InteractiveFly: GeneBrief

scribbler: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References


Gene name - scribbler

Synonyms - brakeless, master of thickveins, CG5580

Cytological map position - 55C3-4

Function - putative transcription factor

Keywords - CNS, axon guidance, segment polarity

Symbol - sbb

FlyBase ID: FBgn0010575

Genetic map position -

Classification - novel protein with a single zinc finger domain

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

scribbler (sbb) was characterized in four different laboratories. Yang (2000) showed that it is indispensable for the normal behavior of larvae. Naming the gene brakeless (bks), Senti (2000) and Rao (2000) showed that it is required for the axonal guidance of the Drosophila visual system, and as master of thickveins (mtv), Funakoshi (2001) showed that the gene shapes the activity gradient of the Dpp morphogen through regulation of thickveins. Scribbler is a novel protein with a single putative zinc finger region and possesses a nuclear localization signal, suggesting that it is a nuclear protein. This overview will concentrate on the behavioral phenotype for sbb described in Yang's especially thorough genetic study, and the first analysis of the possible molecular function of sbb, as described in Funakoshi's 2001 study. The work of Senti and Rao is reviewed in the Effects of Mutation section.

The scribbler mutant was found by screening a collection of P-element-tagged second chromosome pupal lethal lines for abnormal foraging and/or locomotion behavior. One line (l(2)03432) carries a mutation that causes an unusual larval behavioral phenotype on nonnutritive agar. The scribbler larval trail covered a small area and looked as if the larva had scribbled on the agar surface. This was in direct contrast to the relatively straight trails of wild-type larvae. Scribbler behavior was quantitated in two ways. (1) The percentage of larvae in a line that exhibited scribbler behavior was calculated. (2) The number of 6-mm grid squares entered by each larva during its 5-min test on the agar substrate was counted. Scribbler behavior was 64% penetrant in homozygous sbbl(2)03432 larvae. These larvae also entered significantly fewer grid squares than did their heterozygous sibs or larvae of the wild-type forR (rover) control strain. Scribbler behavior is exhibited only when sbbl(2)03432 mutant larvae are traveling on a nonnutritive substrate (agar) and not when mutant larvae forage on a yeast and water paste. Thus, the expression of scribbler turning behavior is conditional on the environment, specifically on the absence of food. The sbbl(2)03432 mutation results in late-stage pupal lethality with 10% of the pupae emerging from the pupal case but dying within 3 days of emergence. These adult escapers lack part of a wing vein at the end of L5 (Yang, 2000).

In situ hybridization revealed that the sbb gene is expressed in the embryonic and larval CNS. The P[GAL4] system was used to manipulate gene expression to determine whether expression of the 3-kb SBB RNA in the nervous system alone is sufficient to rescue scribbler behavior. In this binary system the yeast transcription factor GAL4 directs the expression of any gene fused downstream of the activation sequence UAS, thus permitting ectopic expression of the fused gene. It was found that the expression of the UAS-sbb in the nervous system alone is sufficient to rescue scribbler behavior. When sbb was targeted to the nervous system alone, 60% of the expected number of flies emerged as fertile adults, indicating only partial rescue of the lethal phenotype. Targeted expression of the UAS-sbb to neurons does not rescue the L5 wing vein phenotype, suggesting that this phenotype results from sbb expression outside the nervous system, perhaps in the nonneuronal cells in the imaginal discs (Yang, 2000).

It is of interest that the scribbler turning phenotype is displayed only in the absence of food, since turning rate and localized traveling are known to be important components of food search behavior. Search behavior is the means by which most motile animals find essential resources and hence is a trait that can strongly influence the survival of an individual. Genetic control of search behavior is not unprecedented. It has been shown that larvae carrying the rover allele of the foraging (for) gene exhibit long foraging trails in a large yeast patch and tend to move between depleted food patches, whereas homozygous sitter larvae locate the closest food patch and remain feeding on it. Similarly, adult rover flies walk significantly farther from a recently consumed sucrose drop than sitter flies whose higher turning rate promotes revisiting and keeps the fly near the drop. sbb larvae exhibit characteristic patterns of food search behavior (turning, bending, and feeding movements); however, these patterns are exhibited solely in the absence of food (Yang, 2000 and references therein).

Many developmental genes are known to be pleiotropic; their gene products play multiple roles throughout development. Genes that affect both larval and adult behavior can play important roles in development and sometimes have vital functions (for example, in Drosophila, learning genes such as dunce, latheo, and linotte; courtship mutants such as fruitless and fickle; larval behavioral mutants such as foraging and tamas; and ion channel mutants such as slow poke). One well-studied example of a pleiotropic gene that affects behavior is the learning gene dunce (dnc). Mutations in dnc cause female sterility and abnormal learning. The dnc gene is widely expressed; its expression is not restricted to the mushroom bodies of the fly brain known to play an important role in learning and memory. Pleiotropic genes that play a role in behavior are usually expressed in multiple tissues and during more than one developmental stage. Mutations in pleiotropic genes often affect more than one aspect of neuronal structure and/or function (Yang, 2000 and references therein).

sbb is a pleiotropic gene with a vital function. Four out of five (sbbl(2)03432, sbbl(2)04440, sbbl(2)k00702, and sbbl(2)04525 but not sbbEP(2)0328) P-element insertion alleles of sbb exhibit lethality, primarily in the late pupal stage. Mutations in the sbb gene lead to multiple phenotypic defects that include larval scribbler behavior, pupal lethality, and a defect in the pattern of the L5 wing vein in the adult escapers. sbb transcripts are observed in all developmental stages and in multiple tissue types (the embryonic and larval CNS and the imaginal discs). These expression data support the phenotypic data suggesting that sbb likely functions during multiple developmental stages and in more than one tissue. The four different-sized transcripts suggest the existence of at least four different Scribbler isoforms that may arise from differential RNA splicing or alternative polyadenylation or initiation. It is predicted that some of these isoforms will have different functions and will be found in different tissues. Some initial support for this prediction comes from the finding of a body-specific transcript (7.8 kb) and from the lack of complete rescue of pupal lethality when sbb was only targeted to neuronal cells. Further studies are needed to address the question of how, when, and where sbb acts to accomplish its pleiotropic functions (Yang, 2000).

One of the pleiotropic functions of sbb alluded to by Yang (2000) is an effect of wing morphogenesis. This function has been addressed by Funakoshi (2001), who shows that sbb shapes the activity gradient of the Dpp morphogen through regulation of thickveins. Drosophila wings are patterned by a morphogen, Decapentaplegic, a member of the TGFbeta superfamily, that is expressed along the anterior and posterior compartment boundary. The distribution and activity of Dpp signaling is controlled in part by the level of expression of its major type I receptor, thickveins (tkv). The level of tkv is dynamically regulated by Engrailed and Hedgehog. sbb, termed master of thickveins (mtv) by Funakoshi, downregulates expression of tkv in response to Hh and En. mtv expression is controlled by En and Hh, and is complementary to tkv expression. mtv integrates the activities of En and Hh that shape tkv expression pattern. Thus, mtv plays a key part of regulatory mechanism that makes the activity gradient of the Dpp morphogen (Funakoshi, 2001).

Dpp signaling activity can be visualized by using the antibody against phosphorylated Mothers against dpp (p-Mad): the distribution of the Dpp morphogen activity largely depends on the levels of the Tkv receptor. tkv expression, which is monitored by expression of beta-galactosidase in the tkv-lacZ enhancer trap line, is downregulated by Hh along the A/P border where dpp expression is induced by the same signal. The basal level of tkv is higher in the P compartment than it is in the A compartment. This complex pattern appears to shape the activity gradient of Dpp directly. The p-Mad level is low along the A/P border where tkv is downregulated. The gradient of the p-Mad distribution is steeper in the P compartment than it is in the A compartment, probably because high levels of Tkv limit the movement of Dpp; since the spread of Dpp would be less in the P compartment, its gradient of activity would be expected to be steeper (Funakoshi, 2001).

mtv was identified by characterizing the enhancer trap lines, 1E1 and l(2)k00702, that generate expression patterns largely complementary to that of tkv in wing discs except at the dorsoventral compartment border in the peripheral region, where both genes are expressed at high levels. Distribution of the transcript revealed by in situ hybridization with a probe prepared from the corresponding cDNA is consistent with the pattern of the enhancer trap lines. Only the longer form of bks/sbb/mtv mRNA is predominantly detected in imaginal discs (Funakoshi, 2001).

In order to know whether mtv has a role in regulating tkv, a deletion mutant allele, mtv6, was made by imprecise excision of the P-element and used for clonal analysis. It is believed that mtv6 is a strong hypomorphic allele, because its transcript can only encode a 19 amino acid polypeptide, which lacks most of putative functional domains of the Mtv protein. In mtv6 clones, tkv-lacZ levels are autonomously upregulated indicating that Mtv represses tkv. When a large mtv clone is induced in the area including the A/P border, tkv-lacZ levels become uniform within the clone, suggesting that mtv plays an important role in regulating a dynamic pattern of tkv expression throughout the wing pouch. p-Mad levels are also upregulated in a graded manner. This is consistent with the fact that tkv is derepressed within mtv mutant clones because ectopically induced Tkv upregulates p-Mad levels. No significant changes in dpp transcription levels were observed within mtv mutant clones, thus it is concluded that mtv shapes p-Mad spatial distribution through regulation of Tkv levels (Funakoshi, 2001).

To address whether the increased p-Mad levels produced in mtv clones result in the activation of targets of the Dpp signaling pathway, the expression of spalt (sal) and optomotor blind (omb) were studied. Within the mtv clones, expression of those marker genes is upregulated as expected in both A and P compartments. When clones are located at the edge of the sal-expressing domain, a slight expansion of sal expression is observed, as in the case of p-Mad staining. However, when clones are located at a distance from the sal-expressing domain, no upregulation is observed, presumably because in this location, Dpp signaling activity is below the required threshold for activity (Funakoshi, 2001).

Adult phenotypes of the mtv clones were examined and it was found that they are reminiscent of those caused by overexpression of tkv. When the mtv clone is anterior to the second vein, an ectopic vein is generated anterior to the second vein, probably due to the expansion of the sal expression domain, and triple-row bristles are transformed to double-row bristles. This suggests that the elevated tkv levels in mtv clones cause these cells to differentiate as if they are adjacent to the A/P compartment boundary. Another clone shows a modest overproliferation of the tissue: this is also consistent with the notion that the mtv mutation elevates the level of the Dpp signal, as has been reported by overexpression of Mad. The clone at the A/P border also disrupts the vein pattern. The upregulation of wild-type tkv can generate identical phenotypes to those observed in mtv clones: upregulation of sal expression in the wing pouch, bifurcation of veins and transformation of bristles in the adult wing. Thus, it is concluded that the major target of the mtv activity that organizes the A/P pattern is tkv (Funakoshi, 2001).

The fact that tkv expression is repressed by hh at the A/P border and mtv is highly expressed in the same cells has prompted an examination of whether mtv mediates hh dependent tkv repression. tkv-lacZ levels were examined in clones of cells mutant for patched (ptc), which encodes the Hh receptor. Hh signal transduction is constitutively active in the absence of ptc activity. Anterior compartment ptc clones cause cell-autonomous repression of tkv. In the clones of cells mutant both for mtv and ptc, however, tkv levels are elevated as in the mtv singly mutant clones. This indicates that mtv mediates hh-dependent tkv regulation along the A/P border. mtv-lacZ expression was monitored within clones of cells mutant for smoothened (smo), which encodes a component of the Hh receptor complex and is required for Hh signaling. Within the clones located at the A/P border, mtv-lacZ expression is repressed, indicating that Hh signaling induces the high level of mtv expression along the A/P border. This was also confirmed by the fact that mtv-lacZ levels are elevated within pka mutant clones, in which Hh signaling is constitutively active. These results show that Hh represses tkv levels by upregulating its negative regulator, mtv (Funakoshi, 2001).

As described earlier, the basal tkv level in the P compartment is higher than it is in the A compartment and it is responsible for the asymmetric structure of the wing. The mtv expression pattern is complementary to the tkv expression pattern. The possibility that mtv is also responsible for regulating the basal level of tkv expression was also examined. Initially, it was asked whether the level of tkv is regulated by en in the P compartment. Within clones of cells mutant for en in the P compartment, the tkv-lacZ level is lower than that seen in the A compartment, suggesting that tkv expression is regulated by en. This is confirmed by the observation that, within clones of cells ectopically expressing en in the A compartment, the tkv level is elevated in comparison to that seen in the P compartment in an autonomous way. Within en and mtv double mutant clones, tkv transcription levels are derepressed as in mtv single mutant clones, indicating that mtv mediates en dependent tkv regulation. Ectopically expressed en downregulates mtv-lacZ in the A compartment, implying that low levels of mtv in the P compartment are under the control of en regulation. These results altogether indicate that en regulates the high basal level of tkv expression in the P compartment by downregulating mtv expression (Funakoshi, 2001).

It is concluded that the central region of the wing is patterned by Hh but not by Dpp although Dpp expression is induced by Hh in this region. This is because Dpp signaling is downregulated by Hh by upregulating mtv, which causes repression of tkv expression. The patterning by Hh appears to be ensured by lowering the Dpp signaling, which would otherwise interfere with the Hh morphogen activity, because upregulation of Dpp signaling by overexpressing tkv or by eliminating mtv activity can alter the vein patterns there. Therefore, the mtv-dependent tkv regulation is required both for Hh and Dpp morphogen activities. The patterning along the A/P border between veins 3 and 4 may be more complicated. The dorsal mtv mutant clone at the A/P border disrupts the vein pattern; vein 3 is displaced posteriorly, which is similar to the phenotype associated with sal mutant clones. The fact that mtv is downregulated in sal mutant clones might explain the phenotype if Mtv has a role in mediating Sal activity, which positions vein 3 through regulating target genes such as the iroquois gene complex. Further analysis is required to elucidate whether tkv function is linear or parallel to this regulatory cascade (Funakoshi, 2001).

The mechanism that shapes the tkv pattern and hence the Dpp morphogen gradient is unique in that it makes the mtv expression pattern that is made by integrating En and Hh signals complementary to the tkv pattern. Thus, the mtv expression pattern acts as a 'negative' for generating the tkv pattern. en positive cells initiate the cascade of the patterning along the A/P axis by expressing Hh, which both acts as short-range morphogen and induces the long-range morphogen, Dpp. Here it is proposed that not only does En induce expression of the morphogen, but it also shapes the morphogen activity gradient by regulating its receptor level via Mtv (Funakoshi, 2001).

Drosophila Brakeless interacts with Atrophin and is required for tailless-mediated transcriptional repression in early embryos

Complex gene expression patterns in animal development are generated by the interplay of transcriptional activators and repressors at cis-regulatory DNA modules (CRMs). How repressors work is not well understood, but often involves interactions with co-repressors. Mutations were isolated in the brakeless gene in a screen for maternal factors affecting segmentation of the Drosophila embryo. Brakeless, also known as Scribbler, or Master of thickveins, is a nuclear protein of unknown function. In brakeless embryos, an expanded expression pattern was noted of the Krüppel (Kr) and knirps (kni) genes. Tailless-mediated repression of kni expression is impaired in brakeless mutants. Tailless and Brakeless bind each other in vitro and interact genetically. Brakeless is recruited to the Kr and kni CRMs, and represses transcription when tethered to DNA. This suggests that Brakeless is a novel co-repressor. Orphan nuclear receptors of the Tailless type also interact with Atrophin co-repressors. Both Drosophila and human Brakeless and Atrophin interact in vitro, and it is proposed that they act together as a co-repressor complex in many developmental contexts. The possibility is discussed that human Brakeless homologs may influence the toxicity of polyglutamine-expanded Atrophin-1, which causes the human neurodegenerative disease dentatorubral-pallidoluysian atrophy (DRPLA) (Haecker, 2007).

Repression plays a pivotal role in establishing correct gene expression patterns that is necessary for cell fate specification during embryo development. For example, in the early Drosophila embryo, repression by gap and pair-rule proteins is essential for specifying the positions of the 14 segments of the animal. The mechanisms by which transcriptional repressors delimit gene expression borders are not well understood. However, many repressors require co-repressors for function. In the Drosophila embryo, the CtBP and Groucho co-repressors are required for activity of many repressors. Atrophin has been identified as a co-repressor for Even-skipped and Tll. Still, co-regulators for several important transcription factors in the early embryo have not yet been identified. Therefore a screen was performed for novel maternal factors that are required for establishing correct gene expression patterns in the early embryo (Haecker, 2007).

From this screen, mutations were identified in the bks gene that cause severe phenotypes on gap gene expression and embryo segmentation. The Bks protein is evolutionarily conserved between insects and deuterostomes, but has not been characterized in any species except Drosophila, in which it has been shown to repress runt expression in photoreceptor cells and thickveins expression in wing imaginal discs. However, the molecular function of Bks has been unknown. This study shows that Bks interacts with the transcriptional repressor Tll, is recruited to target gene CRMs, and will repress transcription when targeted to DNA (Haecker, 2007).

Tll has been shown to utilize Atrophin as a co-repressor. Atrophin genetically interacts with Tll and physically interacts with its ligand binding domain. Atrophin binding is conserved in nuclear receptors within the same subfamily, such as Seven-Up in Drosophila as well as Tlx and COUP-TF in mammals. When expressed in mammalian cells, Drosophila Atrophin and mouse Atrophin-2 interact with the histone deacetylases HDAC1 and HDAC2. Histone deacetylation may therefore be part of the mechanism by which Atrophin functions as a co-repressor. Another recent report described genetic interactions among bks and atrophin mutants in the formation of interocellar bristles in adult flies. Furthermore, it was shown that atrophin mutants have virtually identical phenotypes as bks mutants, including de-repression of runt expression in the eye, thickveins expression in the wing, and Kr and kni expression in the embryo (Haecker, 2007).

Both proteins are recruited to the kni CRM, a Tll-regulated target gene, in the embryo. Importantly, Atrophin and Bks interact in vitro and that they can be co-immunoprecipitated from S2 cells. It is proposed that Bks and Atrophin function together as a co-repressor complex, and based on the similar bks and atrophin mutant phenotypes at several developmental stages, the complex may function throughout development. These results are compatible with the existence of a tripartite complex consisting of Tll, Bks, and Atrophin. Bks binding to Tll is enhanced by the Tll DNA binding domain, whereas the interaction of Tll with Atrophin is mediated through the C-terminal ligand binding domain. Tll may therefore simultaneously interact with Bks and Atrophin. Alternatively, Tll interacts separately with Bks and Atrophin on the kni CRM. In either case, both Bks and Atrophin are required for full Tll activity. However, at high enough Tll concentration, Bks activity is dispensable. Some bks embryos misexpressing Tll still repress kni expression, and overexpressing Tll from a heat-shock promoter can repress the posterior kni stripe in both wt and bks mutant embryos. For this reason, it is believed that Bks and Atrophin are cooperating as Tll co-repressors, so that Tll function is only partially impaired by the absence of either one. It was found that Tet-Bks-mediated repression in cells is insensitive to the deacetylase inhibitor trichostatin A (TSA). It is possible, therefore, that whereas Atrophin-mediated repression may involve histone deacetylation, Bks could repress transcription through a separate mechanism (Haecker, 2007).

These results have not revealed any differences between the molecular functions of the two Bks isoforms. Both Bks-A and Bks-B repress transcription when tethered to DNA, and the sequences that mediated binding to Tll and Atrophin are shared between the two isoforms. However, the bks339 allele that selectively affects the Bks-B isoform causes a weaker, but comparable phenotype to the stronger bks alleles that disrupt both isoforms. Therefore, the C-terminus of Bks-B provides a function that is indispensable for embryo development and regulation of kni expression. This part of Bks-B contains two regions (D3 and D4) that are highly conserved in insects and loosely conserved in deuterostome Bks sequences, but does not resemble any sequence with known function. The only sequence similarity to domains found in other proteins is a single zinc-finger motif in Bks-B. Preliminary results indicate that the zinc finger in isolation or together with the conserved D2 domain does not exhibit sequence-specific DNA binding activity. Indeed, multiple zinc fingers are generally required to achieve DNA binding specificity. Instead, Bks is likely brought to DNA through interactions with Tll and other transcription factors (Haecker, 2007).

Atrophins are required for embryo development in C. elegans, Drosophila, zebrafish, and mice. In vertebrates, two atrophin genes are present. Atrophin-1 is dispensable for embryonic development in mice, and lacks the N-terminal MTA-2 homologous domain that interacts with histone deacetylases . However, the homologous C-termini of Atrophin-1 and Atrophin-2 can interact, and it was found that this domain can also bind to the human Bks homolog ZNF608. Atrophin-1 interacts with another co-repressor-associated protein as well, ETO/MTG8, and can repress transcription when tethered to DNA. These data are consistent with the emerging view that deregulated transcription may be an important mechanism for the pathogenesis of polyglutamine diseases. Recent evidence indicates that interactions with the normal binding partners may cause toxicity of polyglutamine-expanded proteins such as Ataxin-1 . It will be interesting to investigate whether the interaction between human Bks homologs and Atrophin-1 is important for the neuronal toxicity of polyglutamine-expanded Atrophin-1 (Haecker, 2007).


REGULATION

Targets of Activity

Drosophila photoreceptor neurons (R cells) project their axons to one of two layers in the optic lobe, the lamina or the medulla. The transcription factor Runt (Run) is normally expressed in the two inner R cells (R7 and R8) that project their axons to the medulla. The relationship between Run and the ubiquitously expressed nuclear protein Brakeless (Bks), which has previously been shown to be important for axon termination in the lamina, has been examined. Bks represses Run in two of the outer R cells: R2 and R5. Expression of Run in R2 and R5 causes axonal mistargeting of all six outer R cells (R1-R6) to the inappropriate layer, without altering expression of cell-specific developmental markers (Kaminker, 2002).

As an axon navigates toward a target region during development, it alters its course based on attractive or repulsive molecular signals in its environment. There are at least two phases in the establishment of neuronal connections. First, axons project to and distinguish between regions or layers and second, once within the target layer, axons fine-tune their projections. This second step involves precise interactions between growth cones and target cells and has been well studied, particularly for the participation of cell-surface molecules and their associated signal transduction machinery. This study investigates the role of two transcription factors, Run and Bks, in the first phase of target layer selection for differentiating R cells in the Drosophila optic lobe (Kaminker, 2002).

The expression pattern of several R cell-specific differentiation markers is normal in bks mutants. In striking contrast to other markers, however, Run is ectopically expressed in two extra R cells per cluster in somatic loss-of-function clones of bks mutant tissue. In bks clones, Run expression is expanded from its normal R7/R8 pattern to also include R2 and R5. This suggests that Bks represses Run in R2 and R5 cells. Cells along the edges of bks clones were analyzed for the expression of Run. In 196 ommatidia counted along clone boundaries, R2/R5 expression of Run was never seen in a cell that is wild type for bks. It is concluded that the repression of run by Bks is cell-autonomous (Kaminker, 2002).

R1-R6 photoreceptor axons misproject to the medulla in bks loss-of-function mutants. To determine whether this axonal targeting defect is due to the relief of Run repression in R2 and R5, the GAL4/UAS system was used to express Run in these cells. When Run is expressed in R2, R5 and R8 using the MT14-GAL4 driver, all innervating R-cell axons bypass the lamina and projected through to the medulla. When Run is misexpressed in R2 and R5 alone, the defect is as severe as when Run is misexpressed in all R cells using the GMR-GAL4 driver. Run over-expression in R8 alone, where it is normally expressed, does not affect axonal projections. In addition, misexpression of Run in R1, R6 and R7 using lz-GAL4 or in R3 and R4 using sal-GAL4 does not give rise to a comparable axonal misprojection phenotype. The severe axonal mistargeting phenotype in MT14-GAL4/UAS-run flies is attributable to Run expression in R2 and R5 (Kaminker, 2002).

Unlike the ordered wild-type array, thick bundles of axons are seen entering the medulla when Run is mis-expressed in R2 and R5. The axons do not project into deeper areas of the brain, but stop within the medulla. The phenotype observed in this genetic background is very similar to that for bks. Therefore, in both bks loss-of-function and MT14-GAL4/UAS-run genetic backgrounds, Run expression in R2 and R5 results in the mistargeting of all retinal axon types to the medulla. This also suggests that the targeting of R2 and R5 axons affects axonal pathfinding of other outer R cells. For technical reasons, it was not possible to generate marked, double-lethal clones of run and bks (Kaminker, 2002).

The mistargeting of R-cell axons could, in principle, result from the conversion of all R-cell fates to R7 and R8. Markers for every R-cell type and for cone cells were therefore analyzed, both in mosaic clones of null bks mutant tissue and in the context of MT14-GAL4/UAS-run. In each of these backgrounds, the Run-expressing R2 and R5 cells did not express the R8-specific antigen, Bride of Sevenless (Boss) or the R7 marker, Prospero (Pros). Therefore, the projection phenotype of R cells to the medulla in these backgrounds does not result from the conversion of these R cells to the R7/R8 type during their development. The R1/R6 marker Bar and the R2/R5/R3/R4 marker Rough are also unaffected in these backgrounds. It is concluded that Run reprograms the projection pattern of outer R cells without affecting the expression of developmental markers of cell identity (Kaminker, 2002).

Consistent with the R cell marker expression in larval tissue, plastic sections of adult eyes show that Run misexpression in R2 and R5 during development does not perturb adult R cells or ommatidial structure. The correct complement and arrangement of R cells was found. Strikingly, these seemingly normal adult R cells misproject their axons to the inappropriate optic layer. Axon termination in the lamina region is virtually absent and all R-cell axons project to the medulla. This adult phenotype is also identical to that reported in bks mutant clones in which R cells are unchanged in the expression pattern of Rhodopsins. These data provide strong evidence that Run expression in R2 and R5 causes mistargeting of all outer R-cell axons without changing their individual R-cell fates, as determined by developmental markers and by the adult morphology of rhabdomeres. In spalt (sal) mutants, cell fate is altered without changes in axonal connectivities. Similarly, in sal-GAL4/UAS-run flies, some change in R3/R4 fate to R7 cell type is evident without a significant perturbation of outer R-cell projections. Hence, transcriptional events that control cell identity are separable from those that control axonal targeting (Kaminker, 2002).

R-cell axons provide critical anterograde signals to the lamina target region to induce the proliferation and differentiation of lamina neurons, and to induce the differentiation and migration of glial cells to their correct position adjacent to the lamina plexus. In turn, the glial cells provide positional information that directs R1-R6 axons to terminate in the lamina, and in the absence of glia these axons project into the medulla. The development of the lamina target region was assessed using neuronal and glial cell differentiation markers. Wild-type brains stained with anti-Dachshund (Dac) antibody show a large area of labeled cells corresponding to maturing lamina precursor cells (LPCs) and differentiated lamina neurons. This remains unchanged in GMR-GAL4/UAS-run brains, although the R-cell axons do not target properly. Additionally, the three rows of glial cells that delineate the lamina plexus in wild type also remain unaltered when Run is misexpressed. It is concluded that the abnormal pathfinding of R cells is due to defects intrinsic to R cells and not to a global disruption of lamina target neurons or glia (Kaminker, 2002).

Thus, this study highlights two important characteristics of neuronal pathfinding in the optic lobe. (1) A mechanism involving Bks exists in wild-type flies for repressing Run expression specifically in R2 and R5 cells. The bks loss-of-function causes relief of run repression in these two cells, which entirely abolishes the distinct targeting of R cells to the two optic ganglia. Perhaps additional genes are responsible for repressing run in the other R cells. (2) The mistargeting of R2 and R5 alone is sufficient for all outer R cells to project to the medulla. It appears that R2 and R5 cells, the first two outer R cells to be specified, provide pioneering axons whose tracts the other axons follow. In rough loss-of-function, R2 and R5 are converted to R8 cells. The resulting phenotype, however, is not as severe as that described in this study. Presumably, a residual pioneering function is maintained. The mechanism underlying the interaction between R2/R5 and R3/R4/R1/R6 axons in regulating target layer selection is unclear, although interaction between R1-R6 axons regulates a later step in axonal pathfinding: the fine-tuning of R-cell connections in the lamina (Kaminker, 2002).


DEVELOPMENTAL BIOLOGY

The 3-kb EST clone LD13770 (called sbb-cDNA LD13770) was used in Northern blot analysis to probe wild-type (forR) poly(A)+ RNA isolated from larvae, pupae, adult head, and adult body. sbb RNA is expressed at all stages of development. Three transcripts (10.5, 3.6, and 1.6 kb) were detected in larvae, pupae, and adult heads, whereas the 7.8-, 3.6-, and 1.6-kb transcripts were detected in adult body. These different-sized transcripts suggested the involvement of alternative splicing, transcript initiation, or termination. Northern analysis using the same sbb-cDNA LD13770 probe has shown a reduction in the abundance of mRNA in homozygous sbbEP(2)0328 compared to larvae of the control strain. Similar results are found for sbbl(2)03432 and sbbJ2, the early larval lethal excision line, which carries a >10-kb deletion (Yang, 2000).

In situ hybridization to CS embyros with antisense RNA from sbb-cDNA LD13770 reveals that sbb is expressed in the embryonic central nervous system. Wild-type sbb expression is strong in early stage embryos (stage 5) before gastrulation. In larvae, wild-type sbb RNA expression is found in the nervous system including the brain, the optic lobes, the ring gland, and the medial region of ventral ganglion. Expression is also found in the eye-antennal, wing, and leg discs. RNA is expressed at normal levels in heterozygous embryos, but expression is reduced in homozygous mutant embryos of sbbl(2)04440, sbbl(2)03432, and sbbEP(2)0328. sbbl(2)04440 appears to have a more severe reduction in transcript abundance than does sbbl(2)03432 and sbbEP(2)0328. Expression of sbb RNA in sbbEP(2)0328 larvae is reduced in the brain and missing in the medial region of the ventral ganglion (Yang, 2000).

Larval

To determine the expression pattern and the subcellular localization of Sbb/Brakeless, antibodies to it were generated. A glutathione S-transferase fusion protein containing a fragment of the Bks protein (amino acids 623-818) was used as an immunogen in rabbits. Western blot analysis of extracts made from wild-type third instar eye-brain complexes using the affinity-purified antibody detected a single band at ~80 kDa, which is close to the predicted size of the Bks protein. That this band corresponds to Bks is supported by its loss in bks mutant extracts. The distribution of the Bks protein in the eye imaginal disk was determined in whole-mount preparations. Neuronal differentiation occurs posterior to a dorsoventral groove called the morphogenetic furrow. Bks was detected in the nuclei of developing R-cells and undifferentiated precursor cells lying in the basal region of the disk. Weak nuclear staining was also seen in cells located anterior to the morphogenetic furrow. This nuclear staining was missing in bks mutants and was greatly enhanced in larvae overexpressing bks, confirming the specificity of the antibody. In addition to the staining in the developing eye disk, Brakeless protein was also detected in the nuclei of glial cells in the optic stalk. High background staining precluded an assessment of Bks protein in the central nervous system. Because bks mutations are lethal, however, bks must be required for functioning outside the eye. Transgene rescue experiments indicate that bks is not required in these glial cells (Rao, 2000).

To examine the cellular localization of Bks proteins, antisera were generated recognizing a region of high sequence complexity at the C terminus of BksA, corresponding to the central region of BksB. These sera detect a widely expressed nuclear antigen in eye-antennal imaginal discs. This antigen is not restricted to R1-R6 cells, or even to photoreceptors, but appears to be present in all cells of the imaginal disc. As the Bks antisera do not distinguish the two Bks isoforms, transgenic animals expressing c-myc epitope-tagged versions of either BksA or BksB were generated under the control of the eye-specific promoter GMR. Using anti-c-myc antibodies to stain the eye discs of animals carrying either of these GMR-myc bksA and GMR-myc bksB trangenes, it was confirmed that indeed both Bks isoforms are predominantly nuclear proteins (Senti, 2000).

Hedgehog (Hh) signaling from posterior (P) to anterior (A) cells is the primary determinant of AP polarity in the limb field in insects and vertebrates. Hh acts in part by inducing expression of Decapentaplegic (Dpp), but how Hh and Dpp together pattern the central region of the Drosophila wing remains largely unknown. The role played by Collier (Col), a dose-dependent Hh target activated in cells along the AP boundary (the AP organizer in the imaginal wing disc) has been examined. col mutant wings are smaller than wild type and lack L4 vein, in addition to missing the L3-L4 intervein and mis-positioning of the anterior L3 vein. These phenotypes are linked to col requirement for the local upregulation of both emc and N, two genes involved in the control of cell proliferation, the EGFR ligand Vein and the intervein determination gene blistered. Attenuation of Dpp signaling in the AP organizer is also col dependent and, in conjunction with Vein upregulation, required for formation of L4 vein. A model recapitulating the molecular interplay between the Hh, Dpp and EGF signaling pathways in the wing AP organizer is presented (Crozatier, 2002).

An unanticipated intricacy of the independent, versus Dpp-mediated, Hh patterning activity in the wing arose from comparison of the gradient of Dpp activity with dpp expression. It revealed that Dpp signaling is downregulated in the AP organizer and upregulated in both A and P flanking cells. This downregulation of Dpp activity was shown to result from the localized transcriptional repression of tkv, which is itself due to activation of the transcriptional repressor Master of thick vein (Mtv: Scribbler) in response to Hh signaling. Col activity is required for the downregulation of Dpp signaling in the AP organizer and this regulation is linked to Col requirement for positioning L3 vein and formation of L4 vein. The relative functions of these two genes remain to be examined in detail, although the observation that mtv transcription is downregulated in col mutant discs and tkv is upregulated in clones of col mutant cells suggests that col may act upstream of mtv in the regulatory cascade, thereby attenuating Dpp signaling in the AP organizer (Crozatier, 2002).

It has been been proposed that Hh does directly control the position of L3 vein, although the molecular mechanisms of this control have not been firmly established. In both col and mtv mutant clones, the position of L3 vein is shifted posteriorwards. That both col and mtv control the position of L3 vein suggests that this position is defined by Hh signaling through the modulation of Dpp signaling. iro is required for rho activation in the L3 primordium and formation of L3 vein. iro is activated by both Dpp and Hh signaling and its anterior border of expression is under control of sal/salr, a target of Dpp. The patterns of col, iro and rho expression are intimately connected. Both an increased number of cells expressing rho and a posterior shift of the anterior border of iro expression are observed in col1 mutant discs. This posterior shift is interpreted as reflecting a modified range of Dpp signaling relayed, at least in part, by sal/salr activity. The increased number of rho-expressing cells, for its part, indicates that Col is able to antagonize rho activation by iro in cells, which express both iro and Col. This correlates well with the wing phenotype – anteriorwards shift of the L3 vein, together with gaps in its distal region – which results from anterior extension of Col expression, in UAS-Col/dpp-Gal4 wing discs. The distal gaps could reflect the complete absence of rho expression close to the DV border, because of the complete overlap between col and iro expression where iro expression is narrower. From col loss- and gain-of function experiments, it is therefore concluded that the primordium of L3 vein corresponds to cells that express iro but not col. Col thus appears to play a dual role in defining the position and width of L3 vein: activating Blistered and repressing EGFR in the wing AP organizer cells, endows these cells with an intervein fate, while attenuating Dpp signaling indirectly positions the anterior limit of iro expression domain, and L3 vein competence anterior to the AP organizer (Crozatier, 2002).


EFFECTS OF MUTATION

The R1-R6 subclass of photoreceptor neurons (R cells) in the Drosophila compound eye form specific connections with targets in the optic ganglia. brakeless (bks: accepted FlyBase name scribbler) is essential for R1-R6 growth cone targeting. In brakeless mutants, R1-R6 growth cones frequently fail to terminate migration in their normal target, the lamina, and instead project through it and terminate in the second optic ganglion, the medulla. Genetic mosaic analysis and transgene rescue experiments indicate that bks functions in R cells and not within the lamina target region. It is proposed that it participates in a gene expression pathway regulating one or more growth cone components controlling R1-R6 targeting (Rao, 2000).

A collection of lethal P element insertions on the second chromosome was screened for mutations that disrupt the R-cell projection pattern. One insertion line, l (2)k06903, causes defects in both R-cell projections and R-cell patterning in the eye, as assessed in preparations of third instar eye-brain complexes stained with an antibody recognizing R-cell bodies and their axons. l (2)k06903 carries two P-element insertions at polytene chromosome bands 47D1-D2 and 55C1-C5. Genetic experiments have demonstrated that the connectivity and patterning defects are genetically separable and that the connectivity defects result from the insertion at 55C1-C5. Initially, the projection, but not the eye, phenotype is uncovered by a deficiency [Df(2R)PC4] that selectively removes 55C1-C5. Next, the two P-element insertions are separated by meiotic recombination; larvae homozygous for the 55C1-C5 insertion exhibit the projection, but not the patterning, defect. Finally, excision of the P element at 55C1-C5 restores R-cell projection pattern in 88 of 90 excision lines analyzed. Because many R1-R6 growth cones fail to stop in the lamina, the mutation was named brakeless (bks) and the initial P allele bksP1. Two other lethal P-element insertions, l (2)k0702 and l (2)04440, have been shown to be allelic to bksP1 and are designated bksP2 and bksp3, respectively. Two imprecise excision mutant alleles are designated bks4 and bks5 (Rao, 2000).

The R-cell axon projection patterns in wild-type and mutant third-instar larval eye-brain complexes were visualized by using mAb24B10. In the wild type, bundles of R-cell axons converge at the posterior end of the eye disk, where they enter the optic stalk. On exiting the stalk, axon bundles elaborate a topographic map in the developing lamina and medulla. The R1-6 growth cones terminate in the lamina with their expanded growth cones, forming a dense continuous layer of immunoreactivity in a region referred to as the lamina plexus. R7 and R8 growth cones project through the lamina and terminate in an array in the medulla neuropil. In bks mutant larvae, R-cell axons project through the optic stalk and into the optic lobe, largely as they do in the wild type. Projection defects in both the lamina and medulla are observed. In contrast to the wild type, the lamina plexus is discontinuous, with regions of dense staining separated by weakly stained regions or gaps. Thick bundles of axons are frequently observed between the lamina and medulla, in striking contrast to the lightly stained thin bundles in the wild type. In the most severe cases, the lamina plexus is largely missing and the medulla neuropil is hyperinnervated. In addition, disruptions in the normally smooth topographic map in the medulla are observed. However, R-cell projections do not extend through the medulla into deeper layers of the optic lobe, suggesting that R8 neurons terminate, as in the wild type, within the medulla neuropil. As R7 axons project into the optic lobe only later, and no early R7-specific axon markers are available, the initial projections of these neurons to their medulla targets could not be assessed. These data are consistent with a strong mistargeting defect, where R1-R6 neurons fail to terminate in the lamina and, instead, project into the medulla neuropil. These phenotypes are completely penetrant in bks4, bksP1, bksP2, and bks5 and partially penetrant in bksP3. Individuals homozygous for bks4 and bksP1 show the most severe phenotype. These phenotypes are not enhanced in trans to a deficiency for the region, Df(2R)PC4, suggesting that bks4 and bksP1 are strong loss-of-function, if not null, mutations. The relative strength of the connectivity defects is bks4>bksP1>bks5=bksP2>bksP3. Stronger alleles not only increase penetrance, they also increase the severity of the mistargeting phenotype. Increasing strength of the alleles also correlates with progressive decrease in larval motility (Rao, 2000).

Although bks mutations lead to severe defects in neuronal connectivity, several markers indicated that development of cells in the target region occurs normally. As in the wild type, lamina neuronal precursor cells are driven through their final division, as assessed by BrdUrd incorporation, and differentiate as assessed by the expression of the lamina neuronal markers, Dachshund and Elav. Lamina glia, the intermediate targets for R-cell growth cones, migrate into the target region and differentiate largely as in the wild type (Rao, 2000).

R-cell projection defects in bks mutants may reflect a role for bks in R cells, in their targets, or both. To distinguish between these possibilities, genetic mosaic analysis was carried out. Mutant eye tissue, homozygous for bksP1, was generated in otherwise heterozygous eyes by x-ray-induced mitotic recombination. R1-R6 targeting from mutant tissue into the lamina was assessed using an R1-R6-specific axonal marker, Rh1-LacZ. Cryostat sections of adult heads prepared from genetic mosaics and wild-type controls were stained with a rabbit anti-beta-galactosidase antibody. In the wild type, all R1-R6 axons terminate in the lamina; no staining is observed in the medulla. In contrast, in all mosaic individuals examined, many R1-R6 axons fail to terminate in the lamina and, instead, project through the lamina and terminate in the medulla. In support of these findings, the bks mutant phenotype is largely rescued by expressing a bks cDNA under the control of an eye-specific promoter, GMR. These data indicate that expression of bks in the eye is not only necessary for normal R1-R6 connectivity, it is also sufficient (Rao, 2000).

In the Senti (2000) study, brakeless/sbb was identified in a large scale genetic screen to isolate mutations that autonomously disrupt photoreceptor axon targeting. Two EMS-induced alleles of bks, bks1 and bks2 were isolated. In this screen, photoreceptor projections were analyzed in genetic mosaics in which the developing eye, but not the optic lobe, was genetically homozygous for a newly induced mutation. Eye-specific mosaics were generated by using the FLP/FRT system for site-specific recombination together with an eyFLP transgene to provide FLP recombinase activity exclusively in proliferating eye imaginal disc cells. In animals of the genotype eyFLP;FRTbks/FRTcl2R11, for example, FLP recombinase activity in a dividing cell in the developing eye induces recombination at the FRT sites to create two daughter cells, one homozygous for the bks mutation and the other homozygous for both bks+ and the cl2R11 mutation. The latter is a recessive cell lethal mutation, introduced to eliminate these homozygous bks+ cells. Thus, a heterozygous bks cell is effectively replaced by its homozygous bks daughter. The continuous high levels of FLP recombinase activity in the developing eye ensure that this occurs with almost 100% efficiency. As a result, almost all heterozygous cells in the developing eye are replaced by homozygous bks cells during the proliferative phase of eye development, which precedes photoreceptor cell fate specification and axon targeting. In the visual system of such animals, axonal connections are therefore established between homozygous bks mutant photoreceptors in the eye and wild-type (bks/+) target cells in the brain. These animals are referred to as bks mosaics (Senti, 2000).

bks mosaics show a striking defect in their photoreceptor axon projections, as visualized in third instar larvae using MAb24B10 to label all photoreceptors and their axons. In wild type, photoreceptor axon fascicles project through the optic stalk to their appropriate topographic locations in the optic lobe. R1-R6 axons terminate in the lamina, where their growth cones expand to form a dense layer of staining, the lamina plexus, that lies between two glial cell layers. R7 and R8 axons continue beyond this layer to terminate in the medulla. In bks mosaics, photoreceptor axons project normally through the optic stalk to the brain, but fail to segregate into their distinct target layers. Only a vestigial lamina plexus is formed, while thick bundles of photoreceptor axons project through to the medulla. These features suggest that the primary defect in these mutants is the failure of ingrowing R1-R6 axons to stop in the lamina, which inspired the name brakeless. The projection defects observed in bks1 and bks2 mosaics, as well as bks1/bks2 transheterozygotes, are indistinguishable (Senti, 2000).

To examine the projection errors of bks mutant photoreceptors in further detail, a series of markers to label the axons of specific photoreceptor subclasses were introduced into bks mosaics. It was first confirmed that R1-R6 axons extend through the lamina to terminate in the medulla in bks mosaics. To label specifically these axons, a rough-taulacZ marker was used to visualize R2-R5 axons in larvae and an Rh1-taulacZ marker to label all R1-R6 axons in adults. In wild-type animals, all photoreceptor axons expressing these markers terminate in the lamina, whereas in bks mosaics most of these axons project through the lamina and terminate instead in the medulla. This observation confirms that many R1-R6 axons are indeed mistargeted to the medulla in bks mosaic larvae, and further demonstrates that these inappropriate projections are maintained in the adult. Within the medulla, mistargeted R1-R6 axons all appear to make R7-like projections (Senti, 2000).

An Rh4-taulacZ marker was used to examine R7 projections and it was found that these axons are correctly targeted to the medulla in bks mosaics. It was also noticed that both mistargeted R1-R6 axons and correctly targeted R7 axons appear to project in topographically correct fashion along the anteroposterior axis. To also examine retinotopic mapping along the dorsoventral axis, an omb-taulacZ reporter expressed in the dorsalmost and ventralmost photoreceptors in the eye imaginal disc was used. These photoreceptors project their axons retinotopically to the dorsal and ventral extremes of the optic lobe, respectively, in both wild-type and in bks mosaic animals. It is therefore concluded that bks is specifically required for the targeting of R1-R6 axons to the lamina; it is not required for targeting of R7 axons to the medulla, nor for the retinotopic mapping of photoreceptor axons along the anteroposterior and dorsoventral axes (Senti, 2000).

The specific failure of R1-R6 axons to terminate in the lamina in bks mosaics might be due to either (a) a transformation of R1-R6 cells towards an R7 cell fate; (b) a failure of lamina cells to issue a 'stop' signal to R1-R6 growth cones, or (c) a failure of R1-R6 growth cones to respond to this stop signal. To distinguish between these possibilities, cell fate specification was examined in both the retina and lamina in bks mosaics. The specification of retinal cell fates was assessed by examining plastic sections of bks mosaic eyes. In both wild-type and bks mutant ommatidia, R1-R6 cells form large-diameter rhabdomeres that are arranged in trapezoidal fashion around the smaller R7 and R8 rhabdomeres. 96% of bks mutant ommatidia examined show the normal complement and arrangement of photoreceptors, consistent with the observation that the R1-R6-specific markers rough-taulacZ and Rh1-taulacZ are still expressed in bks mosaics. Thus, by both morphological and molecular criteria, R1-R6 cells are not transformed towards an R7 cell fate in bks mosaics. Indeed, the most common abnormality in bks mutant ommatidia is a cell fate transformation in the opposite direction: in 40 of the 63 abnormal bks ommatidia observed, the R7 cell appears to be transformed to an R1-R6 cell, as judged by the size and position of its rhabdomere (Senti, 2000).

Lamina precursor cells require retinal innervation for their final differentiation, including, most likely, their ability to instruct R1-R6 growth cones to seek targets within the lamina. Lamina cell fates were examined in bks mosaics using the neuronal marker Elav and the glial marker Repo. Lamina neurons were found to differentiate normally in bks mosaics. More importantly, the glia that are thought to provide targeting signals for R1-R6 growth cones migrate into the developing lamina and express Repo in bks mosaics just as they do in wild type. However, the ordered arrangement of these glia into rows is slightly disrupted in bks mosaics, but it is suspected that this minor irregulatity is a consequence rather than cause of the continued growth of R1-R6 axons through this layer. Thus, the lamina glial cells are in place and appear to be differentiating normally in bks mosaics. Since bks function is not disrupted in these cells, they presumably still issue their normal targeting instructions to incoming retinal axons (Senti, 2000).

It is therefore concluded that the R1-R6 targeting errors in bks mosaics are not due to the transformation of these cells towards an R7 fate, nor the failure of lamina cells to provide targeting instructions to R1-R6 growth cones. Rather, the data strongly suggest that, in bks mosaics, R1-R6 growth cones are unable to sense or respond to a specific stop signal produced in the lamina (Senti, 2000).

Bks proteins are necessary but not sufficient for lamina targeting. The nuclear localization of Bks proteins excludes them from playing a direct role in axon targeting, but raises the interesting possibility that they might control the choice of target layer by regulating the expression of specific guidance receptors. Target layer specificity might even be determined by the specific Bks isoform(s) expressed in each of the different photoreceptor classes. To test such a model, it was asked whether the expression of either BksA or BksB alone would restore lamina targeting of R1-R6 axons in bks mosaics, and possibly even retarget R7 or R8 axons to the lamina. The GMR-mycbksA and GMR-mycbksB transgenes were introduced into bks mosaics and R1-R6 projections were assayed using the Rh1-taulacZ marker. GMR-mycbksA transgene partially rescues the R1-R6 targeting defects in bks mosaics. Most R1-R6 axons now correctly target the lamina. However, a small number of R1- R6 axons in each hemisphere, predominantly those in the posterior regions of the eye, still project through the lamina to terminate in the medulla. Complete rescue of the bks targeting defect was only obtained with the GMR-mycbksB transgene. Identical results were obtained for both the protein null bks1 allele and the protein-positive bks2 allele. Thus, R1-R6 cells expressing only one of the two Bks isoforms still target their axons to the lamina, arguing against models in which the different isoforms specify different target layers. Evidently, the unique regions of BksB, including the putative zinc finger domain, are also largely, though not entirely, dispensible for Bks function in photoreceptor axon targeting (Senti, 2000).

Finally, R8 projections were examined (using MAb24B10 to stain larval eye-brain complexes) and R7 projections were examined (using the adult marker Rh4-taulacZ) in both wild-type and bks mosaic animals carrying either the GMR-mycbksA or GMR-mycbksB transgene. Despite the high levels of expression provided by the GMR promoter, neither BksA nor BksB is sufficient to retarget R7 or R8 axons to the lamina. Thus, Bks proteins are necessary, but not sufficient, for lamina targeting (Senti, 2000).

The Drosophila larva is extensively used for studies of neural development and function, yet the mechanisms underlying the appropriate development of its stereotypic motor behaviors remain largely unknown. Mutations in scribbler (sbb), a gene encoding two transcripts widely expressed in the nervous system, cause abnormally frequent episodes of turning in the third instar larva. Hypomorphic sbb mutant larvae display aberrant turning from the second instar stage onwards. Focus was placed on the smaller of the two sbb transcripts; its pan-neural expression during early larval life, but not in later larval life, restores wild type turning behavior. To identify the classes of neurons in which this sbb transcript is involved, transgenic rescue experiments were carried out. Targeted expression of the small sbb transcript using the cha-GAL4 driver is sufficient to restore wild type turning behavior. In contrast, expression of this sbb transcript in motoneurons, sensory neurons or large numbers of unidentified interneurons is not sufficient. These data suggest that the expression of the smaller sbb transcript may be needed in a subset of neurons for the maintenance of normal turning behavior in Drosophila larvae (Suster, 2004).

Genetic interactions among scribbler, Atrophin and groucho in Drosophila uncover links in transcriptional repression

In eukaryotes, the ability of DNA-binding proteins to act as transcriptional repressors often requires that they recruit accessory proteins, known as corepressors, which provide the activity responsible for silencing transcription. Several of these factors have been identified, including the Groucho (Gro) and Atrophin (Atro) proteins in Drosophila. Strong genetic interactions are seen between gro and Atro and also with mutations in a third gene, scribbler (sbb), which encodes a nuclear protein of unknown function. Mutations in Atro and Sbb have similar phenotypes, including upregulation of the same genes in imaginal discs, which suggests that Sbb cooperates with Atro to provide repressive activity. Comparison of gro and Atro/sbb mutant phenotypes suggests that they do not function together, but instead that they may interact with the same transcription factors, including Engrailed and C15, to provide these proteins with maximal repressive activity (Wehn, 2006; full text of article).

Previous studies demonstrated that Atro acts as a corepressor in Drosophila, the most convincing of these being the demonstration that fusion of Atro to a heterologous DNA-binding domain confers repressive activity to the chimera. Atro has been shown to interact directly with two transcription factors, Even-Skipped (Eve) and Huckebein, and the repressive ability of Eve is compromised in Atro mutants, probably accounting for the loss of en expression in even-numbered parasegments in Atro mutant embryos (Wehn, 2006).

These studies here are consistent with Atro acting as a corepressor since it was shown that several genes, including run, tkv, al, and B, are completely or partially derepressed in Atro mutant clones in imaginal discs, suggesting that transcriptional repressors required to silence these genes recruit Atro. Atro-dependent repression of Bar (B) in the center of the leg disc is very likely due to interaction with the transcription factor C15, which is expressed in the center of the leg and is required for repression of B. Similarly, Atro-dependent repression of al in the posterior of the wing is very likely due to interaction with En, which is expressed in the posterior and required to exclude al from this compartment. At present it is unclear which transcription factors recruit Atro to repress run in the eye or tkv in the wing, although a strong candidate for run would be the Rough homeodomain protein, which is expressed in the same cells, R2 and R5, that exhibit ectopic run expression in Atro mutant clones. Whether Atro can, in fact, bind directly to C15, En, and possibly Rough, needs to be tested biochemically, since previous studies with Eve and Hkb did not identify a possible interaction motif for Atro nor do sequence comparisons among C15, En, Eve, and Hkb suggest a common motif (Wehn, 2006).

The sbb gene encodes a nuclear protein with unknown function. sbb mutations have many different phenotypes affecting multiple tissues. sbb and Atro interact very strongly genetically and that many of the phenotypes of sbb mutants are very similar to those of Atro mutants, including derepession of run, tkv, al, and B in imaginal discs. Thus, Atro is unable to silence these genes in the absence of Sbb, suggesting that it is required for Atro activity either to recruit Atro to transcription factors or possibly to assist binding of these factors to DNA. Since these transcription factors appear to function normally in some respects in the absence of Sbb (or Atro), it appears more likely that Sbb and Atro function together in a corepressor complex (Wehn, 2006).

One problem with the proposal that Atro activity is dependent upon Sbb is that the phenotypes of Atro and sbb mutants are not identical. For example, embryos lacking both maternal and zygotic Atro have a very severe, almost uncharacterizable phenotype, while embryos lacking both maternal and zygotic Sbb have a much less severe phenotype, characterized by a reduced number of abdominal segments, that is similar to that of embryos lacking only maternal Atro. This could be explained if Atro is partially active in the absence of Sbb, or if it is dependent upon Sbb for repression of some genes but not others. Alternatively, the difference between Atro and sbb mutant phenotypes could be related to Atro having functions other than that of a corepressor. It is has been implicated in positive regulation of Hox gene expression, and it also functions in the cytoplasm to control planar cell polarity. This analysis of sbb mutants does not reveal any potential involvement of Hox gene expression or planar cell polarity and, consequently, if Sbb is required only for Atro to act as a corepressor, then it is not surprising that Atro and sbb mutant phenotypes are not identical. Further experiments are required to determine the nature of the Atro dependence on Sbb for transcriptional repression and how direct any interactions might be (Wehn, 2006).

Mutations in sbb and Atro were originally uncovered in a genetic screen for enhancers of al. It is likely that they act as enhancers because they are utilized by the C15 transcription factor to repress genes such as Bar; C15 is expressed in the same cells as Al and it is thought that they bind together to regulate gene expression. Strong genetic interactions were uncovered among sbb, Atro, and en mutations, that could be explained if En also recruits Atro/Sbb (Wehn, 2006).

Curiously, genetic studies also revealed strong interactions among gro, sbb, and Atro. This could be explained if Gro was also required for Atro activity; i.e., all three proteins may form a corepressor complex. However, this appears to be unlikely because, in contrast to the similar phenotypes of sbb and Atro mutants, there are several distinct differences among the phenotypes of gro mutants and those of sbb and Atro mutants. For example, repression of tkv in the anterior of the wing is dependent on both Sbb and Atro but not on Gro, while repression of run in the antennal disc is dependent upon Gro but not upon Atro or Sbb. This suggests that a specific transcription factor recruits Atro/Sbb to repress tkv in the wing and another transcription factor recruits Gro to repress run in the antenna. The identity of these transcription factors remains to be uncovered (Wehn, 2006).

In some cases gro mutants do have a similar phenotype to those of Atro and sbb; this includes partial derepression of al expression in the posterior of the wing and Bar in the center of the leg. This can be explained if C15 (expressed in the center of the leg) and En (expressed in the posterior of the wing) recruit both Gro and Atro/Sbb and if each imparts some but not all the repressive activity to these transcription factors. Consistent with this, both C15 and En possess eh1-type Gro-interaction motifs and previous studies have revealed that En can repress in the absence of Gro. Further biochemical studies are required to determine if C15 and En can indeed recruit Atro (Wehn, 2006).

At present it is unclear whether Atro and Gro provide all the repressive activity to C15 and En; this will await the generation of Atro gro double-mutant clones. sbb gro double-mutant clones have been analyzed and these reveal that some targets of C15 and En are still at least partially repressed, although En activity appears to be somewhat compromised following the simultaneous loss of Sbb and Gro, in comparison to loss of one of these alone. Either Atro has some activity in the absence of Sbb or C15 and En can use mechanisms other than recruitment of Gro and Atro to repress transcription. Many transcription factors have been shown to have the ability to repress by several mechanisms; for example, although Brk recruits both CtBP and Gro, it can repress some genes in the absence of both, using additional repression domains (Wehn, 2006).

Why do C15 and En need to recruit both Gro and Atro? En can repress some genes completely in the absence of either Gro or Atro, for example, ci and dpp in the wing. However, for repression of al, the activity of En is clearly reduced in the absence of either, indicating that it needs to recruit both to completely repress this gene. This would suggest a quantitative explanation; i.e., En recruits both Gro and Atro to increase its activity, rather than to allow it to repress specific genes repressed more efficiently by one or the other. This is consistent with the suggestion that both corepressors function via a similar mechanism: both Gro and a vertebrate homolog of Atro have been shown to recruit a histone deacetylase. The recruitment of both may decrease histone acetylation to a level that cannot be achieved with either alone (Wehn, 2006).

Lines is required for normal operation of Wingless, Hedgehog and Notch pathways during wing development

The regulatory Lines/Drumstick/Bowl gene network is implicated in the integration of patterning information at several stages during development. This study shows that during Drosophila wing development, Lines prevents Bowl accumulation in the wing primordium, confining its expression to the peripodial epithelium. In cells that lack lines or over-expressing Drumstick, Bowl stabilization is responsible for alterations such as dramatic overgrowths and cell identity changes in the proximodistal patterning owing to aberrant responses to signaling pathways. The complex phenotypes are explained by Bowl repressing the Wingless pathway, the earliest effect seen. In addition, Bowl sequesters the general co-repressor Groucho from repressor complexes functioning in the Notch pathway and in Hedgehog expression, leading to ectopic activity of their targets. Supporting this model, elimination of the Groucho interaction domain in Bowl prevents the activation of the Notch and Hedgehog pathways, although not the repression of the Wingless pathway. Similarly, the effects of ectopic Bowl are partially rescued by co-expression of either Hairless or Master of thickveins, co-repressors that act with Groucho in the Notch and Hedgehog pathways, respectively. It is concluded that by preventing Bowl accumulation in the wing, primordial Lines permits the correct balance of nuclear co-repressors that control the activity of the Wingless, Notch and Hedgehog pathways (Benítez, 2009).

The Drosophila wing is a discrete organ that has been used to study the coordination of signaling pathways during development. The developing wing disc is a sac-like structure composed of the columnar epithelium or disc proper cells (DP), the cuboidal marginal cells (MC) and the overlying squamous cells (SC); MC and SC constitute the peripodial epithelium (PE). During larval development, imaginal cells proliferate extensively and are patterned. After metamorphosis, the DP cells differentiate into the cuticle that forms the adult wing and notum, whereas PE cells contribute little to these structures (Benítez, 2009).

The Lin/Drm/Bowl cassette is emerging as an important molecular mechanism with which to coordinate various pathways in different developmental contexts. In all cases, the steady-state accumulation of Bowl is regulated by the relative levels of Drm and Lin proteins. High levels of Drm impede binding of Lin to Bowl and, thus, this transcriptional repressor becomes stabilized in the nucleus. In this study it was found that regulatory interaction Lin/Drm/Bowl also functions during wing development. In lin- or Drm GOF cause ectopic expression of Bowl and dramatic overgrowths within the wing disc. These overgrowths frequently showed altered cell identity, resembling more proximal disc margin cells. Some of the effects can be explained by the ability of Bowl to interact with Gro co-repressor through the eh-1 motif, forming a complex that sequesters Gro from other repressors complexes such as Su(H)/H/Gro and Mtv/Gro (Benítez, 2009).

Although Bowl is ubiquitously transcribed in the wing disc, Bowl protein is present only in the SC and MC, being normally absent from the DP cells. The spatial distribution of nuclear Bowl is dependent on Drm, which causes Lin to relocalize to the cytoplasm. Drm is absent from most of the DP cells and, therefore, Lin turns down the steady-state accumulation of Bowl protein in these cells. In the absence of Lin, Bowl accumulates in the DP cell nuclei and elicits the dramatic alterations observed in lin- mutant cells. Therefore, the main function of Lin is to prevent Bowl accumulation in the DP cells, restricting Bowl protein to MC and SC of the PE (Benítez, 2009).

The main alterations in lin-, Drm GOF or Bowl GOF clones can be classified according to the signaling pathways temporally affected. The earliest defect observed is the repression of Wg pathway responses and the evidence suggests that Bowl functions as a repressor of the Wg pathway. However, activated forms of nuclear Wg pathway components, such as ArmS10 or dTcf, cannot restore the expression of the proximal-distal markers owing to repression of the Wg targets in lin-, indicating that Bowl must act in parallel to or downstream of Arm and dTcf (Benítez, 2009).

Bowl is a zinc-finger protein that can interact with the co-repressor Gro directly through the eh-1 motif. The results indicate that this mechanism is also important under conditions where Bowl accumulates in the wing disc. Most of the alterations observed in lin- or Drm GOF clones can be explained by Bowl sequestering Gro from other repression complexes (causing activation of N targets and Hh). Several results support this model. First, the strong genetic interaction between lin and gro alleles, where trans-heterozygous combinations between lin and gro alleles result in dramatic phenotypes, argue that Gro is a limiting factor. Second, removal of eh-1 motif that recruits Gro, eliminates the effects of Bowl on the Hh and N pathways. Third, ectopic expression of Gro, H or Mtv partially suppress the phenotypes of ectopic Drm or Bowl. These observations imply a 'tug of war' between Bowl, H and Mtv for Gro. Increased H or Mtv would shift the balance back in favor of N target repression and Hh repression (Benítez, 2009).

By contrast, the repression of Wg pathway observed in lin- cells appears to involve a different mechanism. Although the effect is Bowl dependent, repression of Wg targets also occurs with Bowleh1-, indicating that Gro sequestration is not required. Similarly, co-expression of Bowl with H or Mtv cannot re-establish the repression of the Wg targets. These results show that Bowl is able to repress Wg targets independently of Gro and the observation that Bowleh1- VP16 can cause some ectopic expression of Sens suggests that this may involve a direct effect of Bowl on Wg targets (Benítez, 2009).

Wnt/Wg, N and Hh signaling represent major conserved signaling channels to control cell identity and behavior during development. An antagonistic interaction between the Wg and Hh has also been described in the embryo and at the intersection of the D/V and A/P compartment borders of the wing disc. Similarly, Wnt/Wg and N activities are closely entangled in many different systems. Mutual dependent interactions between N and Wnt signaling have been observed in vertebrate skin precursors, in rhombomere patterning and in somitogenesis. It has also been reported that orthologues of the Odd-skipped family, Osr1 and Osr2, function as transcriptional repressors during kidney formation. It is possible therefore that Lin/Bowl/Gro interaction is evolutionary conserved and it will be interesting to discover whether lin is an important regulatory factor in other systems (Benítez, 2009).

By analyzing lin- clones in the wing primordium, this study has uncovered the consequences of stabilizing Bowl in the DP cells. There are, however, two regions where Bowl accumulates normally, in the MC and SC within the PE. Removal of Bowl in the PE might lead to ectopic Wg protein and thus to ectopic activity of the Wg signaling to transform PE from squamous to columnar cells. In this context, recently, it has shown that Bowl inhibition by ectopic expression of Lin results in the replacement of the PE by a mirror image duplication of the DP cells. However, not much alteration has been observed in cell morphology nor in the expression of markers such as Ubx or Hth when Bowl was depleted in PE cells (bowl- clones and UAS-BowlRNAi). It could be that the recovered bowl- clones were not induced early enough or that the levels of Bowl-RNAi were not sufficient to completely eliminate the Bowl function in these cells. Nevertheless, these manipulations revealed that bowl- phenotypes in the proximal wing and notum are consistent with a functional role in MC. Therefore, it is concluded that Lin has an important role in restricting Bowl to the MC (and PE), delimiting a Bowl-free territory that forms the DP cells and enables their responsiveness to key signaling pathways such as Wg (Benítez, 2009).

The Brakeless co-regulator can directly activate and repress transcription in early Drosophila embryos

Previous studies have shown that Brakeless can function as a transcriptional co-repressor. This study performs transcriptional profiling of brakeless mutant embryos. Unexpectedly, the majority of affected genes are down-regulated in brakeless mutants. It was demonstrated that genomic regions in close proximity to some of these genes are occupied by Brakeless, that over-expression of Brakeless causes a reciprocal effect on expression of these genes, and that Brakeless remains an activator of the genes upon fusion to an activation domain. Together, these results show that Brakeless can both repress and activate gene expression. A yeast two-hybrid screen identifies the Mediator complex subunit Med19 as interacting with an evolutionarily conserved part of Brakeless. Both down- and up-regulated Brakeless target genes are also affected in Med19-depleted embryos, but only down-regulated targets are influenced in embryos depleted of both Brakeless and Med19. These data provide support for a Brakeless activator function that regulates transcription by interacting with Med19 (Crona, 2015).

Brakeless has been shown to function as a transcriptional co-repressor. It was identified in a genetic screen for regulators of Drosophila embryo patterning, and was shown to function as a co-repressor for the transcription factor Tailless. Brakeless can associate with another co-repressor, Atrophin, and both Brakeless and Atrophin are required for Tailless repressor function. In brakeless or atrophin mutant embryos, expression of the Tailless-regulated segmentation genes Krüppel and knirps (kni) expand to more cells than normal (Crona, 2015).

Genetic evidence is mostly consistent with Brakeless functioning as a co-repressor. Brakeless was initially identified as required for correct termination of photoreceptor axon projections in the optic lobe of the Drosophila brain, where it represses Runt expression in photoreceptor R2 and R5 neurons. In parallel, it was isolated as scribbler, since when mutated it causes a larval turning behavior phenotype in the absence of food. The gene has also been named master of thickveins (mtv), since it represses expression of the Dpp receptor Thickveins in wing imaginal disks (Crona, 2015).

The brakeless locus encodes two proteins, Brakeless-A and Brakeless-B, generated by alternative splicing. The only sequence similarity to known functional domains is a single C2H2 zinc finger located in the unique region of Brakeless-B. One additional domain (D2) is highly conserved and present also in sequences from other metazoa. In vertebrates, two Brakeless homologs are present, ZNF608 and ZNF609. ZNF608 controls Rag1 and 2 expression during mouse thymocyte development, and has been associated with body mass index in humans. ZNF609 is part of a protein network for pluripotency in mouse embryonic stem cells as a component of a Dax1 complex. Although Brakeless has been suggested to participate in mediating SUMO-dependent transcriptional repression, the mechanisms by which Brakeless controls transcription are not understood. Identification of proteins that interact with Brakeless could provide insight into such mechanisms (Crona, 2015).

The Mediator complex is a multi-subunit co-regulator of RNA polymerase II transcription and is a key link for transducing regulatory signals from enhancer to promoter. It is evolutionarily conserved from yeast to man and contains approximately 25 subunits. Electron microscopy has revealed a four domain structure: head, middle, tail and kinase domains. The Med19 subunit forms a hook at the end of the middle domain, and in the absence of the Med19 subunit, the middle module can be released and a stable complex consisting of only head and tail isolated under stringent conditions. However, purification of Mediator from a Med19 mutant yeast strain under more physiological conditions results in an intact Mediator complex, but that interacts less efficiently with Pol II than wild-type Mediator. Med19 has been implicated both in repression and in activation of transcription. In the yeast Saccharomyces cerevisiae, Med19 is encoded by Rox3, and was first identified as a negative regulator of the CYC7 gene. It is also known as SSN7, since it suppresses the requirement of the Snf1 kinase for expression of glucose-repressed genes. Med19 has also been shown to negatively regulate sporulation-specific genes and a Swi4p-dependent reporter. On the other hand, it is also necessary for full galactose induction of GAL1 expression, Ino2-dependent transcriptional activation, as well as for heat-shock induced gene expression, and when tethered to DNA it activates transcription. In human cells, MED19 is also known as LCMR1 (Lung Cancer Metastasis Related Protein 1), and is overexpressed in a variety of cancers and cancer cell lines. It is recruited by the RE1 silencing transcription factor (REST) together with MED26, and mediates repression of neuronal gene expression. Med19 is also necessary for androgen receptor transcriptional activity in Drosophila S2 and human prostate cancer cells, and for gene activation by Hox transcription factors in Drosophila imaginal disks (Crona, 2015).

Transcriptional profiling of brakeless mutant embryos was performed to identify directly regulated target genes. Unexpectedly, most genes are down-regulated in brakeless embryos, indicating that Brakeless can function both as a co-repressor and as a co-activator. From a yeast two-hybrid screen, Med19 was identified as interacting with the highly conserved Brakeless D2 domain. Interestingly, down-regulated but not up-regulated Brakeless target genes are also affected in embryos depleted of Med19 and Brakeless. Together, these data provide support for a Brakeless co-activator function that regulates transcription through Med19 in Drosophila (Crona, 2015).

Embryos devoid of maternal Brakeless mis-regulate expression of 240 genes. Although potential second-site mutations on the brakeless mutant chromosome may affect gene expression, several targets were confirmed by brakeless RNAi experiments, indicating that many of the genes are affected by lack of Brakeless. Surprisingly, the majority of mis-regulated genes were down-regulated in mutant embryos. A large fraction of these genes are maternally contributed, and may therefore be regulated by Brakeless during oogenesis. Since targets for Brakeless in the embryo were sought, focus was placed on genes with early zygotic transcription. Also among this class, the majority of Brakeless-regulated genes were down-regulated in mutant embryos. That some of these genes are direct Brakeless targets is supported by several observations. First, it was possible to detect occupancy of the Brakeless protein at genomic regions in close proximity to the affected genes. Secondly, over-expression of Brakeless caused a reciprocal effect on expression of these genes, demonstrating that Brakeless levels influence their expression. Thirdly, Brakeless fused to the VP16 activation domain behaves as the unmodified Brakeless in that it up-regulates expression of these target genes. These results argue against a model where Brakeless-mediated repression indirectly leads to gene activation, for example by controlling expression of non-coding RNAs. Taken together, these data suggest that Brakeless can behave either as a repressor or as an activator depending on context (Crona, 2015).

Transcriptional co-regulators are believed to mediate the repressive or activating functions of DNA-binding transcription factors and therefore serve as dedicated co-repressors or co-activators, but recent experiments demonstrate that co-regulators may switch transcriptional activity. In the case of the CtBP co-repressor, the oligomeric state determines its function. The CtBP monomer is a co-activator whereas the dimer acts as a co-repressor. How Brakeless can function as a co-activator at some genes, but as a co-repressor on others will be an important future question (Crona, 2015).

Although Brakeless is believed to associate with transcription factors as a way of recruitment to cis-regulatory DNA, the interactions that result in gene repression or activation are not known. A yeast two-hybrid screen was performed with the most evolutionarily conserved part of Brakeless. Med19, which is a subunit of the Mediator complex, was identified as one of the interacting proteins. The part of Med19 that interacts with Brakeless is also evolutionarily conserved. The Mediator complex is present in all eukaryotes and pivotal for transcription by RNA polymerase II. By interacting both with enhancer-bound transcription factors and with the C-terminal domain (CTD) in the largest subunit of RNA polymerase II, it functions as a co-regulator that recruits RNA polymerase II to the promoter. Mediator can also stimulate CTD phosphorylation and enhance transcriptional elongation. Interestingly, the Med19 subunit has been identified in multiple genetic screens in yeast, suggesting that this subunit has a crucial function in transmitting transcription factor activity to RNA polymerase II. Importantly, in cells lacking Med19, the Mediator complex can remain intact. In contrast to subunits whose absence leads to Mediator complex disassembly, Med19-depleted cells are therefore not expected to cause global gene expression changes, but rather cause specific transcriptional defects (Crona, 2015).

Med19 is implicated both in gene repression and in gene activation in yeast. In Drosophila, Med19 is required for gene activation by Hox transcription factors, but not for Hox-mediated repression (Boube, 2014). In this study it was found that Brakeless-activated, but not Brakeless-repressed genes, are affected by Med19 and Brakeless double knock-down. This indicates that the interaction of Brakeless with Med19 may be important for Brakeless-mediated activation, but less so for Brakeless repressor function. Consistent with this idea, the ability of Brakeless to repress a reporter gene in S2 cells when artificially tethered to DNA was not affected by Med19 knock-down. Taken together, these data suggests that Brakeless activates transcription by interacting with Med19 (Crona, 2015).

Predicted proteins with sequence similarity to Brakeless can be found not only in insects, but also in other Ecdysozoa, in Deuterostomes, in Cnidaria, and even in sponges. However, homologous sequences were not found in unicellular eukaryotes, indicating that Brakeless may have participated in evolution of the complex gene regulatory networks required for multicellularity. In this regard, it is interesting that the Brakeless homolog ZNF609 is part of a protein network for pluripotency in mouse embryonic stem cells. Learning more about Brakeless function could provide novel insights into metazoan gene regulation (Crona, 2015).


REFERENCES

Search PubMed for articles about Drosophila scribbler

Benítez, E., Bray, S. J., Rodriguez, I. and Guerrero, I. (2009). Lines is required for normal operation of Wingless, Hedgehog and Notch pathways during wing development. Development 136(7): 1211-21. PubMed Citation: 19270177

Boube, M., Hudry, B., Immarigeon, C., Carrier, Y., Bernat-Fabre, S., Merabet, S., Graba, Y., Bourbon, H. M. and Cribbs, D. L. (2014). Drosophila melanogaster Hox transcription factors access the RNA polymerase II machinery through direct homeodomain binding to a conserved motif of mediator subunit Med19. PLoS Genet 10: e1004303. PubMed ID: 24786462

Crona, F., Holmqvist, P.H., Tang, M., Singla, B., Vakifahmetoglu-Norberg, H., Fantur, K. and Mannervik, M. (2015). The Brakeless co-regulator can directly activate and repress transcription in early Drosophila embryos. Dev Biol [Epub ahead of print]. PubMed ID: 26260775

Crozatier, M., Glise B. and Vincent, A. (2002). Connecting Hh, Dpp and EGF signalling in patterning of the Drosophila wing; the pivotal role of collier/knot in the AP organiser. Development 129: 4261-4269. 12183378

Funakoshi, Y., Minami, M. and Tabata, T. (2001). mtv shapes the activity gradient of the Dpp morphogen through regulation of thickveins. Development 128: 67-74. 11092812

Haecker, A., et al. (2007). Drosophila brakeless interacts with atrophin and is required for tailless-mediated transcriptional repression in early embryos. PLoS Biol. 2007 Jun;5(6):e145. PubMed citation: 17503969

Kaminker, J. S., et al. (2002). Control of photoreceptor axon target choice by transcriptional repression of Runt. Nat. Neurosci. 5(8): 746-50. 12118258

Rao, Y., et al. (2000). brakeless is required for photoreceptor growth-cone targeting in Drosophila. Proc. Natl. Acad. Sci. 97: 5966-5971. PubMed Citation: 10811916

Senti, K. A., et al. (2000). brakeless is required for lamina targeting of R1-R6 axons in the Drosophila visual system. Development 127: 2291-2301. PubMed Citation: 10804172

Suster, M. L., et al. (2004). Turning behavior in Drosophila larvae: a role for the small scribbler transcript. Genes Brain Behav. 3(5): 273-86. 15344921

Wehn, A. and Campbell, G. (2006). Genetic interactions among scribbler, Atrophin and groucho in Drosophila uncover links in transcriptional repression. Genetics 173(2): 849-61. 16624911

Yang, P., Shaver, S. A., Hilliker, A. J. and Sokolowski, M. B. (2000). Abnormal turning behavior in Drosophila larvae. Identification and molecular analysis of scribbler (sbb). Genetics 155(3): 1161-74. PubMed Citation: 10880478


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date revised:10 October 2015

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