blistered/Serum response factor

DEVELOPMENTAL BIOLOGY

Embryonic

SERF mRNA and protein is uniformly distributed in unfertilized eggs. This maternally provided pool of mRNA and protein slowly disappears during cellularization and germ band elongation. Strong zygotic expression is found to resume only after germ band retraction. The protein forms a complex pattern, mostly representing cells of the tracheal system. Ten clusters of 6-9 cells each are observed on the lateral anterior side of each hemisegment between the second and eighth abdominal segment, while only 3-5 cells are stained in the anterior part of the second thoracic segment. In addition, in each of the hemisegments T1 through A7 [Images], single DSFR-expressing cells are identified ventrally in the vicinity of the developing CNS and dorsally in the vicinity of the amnioserosa. The CNS associated cells are integrated into the CNS at later stages. In stage 15 embryos, the dorsal-most SERF positive cells come to lie close to the midline. A row of cells approximately two cells wide are found along the fused midgut. These cells are loosely attached to the visceral mesoderm and represent cells of the visceral tracheal system. Additional SERF is found in the head region of stage 13 embryos, in rows of nuclei along the pharyngeal muscles, and along the hindgut in the posterior end of the embryos. During stages 15 and 16 weak staining is found in nuclei of all somatic muscle cells (Affolter, 1994).

The staining of CNS, midgut, amnioserosa and mesodermal associated cells represents, in fact, to all be associated with or a part of the visceral tracheal system. All the SERF-positive nuclei in the CNS are in close proximity to the tracheal lumen; ganglionic branches of the trachea only enter the CNS in places where SERF-expressing cells occur. Similarly SERF-positive cells in the head region are intimately linked to the tracheal lumen. Not all tracheal cells express Serf. All the Serf expressing cells are part of the terminal branches of the trachea, that is, in late developing tracheal cells in contact with target tissues (Affolter, 1994).

The Drosophila homologue of SRF acts as a boosting mechanism to sustain FGF-induced terminal branching in the tracheal system

Recent data have demonstrated a crucial role for the transcription factor SRF (serum response factor) downstream of VEGF and FGF signalling during branching morphogenesis. This is the case for sprouting angiogenesis in vertebrates, axonal branching in mammals and terminal branching of the Drosophila tracheal system. However, the specific functions of SRF in these processes remain unclear. This study establish the relative contributions of the Drosophila homologues of FGF [Branchless (BNL)] and SRF [Blistered (BS)] in terminal tracheal branching. Conversely to an extended view, it was shown that BNL triggers terminal branching initiation in a DSRF-independent mechanism and that DSRF transcription induced by BNL signalling is required to maintain terminal branch elongation. Moreover, increased and continuous FGF signalling can trigger tracheal cells to develop full-length terminal branches in the absence of DSRF transcription. These results indicate that DSRF acts as an amplifying step to sustain the progression of terminal branch elongation even in the wild-type conditions of FGF signalling (Gervais, 2011).

The results contribute to clarification of the roles of BNL and DSRF in terminal branch formation. First, DSRF transcription is dispensable for terminal branch initiation but is a crucial requirement for the progression of this process in wild-type embryos. Second, a constitutively activated form of DSRF still requires BNL signalling to achieve terminal branch formation, thereby indicating that an additional outcome from BNL signalling is required for terminal branch development. Third, high levels of BNL signalling give rise to terminal branches independently of DSRF transcription. All these observations indicate that branches with an intracellular lumen can initiate their development in the absence of DSRF activity. As these are the specific features of terminal cells, it can be concluded that DSRF is not a general determinant of terminal cell fate (Gervais, 2011).

On the basis of these results, the following model is proposed for terminal branch development. On the one hand, BNL signalling triggers the initial phases of cell elongation and intracellular lumen formation. This step is independent of DSRF transcription, probably because BNL levels at this stage are high enough to promote terminal branch initiation. On the other hand, BNL signalling activates DSRF, which in turn allows the progression of cell elongation and intracellular lumen formation. Indeed, as mechanical tension has been proposed as a means to active DSRF expression, it could well be that the same elements involved in the triggering of cell elongation by BNL signalling might also mediate activation of DSRF expression in the terminal cell. Irrespective of the mechanism promoting its expression, DSRF activity can be considered to be a boosting mechanism that, together with other outputs from BNL signalling, ensures that the cellular modifications required for elongation and intracellular lumen formation are kept active in the wild-type conditions of BNL signalling. Consequently, this process appears to spatially restrict induction of terminal branching to places of high BNL signalling, which are often found at the tip of the branches. Finally, although high levels of BNL signalling do not reproduce physiological wild-type conditions, the observation that such high levels bypass the requirement of DSRF transcription for the growth of terminal branches can be of relevance in stress conditions, such as in hypoxia. Likewise, a similar mechanism could be of significance for the induction of angiogenesis in disease (Gervais, 2011).

Larval

Serf is expressed in the future intervein issue of the wing imaginal disc. SERF is distibuted in a specific patched pattern in the wing disc of third instar larvae. The patched staining is restricted to the wing pouch and the hinge region. Within the developing wing pouch, SERF protein appears to be absent from the future wing margin, and also absent from four stripes of cells extending at a right angle from the wing margin towards the proximal regions. As the intervein tissue of the wing is separated into five areas of the wing veins, the expression domains of Serf suggest that the gene is expressed exclusively in those cells that correspond to the future intervein tissue (Montagne, 1996).

In order to understand the role of blistered in wing development, blistered expression was examined in detail. The expression of a blistered reporter is first detected in imaginal wing discs by early third instar larva (70-80 hours AEL). At this stage, it is expressed homogeneously at low levels throughout the wing pouch, except in the presumptive wing margin. Mid-third instar imaginal wing discs (80-100 hours AEL) reveal increasing blistered levels, except in the wing margin; three perpendicular stripes of cells occur, corresponding to veins, where blistered expression begins to fade. At approximately the same developmental stage, veinlet (common alternative name rhomboid) is expressed in stripes corresponding to the gaps in blistered expression. The future veins L3, L4 and L5 will arise from these gaps. In late third instar imaginal wing discs (100-120 hours AEL), a further gap appears in the blistered expression, revealing the presence of the L2 vein. At this stage, a complex modulation of blistered expression is detected in the hinge region, possibly corresponding to the proximal vein trunks and interveins. There is also no expression in the notum. These gaps in the expression of blistered become more conspicuous in everted discs of pupae and, by 24-30 hours APF, all the interveins are apparent. blistered expression must be further refined after this stage, since the stripes lacking blistered are now about 6-8 cells wide, whereas in the adult wings they are only 3-5 cells wide. In adult wings, all vein cells lack blistered expression, which is present in all the intervein cells. The haltere is the only other imaginal disc to express blistered. blistered is expressed at high levels in the region corresponding to the pedicellum and scabellum, but is not present in the presumptive capitellum, the homologous region to the wing pouch. It is thus tempting to speculate that the absence of transalar connections and lack of apposition of dorsal and ventral surfaces in the haltere capitellum might be related to this non-appearance of blistered expression, such as occurs in the hollow wing veins (Roch, 1998).

Effects of Mutation or Deletion

Tracheal development appears normal in mutant embryos until germ band retraction (stage 13), the time when zygotic Serf expression is first detectable in the tracheal system. In more than 90% of stage 14/15 embryos, the 5-8 tracheal cells that presumably would have formed the dorsal branch in wild-type embryos have migrated to positions close the the dorsal midline in the mutants. This abnormal migration behavior is consistent with the tracheal phenotype seen with the Crumbs antibody, revealing the apparent absence of a dorsal branch except for an accumulation of Crumbs in the dorsal portion of each hemisegment. While the gangleonic branch is disrupted, cells of the dorsal trunk, the most ventral anastomosis, and the first dorsal branch are not affected. The defect seems to be manifested by a decay of a rather normal looking tracheal network. It seems that cells do not stop migrating, but instead follow the cell leading the tip of the outgrowth branch (Affolter, 1994).

To establish that the Serf gene and blistered correspond to the same genetic locus, Serf coding sequences from blistered alleles were cloned and sequenced. Point mutations altering Serf coding sequences were identified in three blistered alleles. There is a strong correlation between the severity of the different blistered phenotypes and the predicted molecular defects in the encoded SERF proteins. A single amino acid change in the MADS-box is sufficient to induce a homozygous wing phenotype, but causes neither haploinsufficiency or larval lethality. Trucnation of the SERF protein affects the function much more dramatically and causes a haploinsufficiency wing phenotype and homozygous lethality. The most severe truncation results in a tracheal phenotype phenotype similar to that of pruned1 (Montagne, 1996).

Integrins are evolutionarily conserved transmembrane alpha,beta heterodimeric receptors involved in cell-to-matrix and cell-to-cell adhesions. In Drosophila, the position-specific (PS) integrins (see Myospheroid) mediate the formation and maintenance of junctions between muscle and epidermis and between the two epidermal wing surfaces. Besides integrins, other proteins are implicated in integrin-dependent adhesion. In Drosophila, somatic clones of mutations in PS integrin genes disrupt adhesion between wing surfaces to produce wing blisters. To identify other genes whose products function in adhesion between wing surfaces, a screen was conducted for autosomal mutations that produce blisters in somatic wing clones. 76 independent mutations were isolated in 25 complementation groups, 15 of which contained more than one allele. Chromosomal sites were determined by deficiency mapping, and genetic interactions with mutations in the beta PS integrin gene myospheroid were investigated. Mutations in four known genes (blistered [Drosophila's Serum response factor implicated in the specification of intervein cells], Delta, dumpy and mastermind) were isolated. Mutations were isolated in three new genes (piopio, rhea and steamer duck) that affect myo-epidermal junctions or muscle function in embryos. Mutations in three other genes (kakapo, kiwi and moa) may also affect cell adhesion or muscle function at hatching. These new mutants provide valuable material for the study of integrin-dependent cell-to-cell adhesion. It is thought that blisters arise in Delta and mastermind clones because of a failure to maintain the normal properties of ectodermal cells within the clonal boundaries (Prout, 1997).

The blistered function in wing vein development was examined by studying genetic mosaics of mutant cells, genetic interactions with other genes affecting vein development and blistered expression in several mutant backgrounds. Clones of blistered mutant cells proliferate normally but tend to grow along veins and always differentiate as vein tissue. These observations indicate that vein-determined wing cells show a particular behaviour that is responsible for their allocation to vein regions. Strong genetic interactions are observed between blistered, veinlet and genes of the Ras signaling cascade, in particular Egf receptor, rolled (rl) (MAPK), and a putative ligand of Egf receptor, vein, that codes for a neuregulin secreted protein. Hemizygosity for blistered totally suppresses the lack of vein L4 phenotype in Egfr, rolled and vein homozygous mutants, while greatly enhancing the amount of ectopic vein observed in a gain-of-function rolled allele. blistered hemizygosity also suppresses the lack of veins in veinlet hypomorph conditions. Conversely, it dramatically enhances the amount of ectopic vein tissue obtained after ubiquitous expression of veinlet + product (Roch, 1998).

The observed interaction between Egf receptor and blistered in hypomorphic conditions led to the study of double mutants for strong alleles of both genes in mosaic clones. Double mutant clones were generated at 48-72 hours AEL for the top 4A and bs P1292 alleles. top 4A clones appear with a reduced frequency, are smaller, narrower and more elongated than controls, and are composed of small cells that are unable to differentiate vein histotype, leaving a gap of intervein tissue wherever they touch a vein, except in the anterior wing margin vein (L1). Double mutant top 4A;bs P1292 clones tend to occupy vein territories like bs P1292 clones, a preference never observed in top 4A clones, but one which appears with a frequency and size similar to top 4A controls. Double mutant cells differentiate autonomously, in all cases, into pigmented, corrugated and compacted tissue with smaller cells than those characteristic of torpedo. The observation of these typical vein features leads to the conclusion that this tissue has a vein histotype indicating that the blistered extra vein phenotype is epistatic to torpedo (Egfr) lack of veins. It is concluded that during disc proliferation, blistered expression is under the control of the Ras signal transduction pathway, but its expression is independent of veinlet. During the pupal period, blistered and veinlet expression become interdependent and mutually exclusive. These results link the activity of the Ras pathway to the process of early determination of intervein cells, by the transcriptional upregulation of the blistered nuclear factor (Roch, 1998).

Alleles of the Stubble-stubbloid locus at 89B9-10 act as dominant enhancers of broad alleles of the BR-C. Sb-sbd wild-type products are necessary for appendage elongation. Three other loci are implicated in imaginal disc morphogenesis based on their genetic interactions with both BR-C and/or Sb-sbd mutants. Enhancer of broad [E(br)] was identified as a dominant enhancer of the br1 allele of the BR-C and is a recessive lethal. Mapping of E(br) has led to the identification of two loci, blistered and l(2)B485, mutants of which interact with E(br) and the Sb-sbd locus. Blistered, but not l(2)B485, interacts strongly with the BR-C. Alleles of the blistered locus are viable and disrupt proper wing disc morphogenesis independent of genetic interactions. All three loci map within the 0.6-map unit interval between the genetic markers speck and Irregular facets and to the cytological region 60C5-6; 60E9-10 at the tip of chromosome 2R. Genetic evidence is consistent with the view that the BR-C regulates blistered (Gotwals, 1991).

The function of extra macrochaetae is required during wing morphogenesis. Mitotic recombination clones of both null and gain-of-function alleles of emc, indicate that during wing morphogenesis, emc participates in cell proliferation within the intervein regions (vein patterning), as well as in vein differentiation (de Celis, 1995). The study of relationships between emc and different genes involved in wing development reveal strong genetic interactions with genes of the Ras signaling pathway (torpedo, vein, veinlet and Gap), and with several other genes (blistered, plexus and net) in both adult wing phenotypes and cell behaviour in genetic mosaics. These interactions are also analyzed as variations of emc expression patterns in mutant backgrounds for these genes. In addition, cell proliferation behaviour of emc mutant cells varies depending on the mutant background. The results show that genes of the Ras signaling pathway are co-operatively involved in the activity of emc during cell proliferation, and later antagonistically during cell differentiation, repressing EMC expression (Baonza, 1999).

Genetic interactions have also shown synergistic mutant effects on venation between emc, plexus (px whose molecular nature is unknown) and net, which codes for a bHLH transcription factor. The net gene is required for intervein fate in wings. Furthermore, emc expression, which is absent in normal veins, also disappears in pupal extra veins caused by px and net. Given the molecular nature of net, the co-operative behavior wth emc could reflect direct molecular interactions. Similarly, genetic interactions and changes in expression pattern of emc are found with blistered (bs) mutants. blistered, coding for the Serum response factor of Drosophila, is expressed in the future intervein issue of the wing imaginal disc, in a complementary pattern to Ras pathway genes. In wing differentiation, bs plays a dual role in wing development. Two fully active copies of bs are required to ensure that the formation of wing veins is limited to vein territories. In addition Bs protein is essential for proper terminal differentiation of intervein cells. bs causes strong phenotypic interactions with mutants of the Ras pathway. Thus, it is proposed that emc, bs, px, net and the Ras signaling pathway set of genes are intimately related in vein/intervein patterning and differentiation. The Ras signaling pathway is thought to be involved in maintaining low levels of emc expression during vein pattern differentiation in cells that will differentiate as veins. This is consistent with observations of the expression pattern of emc. Emc protein and mRNA are found at highest levels in intervein regions (Baonza, 1999).

Drosophila integrins have essential adhesive roles during development, including adhesion between the two wing surfaces. Most position-specific integrin mutations cause lethality, and clones of homozygous mutant cells in the wing do not adhere to the apposing surface, causing blisters. FLP-FRT induced mitotic recombination to generate clones of randomly induced mutations in the F₁ generation was carried out, as well as a screen for mutations that cause wing blisters. This phenotype is highly selective, since only 14 lethal complementation groups were identified in screens of the five major chromosome arms. Of the loci identified, three are PS integrin genes (mys, mew, and if); two are blistered and bloated, and the remaining nine appear to be newly characterized loci. All 11 nonintegrin loci are required on both sides of the wing, in contrast to integrin alpha subunit genes. The nine novel genes were named either on the basis of the wing blister phenotype [papillote (pot), bladderwrack (bad), pompholyx (pomp), kopupu (kop), puri (puri), sac (sac), gonfle (gon)] or their embryonic phenotype [beerbelly (bee) and scorpion (sci)]. Mutations in eight loci only disrupt adhesion in the wing, similar to integrin mutations, while mutations in the three other loci cause additional wing defects. Mutations in four loci, like the strongest integrin mutations, cause a 'tail-up' embryonic lethal phenotype, and mutant alleles of one of these loci strongly enhance an integrin mutation. Thus several of these loci are good candidates for genes encoding cytoplasmic proteins required for integrin function (Walsh, 1998).

The two integrins PS1 and PS2 are each required only on one side of the developing wing, with PS1 on the dorsal side and PS2 on the ventral side. If one of the new genes isolated here were involved only in the function of one of the integrins, then it could be expected that it would also be required only on one side of the wing. Conversely, if it were required for the function of both integrins then it would be required on both sides of the wing. Each of the new genes was tested by making mutant clones marked with the trichome marker forked. One allele from each complementation group was tested and mutant clones were scored for the presence or absence of blisters. Blisters were found to be associated with both dorsal and ventral clones for all of the new genes. Therefore, the new genes are required on both sides of the wing to mediate adhesion between the dorsal and ventral epithelial cell layers and may be required for the function of both integrins (Walsh, 1998).

A similar, independent, FLP-FRT screen has been carried out for mutations that cause wing blisters (Prout, 1997). To determine the extent of overlap between the two screens, simple complementation tests were performed with their mutations, which were isolated from the autosomes. On 2 L, bladderwrack appears to be allelic to cassowary, and pompholyx appears to be allelic to pygoscelis (previously called penguin). The single alleles on this arm, termed 2L-A and 2L-F by Prout (1997) each fail to complement one of the four single mutant alleles that have been isolated in the current study, resulting in the identification of two additional complementation groups, which have been renamed bubblewing (bub, replacing 2L-A) and blisterwing (blis, replacing 2L-F). Mutations in many of the loci on 2 R were recovered with a similar frequency in the two screens: both isolated blistered alleles; bloated appears to be allelic to kitikete ; gonfle appears to be allelic to auk; and sac, to moa. A single mutant allele in the current study appears to be allelic to piopio. Mutations in other loci on 2 L were recovered at quite different rates: one single-mutant allele appears to be allelic to kiwi; kopupu appears to be allelic to kakapo; and beerbelly, to takehe. The high degree of overlap between the two screens on the second chromosome emphasizes the selectivity of the screen and suggests that identification may be forthcoming of all of the loci on this chromosome that can be mutated to give this phenotype. The small number of mutations isolated on the third chromsome and lack of overlap suggests that additional genes are likely to exist. Nonetheless, these screens have provided a large set of genes required for adhesion between the two wing surfaces (Walsh, 1998).

When the homozygous phenotypes caused by the mutations were examined, they could be separated into three groups. Members of the first group, consisting of mutations in bad/cass, bee/tak, kop/kak, and sci, cause embryonic lethality, as well as one prominant phenotype that is also caused by mutations in mys: a failure in germ-band retraction. Mutations in the second group also cause embryonic lethality, although tissue morphogenesis appears to occur normally, and this group includes the loci pot, puri, and sac/moa. The third group, consisting of pomp/pyg, blo/kit, bs, and gon/auk, have mutant alleles that cause larval lethality and also do not cause obvious defects in tissue morphogenesis. These latter two classes are more similar to the phenotypes caused by mutations in mew, which are largely larval lethal although they cause a clear defect in gut morphogenesis. Mutations in the first group cause additional phenotypes, such as defects in head involution, which are not caused by embryonic lethal if and mys mutations, suggesting that the products of this group are required for other functions in addition to PS integrin-mediated adhesion (Walsh, 1998).

The isolation of mutations in the gene blistered in this screen shows that the products of the loci recovered in this screen may be involved in the specification of intervein cell fate. The product of the blistered gene is the Drosophila homolog of serum response factor transcription factor, which is required to promote intervein cell development. In bs mutant wings, intervein tissue is converted toward vein, which normally does not show adhesion between dorsal and ventral surfaces of the wing, and this results in blisters. Genetic interactions between mutant alleles of bs and mys and if and mew cause an increase in the penetrance of intervein blisters, but do not enhance vein defects. The dominant genetic enhancement of integrin mutations by bs may occur through a partial transformation to vein fate in the absence of one copy of bs, which results in a reduction in adhesion in the transformed interveins, possibly due to a reduction of integrin expression. In pupal wings, PS integrin expression is normally absent from the veins. This suggests that blistered is upstream of the integrins, and the maintenance of integrin expression is one important aspect of intervein fate. None of the mutations in the other genes identified in this screen appears to be causing blisters due to a similar change of fate from intervein to vein, because they do not show a detectable transformation in the mutant clones (Walsh, 1998).

The link between the two sheets of cuticle that form the mature wing is constructed during puparation. At the apical surface of each epidermal cell, the plasma membrane is linked to the cuticle by an apical hemiadherens junction. This junction serves as an organizing center for the microtubules that extend from the apical to basal surfaces, which with their associated actin filaments form transalar arrays. The PS integrins are localized at the basal surface, where it is thought that they link to the opposite layer of cells via components of the extracellular matrix. The genes identified in this screen could encode any part of this link, including components of the apical junction as well as those more directly connected to integrins at the basal surface. Integrin-mediated adhesion between the two basal surfaces of the wing could potentially require proteins that have a wide variety of functions: (1) extracellular ligands, which may be secreted proteins or other transmembrane proteins; (2) intracellular proteins that link the integrins to the cytoskeleton; (3) intracellular proteins that signal to activate integrins into a high affinity state; and (4) intracellular proteins involved in transmitting signals that are essential for adhesion. The screen described here can only lead to the isolation of some of these proteins. For example, mutations in genes encoding secreted extracellular ligands are likely to cause nonautonomous phenotypes: a mutant clone of cells would be rescued by secretion of the ligand from the surrounding wild-type cells. Because mutant clones of cells would not be produced, the screen would also not identify genes that encode products that are involved in any process that is essential for cell survival or division, in addition to integrin adhesion. This screen should succesfully identify genes that encode cytoplasmic proteins that are essential and fairly specific for integrin functions. Thus it is anticipated that the eight loci identified in this screen that appear to be specifically involved in adhesion of the two surfaces will encode proteins involved in several processes. The most likely types are proteins that link the PS integrins to the cytoskeleton and proteins essential for activating integrins to a high affinity state. If integrin signaling should prove to be an essential part of adhesion in the wing, then isolation of mutations in the genes encoding this signaling pathway would be expected. Finally, proteins that are transmembrane ligands for the PS integrins or proteins that link the apical surface of the wing to the cuticle may be recovered. Therefore, the next goal in this study is to clone these genes to allow a molecular characterization of their roles in adhesion between the two surfaces of the wing (Walsh, 1998).

Signaling by receptor tyrosine kinases (RTKs) is critical for a multitude of developmental decisions and processes. Among the molecules known to transduce the RTK-generated signal is the nonreceptor protein tyrosine phosphatase Corkscrew (Csw). Csw functions throughout the Drosophila life cycle and, among the RTKs tested, Csw is essential in the Torso, Sevenless, EGF, and Breathless/FGF RTK pathways. While the biochemical function of Csw remains to be unambiguously elucidated, current evidence suggests that Csw plays more than one role during transduction of the RTK signal and, further, the molecular mechanism of Csw function differs depending upon the RTK in question. The isolation and characterization of a new, spontaneously arising, viable allele of csw, csw^lf, has allowed a genetic approach to identify loci required for Csw function. The rough eye and wing vein gap phenotypes exhibited by adult flies homo- or hemi-zygous for csw^lf has provided a sensitized background from which a collection of second and third chromosome deficiencies have been screened to identify 33 intervals that enhance and 21 intervals that suppress these phenotypes. Intervals encoding known positive mediators of RTK signaling, e.g., drk, dos, Egfr, E(Egfr)B56, pnt, Ras1, rolled/MAPK, sina, spen, Src64B, Star, Su(Raf)3C, and vein, as well as known negative mediators of RTK signaling, e.g., aos, ed, net, Src42A, sty, and su(ve), have been identified. Of particular interest are the 5 lethal enhancing intervals and 14 suppressing intervals for which no candidate genes have been identified (Firth, 2000).

Among the suppressor loci identified are net, a mutation with a vein promoting phenotype; ed, which has previously been shown to be a suppressor of reduced Egfr signaling, and sty, a known negative regulator of multiple RTK pathways throughout development. In addition, two strong suppressors are contained within overlapping deficiencies that remove the blistered (bs) gene. The latter deficiencies result in dominant wing blistering and ectopic vein phenotypes, which in turn are suppressed by csw^lf, suggesting that the interacting allele is indeed bs, a finding that has been confirmed by testing interactions with a number of bs alleles. Bs has previously been shown to act autonomously in the intervein cells of the pupal wing in order to limit the width of the wing veins and is a Drosophila homolog of the mammalian serum response factor, a MADS-box containing transcriptional regulator. Mutual suppression between the csw^lf and bs alleles may indicate that the balance of vein differentiation observed reflects antagonistic activity between Bs and the Egfr pathway. This is supported by the observation that in the pupal wing the activities of bs and veinlet mutually repress the expression of the other. The finding that strong alleles of bs suppress the eye phenotypes of csw^lf is interesting because this is the first report of a role for Bs in the developing eye (Firth, 2000).

Merlin, the Drosophila homolog e of the human tumor suppressor gene Neurofibromatosis 2 (NF2), is required for the regulation of cell proliferation and differentiation. To better understand the cellular functions of the NF2 gene product, Merlin, recent work has concentrated on identifying proteins with which it interacts either physically or functionally. Genetic screens designed to isolate second-site modifiers of Merlin phenotypes are described from which five multiallelic complementation groups have been identified that modify both loss-of-function and dominant-negative Merlin phenotypes. Three of these groups, Group IIa/scribbler (also known as brakeless), Group IIc/blistered, and Group IId/net, are known genes, while two appear to be novel. In addition, two genes, Group IIa/scribbler and Group IIc/blistered, alter Merlin subcellular localization in epithelial and neuronal tissues, suggesting that they regulate Merlin trafficking or function. Mutations in scribbler and blistered display second-site noncomplementation with one another. These results suggest that Merlin, blistered, and scribbler function together in a common pathway to regulate Drosophila wing epithelial development (LaJeunesse, 2001).

While sbb encodes novel proteins with unknown function, the bs gene product, also known as the Drosophila serum response factor (BS/DSRF), is a well-characterized transcription factor. bs is required for formation of terminal tracheal branches and differentiation of the adult wing. BS/DSRF activity, like that of its mammalian homolog, is regulated by the epidermal growth factor receptor (Egfr) signaling pathway. During development of the wing imaginal disc, cells can adopt one of two fates; most cells form wing blade (intervein tissue), while a subset form the characteristic longitudinal veins. BS/DSRF is believed to promote the intervein cell fate -- loss-of-function bs mutations result in wings in which all cells develop as vein tissue. Activity of the Egfr pathway is believed to promote the vein cell fate by downregulating BS/DSRF function in the vein primordia and promoting the expression of vein-specific genes. Thus interactions between the Egfr pathway and BS/DSRF play a crucial role in wing development (LaJeunesse, 2001 and references therein).

The identification of bs as a dominant modifier of Merlin phenotypes suggests that Merlin, like Blistered, is involved in Egfr signaling. Specifically, the observation that bs mutations enhance Merlin dominant-negative and loss-of-function phenotypes suggests that Merlin may function antagonistically to Egfr pathway function. Although this hypothesis should be considered as tentative, several lines of evidence support this notion: (1) developing wing cells that have lost both Merlin and expanded, which appear to function redundantly, produce abundant ectopic vein material adjacent to endogenous veins; (2) net, which was also identified as a Merlin modifier, has been shown to modify phenotypes of components of Egfr signaling in the wing; (3) a role for Merlin in negatively regulating Egfr function is consistent with the observation that Merlin mutations result in overproliferation phenotypes and (4) a hypermorphic Egfr mutation called Ellipse enhances phenotypes expressed by dominant-negative and hypomorphic Merlin alleles. However, despite these intriguing indications that Merlin may function to regulate Egfr pathway activity, it should be noted that Merlin does not interact genetically with several other known pathway members (Star, asteroid, and rhomboid), nor does it interact with hypomorphic Egfr mutations. In addition, because other signaling pathways, including dpp, wingless, and Notch, are involved in vein specification, it is possible that Merlin functions to regulate one or more of these either instead of or in addition to the Egfr pathway. In support of this notion, Merlin and expanded have both been shown to genetically interact with dpp. Further experiments are required to determine the significance of these genetic interactions. Nonetheless, the identification of Merlin modifiers suggests testable hypotheses regarding Merlin cellular functions and opens new avenues for further investigation of the molecular basis of the NF2 disorder (LaJeunesse, 2001).

The synthesis of dorsal eggshell structures in Drosophila requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. Advantage of this process was taken to identify genes that link patterning and morphogenesis; lethal mutations on the second chromosome were screened for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. These genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61ß, implicated in ligand secretion. Although the functional requirements of bs in dorsal appendage formation has not been tested, it is likely that SRF acts in follicle cell nuclei (Schnorr, 2001).

Genetic observations in wing and tracheal development reveal a role for SRF in processes regulated by Egfr and FGF-R signaling pathways. In Drosophila wing imaginal discs, bs is expressed in the future intervein tissue in a pattern complementary to that of Rhomboid, an Egfr accessory protein that facilitates presentation of ligand. Loss-of-function mutations in bs interact strongly with Egfr and other Ras1 signaling components in the wing and suppress the effects of disruptions in that pathway. In contrast, bs mutations enhance Ras1 defects in the egg, revealing important differences in the regulation of these two processes (Schnorr, 2001).

In tracheal development, bs functions in the terminal branching process that results from activity of breathless (FGF-R), an RTK that can employ the Ras signaling cascade. Lack of bs or breathless function eliminates cellular outgrowths and terminates tracheal branching prematurely. Thus, SRF and FGF-R act in concert to regulate cellular morphogenesis and in these ways resemble SRF and Ras1 function in dorsal appendage formation (Schnorr, 2001).

LIM kinase and Diaphanous cooperate to regulate serum response factor and actin dynamics

The steroid hormone 20-hydroxyecdysone (ecdysone) is the key regulator of postembryonic developmental transitions in insects and controls metamorphosis by triggering the morphogenesis of adult tissues from larvae. The Rho GTPase, which mediates cell shape change and migration, is also an essential regulator of tissue morphogenesis during development. Rho activity can modulate gene expression, in part, by activating LIM kinase (LIMK) and consequently affecting actin-induced SRF transcriptional activity. A link has been established between Rho-LIMK-SRF signaling and the ecdysone-induced transcriptional response during Drosophila development. Specifically, Rho GTPase, via LIMK, regulates the expression of several ecdysone-responsive genes, including those encoding the ecdysone receptor itself, a downstream transcription factor (Br-C), and Stubble, a transmembrane protease required for proper leg formation. Stubble and Br-C mutants exhibit strong genetic interactions with several Rho pathway components in the formation of adult structures, but not with Rac or Cdc42. In cultured SL2 cells, inhibition of Rho, F-actin assembly, or SRF blocks the transcriptional response to ecdysone. Together, these findings indicate a link between Rho-LIMK signaling and steroid hormone-induced gene expression in the context of metamorphosis and thereby establish a novel role for the Rho GTPase in development (Chen, 2004).

Metamorphosis in Drosophila is stringently controlled by pulses of the steroid hormone ecdysone at discrete developmental stages. During larval-pupal transition, ecdysone triggers coordinated changes in tissue morphology that involve histolysis of larval tissues and the initiation of adult structures. Rho GTPase-mediated signaling pathways have been implicated in several aspects of morphogenesis during Drosophila embryo formation. However, a role for Rho signaling in metamorphosis has not yet been reported. Among the downstream mediators of Rho signaling are the LIM kinases, and a closely related Drosophila ortholog of mammalian LIM kinases (designated Dlimk) is specifically expressed at relatively high levels in late larval and pupal stages, suggesting a potential role in Rho-LIMK signaling during this transition. In adult flies, Dlimk is expressed at substantially higher levels in males than in females, consistent with a potential evolutionarily conserved role in spermatogenesis, a process in which mammalian LIMK2 has been implicated. Dlimk mRNA is uniformly expressed throughout eye, wing, and leg imaginal discs (Chen, 2004).

The malformed legs in Dlimk^D522A flies closely resemble leg defects in flies in which Rho signaling is perturbed through genetic disruption of Rho1, DrhoGEF2 (a guanine nucleotide exchange factor for Rho1), sqh (myosin light chain), and zipper (nonmuscle myosin heavy chain). Sqh and zipper are downstream targets of Drok and regulate actomyosin contractility. Loss-of-function mutants of Rho1 or DrhoGEF2 strongly suppress the severity of wing defects associated with Dlimk expression. Reducing Rho activity by overexpressing the potent Rho inhibitor, p190 RhoGAP, also efficiently suppresses Dlimk-induced wing defects. Moreover, reducing levels of Diaphanous or Drok, two Rho targets that promote actin assembly, also substantially reduces the severity of Dlimk-induced wing defects. A loss-of-function allele of blistered, the Drosophila SRF ortholog, also suppresses the Dlimk-induced wing defects, suggesting that regulation of SRF-dependent transcription by Rho-LIMK signaling plays a role in wing morphogenesis. Significantly, in mammalian cells, LIMK and Diaphanous cooperate to regulate SRF activity (Geneste, 2002). Reducing levels of the Rho-related GTPases, Rac1, Rac2, and Cdc42, or the Rac activator, Myoblast city (Mbc), or the Rac/Cdc42 effector target, PAK, has very little effect on the Dlimk-induced wing phenotype. Thus, it appears that in the developing leg and wing, Dlimk specifically mediates a Rho-actin signaling pathway required for imaginal-disc morphogenesis (Chen, 2004).

The observed interactions among Rho1, Dlimk, br, and Sb support a role for Rho signaling in ecdysone-regulated metamorphosis. However, neither Rho1 expression nor activation is ecdysone inducible. In light of studies linking Rho-LIMK signaling to effects on gene expression (Sotiropoulos, 1999), BR-C and Sb expression were examined in flies overexpressing Rho1, Dlimk, or p190 RhoGAP during early puparium stages, when disc morphogenesis is underway. Expression of BR-C and Sb mRNA normally peaks approximately 2-4 hr after puparium formation. However, in flies overexpressing Rho1 or Dlimk, expression of these genes persists well beyond the normal peak of expression seen in 'driver-only' control flies (approximately 8-10 hr after puparium formation. Moreover, expression of these genes is greatly reduced at all stages of pupation in flies expressing p190 RhoGAP. Significantly, although most of the transgenic flies that overexpress p190 RhoGAP die at a late pupal stage, the few 'escapers' that eclose exhibit malformed wings and twisted and bent leg phenotypes that are very similar to those seen in flies expressing Dlimk^D522A . In addition, the pupal lethality that is frequently observed with overexpression of p190 RhoGAP is efficiently rescued by coexpressing Dlimk, indicating that the late developmental defects that arise as a consequence of Rho inactivation largely reflect defects in Rho-LIMK signaling (Chen, 2004).

To examine more directly a requirement for a Rho-actin-SRF pathway in the transcriptional response to ecdysone, Drosophila SL2 cells were used. In SL2 cells, as in developing discs, ecdysone induces the expression of EcR mRNA. Transfection of cells with the Rho-inhibitory C3 toxin or pretreatment with the actin polymerization inhibitor, latrunculin B, substantially reduces the ecdysone-induced increase in EcR mRNA but does not affect transcription of the ecdysone-insensitive gene rp49 or the Rho1 gene. As expected, latrunculin B completely inhibits morphogenesis of leg appendages, indicating a requirement for F-actin assembly. To examine the role of SRF in ecdysone-induced EcR expression, SL2 cells were treated with RNAi corresponding to the blistered gene. RNAi-treated cells exhibit reduced SRF expression and an absence of ecdysone-induced EcR mRNA expression. Together, these results suggest that the ability of Rho and Dlimk to promote F-actin assembly and SRF activation is responsible for their effects on ecdysone-responsive gene expression and tissue morphogenesis. In addition, the findings in SL2 cells indicate that the observed effects of Rho-SRF signaling on the ecdysone response are cell-autonomous effects. Interestingly, genetic interactions have been observed between zipper and sb and between zipper and br, suggesting that Rho-regulated actomyosin contractility, in addition to F-actin assembly, may also influence the ecdysone response. In this regard, it is interesting to note that mechanical stretching of cells reportedly promotes SRF activity. Alternatively, actomyosin contractility may play a parallel role in disc morphogenesis that is independent of any direct regulation of the ecdysone response (Chen, 2004).

No motif has been identified within the 5' and 3' regulatory sequences (2 kb each) of the EcR gene has been identified that matches the reported SRF binding consensus site. Hence, it remains possible that an SRF-regulated coactivator of ecdysone receptor gene expression is a primary target of Rho-Dlimk signaling. It is interesting to note that the Drosophila transcription factor, Crooked legs, regulates expression of ecdysone receptor mRNA and is encoded by an ecdysone-inducible gene that is also required for wing and leg morphogenesis. Such findings highlight the complexity of the gene expression hierarchy involved in the morphogenetic response to ecdysone and indicate a likely role for transcriptional feedback mechanisms (Chen, 2004).

Use-dependent plasticity in clock neurons regulates sleep need in Drosophila

Sleep is important for memory consolidation and is responsive to waking experience. Clock circuitry is uniquely positioned to coordinate interactions between processes underlying memory and sleep need. Flies increase sleep both after exposure to an enriched social environment and after protocols that induce long-term memory. This study found that flies mutant for rutabaga, period, and blistered were deficient for experience-dependent increases in sleep. Rescue of each of these genes within the ventral lateral neurons (LNVs) restores increased sleep after social enrichment. Social experiences that induce increased sleep were associated with an increase in the number of synaptic terminals in the LNV projections into the medulla. The number of synaptic terminals was reduced during sleep and this decline was prevented by sleep deprivation (Donlea, 2009).

Although sleep is a process that is necessary for survival, the functions of sleep are unknown. Sleep is regulated by circadian influences and is important for consolidation of long-term memory (LTM). Additionally, LTM is modulated by circadian mechanisms. Because the relationship between sleep, memory, and circadian rhythms seem to be phylogenetically conserved, Drosophila can be used to explain mechanisms that coordinate these processes. Drosophila show an increase in daytime sleep after exposure to socially enriched environments. Similarly, an increase in sleep after courtship conditioning is necessary for LTM (Donlea, 2009).

Increased sleep after social enrichment is dependent upon genes that are required for learning and memory, including genes that alter cyclic adenosine monophosphate signaling. Although newly eclosed flies that are mutant for the adenylyl cyclase rutabaga (rut²⁰⁸⁰) show increased sleep after social enrichment, 3 to 4 day-old adult rut mutants do not respond to changes in the social environment. Elevating wild-type rut in adult flies with an RU486-inducible driver rescued experience-dependent increases in sleep in adult rut mutants; vehicle-treated siblings showed no increase in sleep. To identify circuits that mediate experience-dependent increases in sleep, a series of GAL4 lines was used to drive wild-type rut expression in brain circuits. Expression of UAS-rut using pdf-GAL4 restored the increase in daytime sleep and daytime sleep-bout duration, although to a lesser extent than GSelav. The expression pattern of pdf-GAL4 is limited to the ventral lateral neurons (LN_Vs), a group of clock neurons that express pigment-dispersing factor (pdf). Although pdf is the only known output from the LN_Vs, flies mutant for pdf show a wild-type increase in sleep (Donlea, 2009).

Given this role of clock cells, the clock gene period (per), which is expressed in the LN_Vs and is required for LTM, was examined. Rescue of wild-type per using a 7.2-kb fragment of the per genomic sequence (per⁰¹; per+7.2-2) restored expression of PER at CT0 within the LN_Vs as well as the dorsal lateral neurons, LN_Ds; mutant flies carrying a null mutation, per⁰¹, expressed no PER. Although per⁰¹ mutants showed no increase in sleep after social enrichment, per⁰¹;per+7.2-2 flies displayed normal experience-dependent increases in sleep. per⁰¹ mutants have no LTM when tested 48 hours after training and only show a transient increase in sleep. per⁰¹;per+7.2-2 flies displayed LTM and increases in sleep. Although per levels are low in mutants for Clock and cycle, both acquire LTM and increase sleep after social enrichment. Thus, only a very small amount of per may be required to support increased sleep and LTM (Donlea, 2009).

To further investigate the role of synaptic plasticity in clock cells, the Drosophila homolog for serum response factor (SRF), blistered (bs), was used. In mice, SRF is essential for activity-induced gene expression and plays an important role in synaptic long-term potentiation (Ramanan, 2005) and in contextual habituation (Etkin, 2006). bs retains a 93% identity with SRF within the DNA-binding MCM1-ARG80-Agamous-Deficiens-SRF (MADS) domain. Social enrichment elevated the transcription of bs in wild-type Canton-S (Cs) flies. Mutants carrying a P element inserted into the bs gene (P{GAL4}bs¹³⁴⁸) do not increase sleep after social enrichment. This deficit was also found in flies carrying either of two other mutant alleles for bs (bs² and bs³) and was present in flies that are homozygous for mutant bs alleles and flies that have been outcrossed to either Cs or to flies carrying the In(2LR)Px⁴ deficiency. The P-element insertion in bs¹³⁴⁸ preserves the MADS domain; similar N-terminal truncated mutant SRF acts as dominant negative. BS is expressed throughout the brain, including pdf-expressing LN_Vs. When UAS-egfp was driven by P{GAL4}bs¹³⁴⁸, expression was restricted to a small number of neurons, including the LN_Vs. Expression of bs using P{GAL4}bs¹³⁴⁸ to drive either of two wild-type bs (UAS-bs) constructs rescued experience-dependent increases in sleep. Moreover, inducing bs expression within the LN_Vsusing pdf-GAL4 increased sleep after social enrichment (Donlea, 2009).

To establish whether expression of bs is required for LTM, flies carrying the P{GAL4}bs¹³⁴⁸ mutant allele were tested using courtship conditioning. Although P{GAL4}bs¹³⁴⁸/+ flies acquire short-term memory, LTM was impaired. Rescue of wild-type bs using P{GAL4}bs¹³⁴⁸ restored LTM. Next, the GAL4 repressor cry-GAL80 was used to block UAS-bs expression within the LNs. Although UAS-bs/+;cry-gal80/+ control flies showed significant courtship suppression, P{GAL4}bs¹³⁴⁸/UAS-bs;cry-GAL80/+ flies had no LTM, which suggests a role for the LNs, although a role for the dorsal neurons (DNs) cannot be excluded. Although SRF deletion in mouse forebrain results in neurons with abnormal morphology (Knoll, 2006), the morphology of LN_Vs in mutant P{GAL4}bs¹³⁴⁸/+ flies did not differ from that of LN_Vs in P{GAL4}bs¹³⁴⁸/UAS-bs rescue flies. All three mutants for bs had intact circadian rhythms and showed anticipatory activity before light-dark transitions; only bs³ flies show an altered period under constant darkness. These findings suggest that there are no developmental abnormalities in the LN_Vs in bs mutants (Donlea, 2009).

Hypomorphic alleles for bs prevent proper wing development through interactions with Epidermal growth factor receptor (Egfr) signaling. Because Egfr alters sleep in Drosophila (Foltenyi, 2007), interactions between bs and Egfr may regulate responses to social experience. After social enrichment, transcription of Egfr was significantly elevated in Cs flies. The Egfr genomic sequence contains several CC(A/T)6GG CArG elements that can be bound by bs to promote transcription, and transcription of Egfr was significantly reduced in bs mutants. Thus, P{GAL4}bs¹³⁴⁸ was used to drive expression of a constitutively active Egfr construct (UAS-Egfr*). Although P{GAL4}bs¹³⁴⁸/+ mutants showed no change in sleep after social enrichment, activation of Egfr in P{GAL4}bs¹³⁴⁸/+;UAS-Egfr*/+ flies increased sleep. Conversely, the expression of a dominant-negative construct for Egfr (UAS-Egfr^DN) using pdf-GAL4 prevented increases in sleep after social enrichment (Donlea, 2009).

A recent theory proposes that a function of sleep is to downscale synaptic connections. Moreover, structural plasticity can be induced by environmental manipulation in Drosophila. To quantify the effect of social enrichment on the number of post-synaptic terminals in LN_V projections, pdf-GAL4 was used to drive expression of a green fluorescent protein (GFP)-tagged construct of the postsynaptic protein discs-large (UAS-dlgWT-gfp). After 5 days of social enrichment, LN_V projections into the medulla of pdf-GAL4/+;;UAS-dlgWT-gfp/+ flies contained significantly more GFP-positive terminals. Although it has not been demonstrated that the labeled synaptic terminals are functional, these tools have been used to quantify synapses. The expression of the UAS-dlgWT-GFP marker did not alter synaptic function in a wild-type background and did not prevent the increase in sleep when expressed using pdf-GAL4 after social enrichment. To determine the effect of waking on synapse number, socially isolated pdf-GAL4/+;;UAS-dlgWT-gfp/+ flies and their enriched siblings either were allowed to sleep ad libitum or were sleep deprived for 48 hours after social enrichment. Although the number of dlg-GFP positive terminals remained elevated in sleep-deprived socially enriched flies, terminal number was significantly reduced in siblings that were allowed to sleep. Similarly, the number of presynaptic terminals in LN_V projections into the medulla using a GFP-tagged construct of the presynaptic protein synaptobrevin (UAS-VAMP-GFP) in pdf-GAL4/+;UAS-VAMP-GFP/+ flies was increased. After 48 hours of recovery, socially enriched pdf-GAL4/+;UAS-VAMP-GFP/+ flies had a reduced number of VAMP-GFP-positive presynaptic terminals relative to their sleep-deprived siblings. A recent study has reported a clock-dependent remodeling in the axonal terminals of the PDF circuit that is highest during the day. Recent data indicates that hyperexcitation of a subset of the LN_Vs suppresses sleep in Drosophila. Together with the current results, these data suggest that the PDF circuit is well suited to test the hypothesis that sleep acts to downscale synaptic connections that are potentiated during waking experience (Donlea, 2009).

Genetic rescue of functional senescence in synaptic and behavioral plasticity

Aging has been linked with decreased neural plasticity and memory formation in humans and in laboratory model species such as the fruit fly, Drosophila melanogaster. This study examined plastic responses following social experience in Drosophila as a high-throughput method to identify interventions that prevent these impairments. Young (5-day old) or aged (20-day old) adult female Drosophila were housed in socially enriched or isolated environments, then assayed for changes in sleep and for structural markers of synaptic terminal growth in the ventral lateral neurons (LNVs) of the circadian clock. When young flies are housed in a socially enriched environment, they exhibit synaptic elaboration within a component of the circadian circuitry, the LNVs, which is followed by increased sleep. Aged flies, however, no longer exhibit either of these plastic changes. Because of the tight correlation between neural plasticity and ensuing increases in sleep, sleep after enrichment was used as a high-throughput marker for neural plasticity to identify interventions that prolong youthful plasticity in aged flies. To validate this strategy, three independent genetic manipulations were used that delay age-related losses in plasticity: (1) elevation of dopaminergic signaling, (2) over-expression of the serum response factor transcription factor blistered (bs) in the LNVs, and (3) reduction of the Imd immune signaling pathway. These findings provide proof-of-principle evidence that measuring changes in sleep in flies after social enrichment may provide a highly scalable assay for the study of age-related deficits in synaptic plasticity. These studies demonstrate that Drosophila provides a promising model for the study of age-related loss of neural plasticity and begin to identify genes that might be manipulated to delay the onset of functional senescence (Donlea, 2014).

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