to fewer precursors adopting an R7 fate and more adopting a cone fate, Again, these data fit with a model in which Lola sharpens the threshold response to levels of Dl-N signaling. It helps block strong N-dependent expression in the R7 precursor receiving a weak Dl-N signal, while it is insufficient to block the expression of the same genes in the cone precursors that receive a stronger Dl-N signal. However, in contrast to Lola's role in the R3/R4 fate switch, Lola has less effect on the R7/cone fate switch. Loss of lola causes infrequent R7 cell loss, and there is no significant correlation between the lola genotype of R7 cells in mosaic ommatidia and their normal development. Moreover, flies overexpressing Lola in cone cells only occasionally exhibit ectopic R7 formation. The reason why Lola has less impact on the R7/cone cell fate choice is unclear. Perhaps other factors play a redundant role with Lola in this fate switch (Zheng, 2008).

Genome-wide analysis of self-renewal in Drosophila neural stem cells by transgenic RNAi

The balance between stem cell self-renewal and differentiation is precisely controlled to ensure tissue homeostasis and prevent tumorigenesis. This study use genome-wide transgenic RNAi to identify 620 genes potentially involved in controlling this balance in Drosophila neuroblasts in the larval CNS. All phenotypes and derive measurements were quantified for proliferation, lineage, cell size, and cell shape. A set of transcriptional regulators essential for self-renewal was identified and hierarchical clustering and integration with interaction data were used to create functional networks for the control of neuroblast self-renewal and differentiation. The data identify key roles for the chromatin remodeling Brm complex, the spliceosome, and the TRiC/CCT-complex and show that the alternatively spliced transcription factor Lola and the transcriptional elongation factors Structure specific recognition protein (Ssrp) and Barricade (Barc) control self-renewal in neuroblast lineages. As these data are strongly enriched for genes highly expressed in murine neural stem cells, they are likely to provide valuable insights into mammalian stem cell biology as well (Neumuller, 2011).

As CG6049 had not been characterized before, this gene was chosen for in-depth analysis. CG6049 was named barricade (barc) to indicate the block in Nb lineage progression observed upon RNAi. Barc is conserved from yeast to humans. Like its vertebrate homolog Tat-SF1, it contains two RNA recognition modules (RRM), a nuclear localization signal, and a conserved region that contains two motifs that are known to bind to FF domains and that was named the Barc-Tat-SF1 (BTS) motif. To determine the specificity of the barc-RNAi phenotype, an RNAi-resistant barc construct was generated. When expressed together with barc-RNAi, this construct can rescue both lethality and the Nb phenotype. In addition, the barc-RNAi phenotype could be confirmed by a second, nonoverlapping RNAi line. Thus, barc is a regulator of lineage progression in Drosophila Nbs (Neumuller, 2011).

While barc-RNAi in type II lineages using wor-Gal4; ase-Gal80 causes overproliferation, barc-RNAi induced by ase-Gal4 has no overproliferation phenotype. The additional CD8::GFP-positive cells in the type II lineages express Cyclin E, indicating active proliferation, and do not express the neuronal marker Elav. More cells positive for Mira and Dpn were observed, that are expressed both in Nbs and in INPs. On average, the number of Mira-positive cells is approximately 4-fold increased. Since only one large Ase-negative type II Nb was observed, and the extra cells express the INP marker Ase, it is concluded that barc is required for INPs to generate differentiating neurons. Upon barc-RNAi, the daughter cells retain the INP fate, and this results in the overproliferation phenotype. Although barc-RNAi does not cause a similar overproliferation phenotype in type I lineages, it was observed that the diameter of type I Nbs is increased from 15 ± 0.31 μm to 17.16 ± 0.27 μm. This phenotype could either indicate an increase in growth rate or a decrease in cell cycle progression. Thus, barc is required for lineage progression in type II Nb lineages, but might also have a function in mitotic progression of type I Nbs (Neumuller, 2011).

To test Barc expression and subcellular localization, a peptide antibody was generated. The antibody detects a single band of approximately 75 kD on a western blot, which can be blocked by the antigenic peptide. The anti-Barc immunofluorescence signal is absent after barc-RNAi and increases upon Barc overexpression. Barc antibody staining revealed that Barc is a nuclear protein that is predominantly expressed in both type I and type II Nbs and to a lesser extent in INPs, GMCs, and differentiated neurons. Thus, this study has identified a nuclear regulator of type II Nb lineages that allows INPs to generate daughter cells, which undergo terminal neural differentiation (Neumuller, 2011).

This screen has identified a total of 620 genes that are potentially involved in controlling self-renewal in Drosophila neural stem cells. It was demonstrated that precise quantification of phenotypic data allows for a computer analysis that can lead to biological insights that are not easily obtained through classic single-gene approaches. Through network analysis, splicing control was identified as a key regulator of Nb self-renewal. Alternative splicing of lola might be one of the targets of this machinery as different isoforms of this transcription factor are differentially expressed and phenotypically distinct. It was also shown that duplicated forms of ribosomal subunits are functionally distinct, with one form being more specifically required in Nbs. Finally, it was demonstrated that genes involved in transcriptional elongation and chromatin remodeling are important regulators of Nb self-renewal and differentiation. It is known that more than one third of all Drosophila genes are in a poised state where active RNA polymerase is stalled in a promoter proximal position. Release of stalled polymerases might contribute to the rapid activation of differentiation genes during Nb ACD. Transcriptional elongation is important for controlling vertebrate stem cell lineages as well, but how stalled promoters are released in a cell-type-specific manner is currently unknown. Analysis in the simple Drosophila Nb lineage could shed some light on this important question in stem cell biology (Neumuller, 2011).

Transcriptional regulation of Drosophila gonad formation

The formation of the Drosophila embryonic gonad, involving the fusion of clusters of somatic gonadal precursor cells (SGPs) and their ensheathment of germ cells, provides a simple and genetically tractable model for the interplay between cells during organ formation. In a screen for mutants affecting gonad formation a SGP cell autonomous role was identified for Midline (Mid) and Longitudinals lacking (Lola). These transcription factors are required for multiple aspects of SGP behaviour including SGP cluster fusion, germ cell ensheathment and gonad compaction. The lola locus encodes more than 25 differentially spliced isoforms, and an isoform specific requirement was identified for lola in the gonad, that is distinct from that in nervous system development. Mid and Lola work in parallel in gonad formation and surprisingly Mid overexpression in a lola background leads to additional SGPs at the expense of fat body cells. These findings support the idea that although the transcription factors required by SGPs can ostensibly be assigned to those being required for either SGP specification or behaviour, they can also interact to impinge on both processes (Tripathy, 2014).

Embryonic gonad formation involves a complex interplay between two cell types and is a good model system for studying changes in cellular behaviors and cell-cell interactions, required for organogenesis. This study has identified a role for two transcription factors, Lola and Mid, in gonad development. A previous study had also identified a role for Lola in gonad formation (Weyers, 2011), and this work extends this finding in several respects. First, lola-R was identified as a specific isoform that is required by the gonad during its development. Second, Lola-R was shown to be expressed by the SGPs, and mesodermal expression of this isoform can rescue the gonad formation defects of lola null embryos. This indicates that Lola-R is required mesodermally, and that this lola isoform is sufficient to provide all Lola function in the gonad. Another zinc finger containing Lola isoform was unable to rescue the gonad defects of lola mutant embryos indicating functional differences in the distinct Lola isoforms. Whether Lola is required in the SGPs or the surrounding mesodermal cells remains an open question. It remains possible that Lola has cell autonomous functions in the SGPs as well as non-cell autonomous functions in the mesoderm, such as repressing mid or Mid function (Tripathy, 2014).

Although germ cell lola-R is not required for germ cell migration or gonad formation during embryogenesis and the Lola-R protein cannot be detected in these cells; other Lola isoforms are expressed and required in adult germ cells. In testes, lola-B and lola-I are required cell autonomously for germline stem cell maintenance and differentiation (Davies, 2013). In ovaries Lola-I is required for programmed cell death of late stage nurse cells (Bass, 2007). However, this requirement for Lola during oogenesis blocks the production of eggs from germ line clones of lola null alleles, which prevents testing whether other Lola isoforms play a role in embryonic germ cells (Tripathy, 2014).

In addition to being required for gonad formation, lola is required in the CNS. Mutants for lola null alleles show disrupted axonal tracts, however mutants in lola-R have wild-type axonal tracts. This reiterates the isoform-specific function of lola and demonstrates the ability to genetically uncouple the role of Lola in nervous system and gonad development. The lethality of flies containing the lola-R specific mutation in trans to a lola null indicates that Lola-R is also required in tissues other than the gonad, as defects in the latter would not be expected to lead to lethality (Tripathy, 2014).

The second transcription factor identified in this study was mid. Mid is a T box containing transcription factor of the tbx20 subclass with roles in embryonic patterning and axonal pathfinding. This study shows that mid mutants also have defects in gonad formation and that mid is required tissue autonomously by the SGPs (Tripathy, 2014).

To search for targets downstream of Mid and Lola in the gonad the expression was tested of genes either already identified as being important for SGP behaviour or known downstream targets in other tissues. Mid and Lola are both reported to be upstream of the cell surface receptor Robo, in the CNS. A Mid consensus binding site in the promoter region of Robo was identified with demonstrated Mid binding by chromatin immunoprecipitation (Liu, 2009). However, in the gonad of both mid and lola mutants Robo expression appeared normal. Furthermore no observable differences were found in Robo levels in the CNS of mid[B23] or mid[1] mutants compared to their heterozygous siblings in the same embryo collection. Moreover, a recently published study questioned the binding site proposed by Liu (2009) and identified a Mid consensus motif closer to the that of its vertebrate homologue, Tbx20 (Najand, 2012). Thus, although Robo is clearly required for gonad formation, whether it is downstream of Mid remains a matter of controversy (Tripathy, 2014).

Besides having demonstrated the role of two genes in gonad formation, this study has further built upon the transcriptional regulatory map in this tissue. The early SGP expression of Traffic jam (Tj) was identified as being Mid-dependent. Although Tin was not detected in late SGPs, this expression was not dependent on Mid. However, the loss of Mid and Tj expression in tin mutant SGPs, revealed a cascade of transcription factors functioning in a hierarchical and stage dependent fashion. Although, a reciprocal relationship exists between Mid and Tin in the heart, the current data demonstrates how tissues derived from the same germ layer can have different regulatory networks between the same genes (Tripathy, 2014).

Since Lola and Mid are both transcription factors, they could potentially regulate a common pool of downstream targets. Mesodermal expression of Lola-R-GFP in a mid mutant background did not rescue the mid mutant phenotype. This indicates that lola is not the sole downstream target of mid in the gonad. However, Mid over-expression in a lola mutant background results in a 'super-elongated' gonad consisting of supernumerary SGPs that span several parasegments even at late embryonic stages. This 'super-elongated' gonad results from additional SGPs being specified at the expense of fat body cells, and mirrors the effect of overexpression of the homeobox containing transcription factor Abd-A (Boyle and DiNardo, 1995 and Greig and Akam, 1995). This data raises the possibility that Abd-A balances the relative expression of Mid and Lola and suggests that the number of direct Abd-A targets is rather limited as its over-expression phenotype can be recapitulated by affecting their expression (Tripathy, 2014).

Given that Mid and Lola do not contain homeoboxes and are not required for SGP specification or maintenance, the ‘super-elongated’ phenotype seen upon over-expression of Mid in a lola background is surprising. These data argue that Lola functions to oppose Mid. Thus in the presence of wild-type Lola, overexpression of Mid does not affect SGP specification, however, in the absence of Lola, Mid overexpression results in additional SGPs (Tripathy, 2014).

A similar situation, of cell fate changes requiring shifts in expression of multiple transcription factors, occurs in the Drosophila heart. Heart cell specification requires Nkx (tin), GATA (pannier) and T box (mid, or Dorsocross) transcription factors. Whilst mis-expression of each factor alone is not sufficient to induce extra cardiac cells, combinations of these transcription factors (for example over-expression of Doc2 and pnr) can induce numbers of extra cardiac cells (Tripathy, 2014).

The results suggest that although the transcription factors required by SGPs can ostensibly be assigned to those being required for either SGP specification (such as Tin, Abd-A, Abd-B and Zfh-1) or behaviour (including D-Six4, Tj, Mid and Lola), such transcription factors can also interact to impinge on both processes. Investigating the downstream targets of Mid and Lola will provide new players and clues into how SGPs are specified and then programmed to interact with germ cells and each other to form a functional gonad (Tripathy, 2014).

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date revised: 19 June 2024

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