Antibodies were raised against a common fragment of the Spen proteins (amino acids 3203-3714) to assay the expression pattern and subcellular localization. Immunostaining of whole-mount embryos shows that Spen antigen is expressed in most or all cell types, and is concentrated in nuclei. Spen protein staining is first detectable prior to cellularization in stage 3 embryonic nuclei, and is present in all blastoderm cells, including the pole cells. The protein is expressed throughout the rest of embryogenesis, and is concentrated in nuclei. At later embryonic stages (9 through 14), nuclear Spen staining appears to be most abundant in CNS and epidermal cells, but most tissues exhibit nuclear staining at detectable levels (Wiellette, 1998).
Antibodies against two different C-terminal epitopes reveal that Spen proteins are nuclear and expressed in most, if not all, cells. The Spen proteins are detectable as early as cellular blastoderm and are ubiquitously nuclear during early development. Spen expression in the CNS appears to be at higher levels than in the surrounding epidermis after stage 15. Spen is also expressed in non-neuronal cells, most likely glia, within the CNS. Spen proteins are also detected in muscle nuclei. Maternal protein persists in spen mutant embryos until the onset of terminal neuronal differentiation (Kuang, 2000).
The receptor tyrosine kinase (RTK) signaling pathway is used reiteratively during the development of all multicellular organisms. While the core RTK/Ras/MAPK signaling cassette has been studied extensively, little is known about the nature of the downstream targets of the pathway or how these effectors regulate the specificity of cellular responses. Drosophila yan is one of a few downstream components identified to date, functioning as an antagonist of the RTK/Ras/MAPK pathway. Ectopic expression of a constitutively active protein (yanACT) inhibits the differentiation of multiple cell types. In an effort to identify new genes functioning downstream in the Ras/MAPK/yan pathway, a genetic screen was performed to isolate dominant modifiers of the rough eye phenotype associated with eye-specific expression of yanACT. Approximately 190,000 mutagenized flies were screened, and 260 enhancers of Yan (EY) and 90 suppressors were obtained. Among the previously known genes recovered are four RTK pathway components [rolled (MAPK), son-of-sevenless, Star, and pointed], and two genes (eyes absent and string) that have not been implicated previously in RTK signaling events. Mutations in five previously uncharacterized genes were also recovered. One of these, split ends, has been characterized molecularly and shown to encode a member of the RRM family of RNA-binding proteins (Rebay, 2000).
The isolation of spen as an enhancer of yanACT suggests it may play a role as a positive regulator of the RTK/Ras pathway. Preliminary results indicate spen is a nuclear protein broadly expressed in most tissues and enriched in neuronal lineages. It is currently not known whether spen functions upstream or downstream of yan. One possibility is that spen might regulate the stability of the yan transcript. It has been postulated that the mechanism for downregulating yan activity involves post-translational modifications of the protein, namely phosphorylation by activated MAPK, that subsequently targets yan for degradation. Such post-translational regulation of yan would presumably need to be reinforced at the transcriptional and/or translational level. Thus, spen might play a role in destabilizing yan mRNA in response to Ras signaling. This would be consistent withthe isolation of mutations in spen as enhancers of yanACT. Alternatively, spen could be transcriptionally regulated by yan, and could play a role in splicing, stability, or transport of other downstream effector genes. Future phenotypic, genetic, and biochemical characterization of spen will be necessary to understand its role in Ras/yan signaling events (Rebay, 2000).
In MZspen embryos, the number of many PNS and CNS cell types is altered, and the development of other organs is affected. In order to analyze the molecular basis of these defects, focus was placed on the well-marked development of the lateral chordotonal (lch) sensory neurons. Wild-type embryos contain a cluster of five lch neurons in each abdominal hemisegment. In MZspen embryos, this number varies from none to six, and is typically four. Clusters containing the normal number are often disorganized (Kuang, 2000).
Other mutations have been previously shown to affect lch neuron number by affecting either precursor formation or EGF receptor signaling. In wild-type development, three Atonal-expressing cells become lch precursors. These cells activate EGFR signaling in two neighboring ectodermal cells, recruiting these cells to the lch neuronal fate. Mutants that block either precursor formation or recruitment therefore produce characteristic numbers of lch neurons. atonal, which blocks lch proneural precursor formation, produces occasionally one lch neuron. Mutations in the EGF/Spitz group, which block the recruitment process, produce only three lch neurons. Gain-of-function mutations in the EGF pathway yield 6-7 lch neurons. By contrast, the lch neuron phenotypes in MZspen embryos spans the range observed in loss-of-function mutations that affect precursor formation or recruitment and gain-of-function mutations in EGFR signaling (Kuang, 2000 and references therein).
The variable number of lch neurons could therefore reflect defects in precursor formation, EGFR signaling or another pathway. The formation and refinement of the chordotonal (ch) proneural cluster was followed in wild-type and MZspen embryos using an antibody directed against Atonal, the helix-loop-helix transcription factor that specifies ch neuronal fate. Atonal is initially expressed in the nuclei of a patch of ectodermal cells in each abdominal hemisegment, the proneural cluster. Atonal expression becomes progressively restricted to the precursor cells that contribute to both lateral and ventral chordotonal clusters. At any stage of this dynamic process, abdominal hemisegments in wild-type embryos contain similar numbers of Atonal-expressing cells. By contrast, in MZspen embryos, abdominal hemisegments contain variable numbers of these ch precursor cells. The variability in precursor number suggests possible defects in Notch signaling because Notch is required to generate the appropriate numbers of precursors from proneural clusters. Notch expression is normal in embryos lacking Spen, but expression of Su(H), the key known transcriptional effector of Notch signaling, is dramatically reduced throughout the embryo. Not surprisingly, the expression of Enhancer of Split Complex (En(Spl)-C) proteins, which depends on Su(H), is also reduced in the embryos that lack Spen (Kuang, 2000).
The cell-fate changes in MZspen embryos are not observed in embryos that lack only zygotically contributed Spen. Maternally contributed spen can be detected in zygotic mutant embryos until late stage-12, at which point most cell-fate decisions have been made. No defects are detected in zygotic spen mutant embryos with respect to neuronal cell fate as assayed with mAbs that recognize CNS neuronal subsets [anti-Eve, anti-Engrailed, anti-Ftz, PNS neurons (22C10), glial development (anti-Repo and anti-Sim), and muscle development (anti-myosin heavy chain and anti-connectin)]. spen1 affects the growth and guidance of a subset of CNS and sensory axons (Kolodziej, 1995), but whether the apparent specificity of these phenotypes reflects residual protein function has not been determined. spen3 and spen5 are predicted to produce proteins one-sixth and one-half the size of spen respectively, and so could be nulls. Whether residual spen function is present in zygotic mutant embryos was investigated by comparing CNS and motor axon development in wild-type, spen3 and spen5 zygotic mutant embryos, and in embryos that lack both maternal and zygotic Spen. In zygotic mutant embryos, defects were identified in the elongation and pathfinding of axons in a subset of longitudinal CNS axon tracts, in the intersegmental nerve b (ISNb) and segmental nerve a (SNa) motor axon pathways, in the transverse nerve (TN), but not in the commissural CNS axon tracts, nor in the intersegmental nerve (ISN) motor axon pathways. The frequency of motor axon defects is similar in spen3 homozygous embryos and embryos heterozygous for spen3 and a chromosomal deficiency that removes the 21B region, suggesting that spen3 is a null allele. Defects in axon extension and guidance are more pronounced when maternally contributed spen is also removed, indicating that maternally contributed spen provides residual function in zygotic mutants (Kuang, 2000).
mAb 1D4 reveals three parallel longitudinal axon tracts that extend continuously in the CNS on each side of the ventral midline in wild-type late stage-16 or stage-17 embryos. In spen mutant embryos, the outermost fascicle is discontinuous in some segments, and these axons occasionally invade the middle fascicle. Axon defects are more severe, and extend to all axon tracts in the CNS in spen mutant embryos derived from spen germ line clones (Kuang, 2000).
mAb 1D4 also labels motor axons. In each abdominal hemisegment of the Drosophila embryo, approximately 30 motor axons innervate 30 muscle fibers in a stereotyped pattern. Development was examined of the intersegmental nerve (ISN), which innervates the dorsal muscles, the ISNb, which innervates ventral muscles, and the SNa, which innervates the lateral muscles. The ISN appears normal in zygotic spen mutants. In late stage-16 wild-type embryos, the ISNb has defasciculated from the ISN and forms three connections with ventral muscles: at muscles 12 and 13 and the cleft between muscles 6 and 7. In late stage-16 zygotic spen mutant embryos, most ISNb motor axons defasciculate from the ISN, but stall short of their ventral muscle targets. In the 4%-18% of cases where three connections can be detected in spen mutant embryos, they appear generally smaller than wild type. The SNa motor axons are also abnormal in spen mutant embryos. In stage-16 wild-type embryos, the SNa motor axons have traversed the ventral muscle field and bifurcated just above the ventral muscles. The dorsal branch extends away from the ventral muscles, and the lateral branch extends posteriorly, roughly parallel to the dorsal edge of muscle 12. In stage-16 zygotic spen embryos, most SNa motor axons either stall near the initial entry into the lateral muscle field, or make shorter than wild-type dorsal or lateral extensions (Kuang, 2000).
In MZspen embryos, defective in both maternal and zygotic spen, the development of all motor axon pathways is defective. Motor axons exit the CNS, pick the correct pathways, but fail to innervate their muscle targets. In the cases of the SNa and ISNb motor axons, these axons must first cross the midline, and the occasional absence of these axon tracts may reflect midline defects. However, in hemisegments where distinct ISN, SNa and ISNb fascicles are observed, the motor axons still fail to reach target muscle, and even occasionally cross over segmental boundaries. Some of these defects may also reflect the disorganized and missing muscle fibers in these embryos. Thus, spen appears not to be required for motor axons to distinguish dorsal, lateral and ventral muscles, but is required for later steps in motor axon development in the neuron, muscle or both (Kuang, 2000).
Split ends (Spen) is a protein that acts in parallel with Hox proteins to regulate different segmental morphologies. spen plays two important segment identity roles. One is to promote sclerite development in the head region, in parallel with Hox genes; the other is to cooperate with Antennapedia and teashirt to suppress head-like sclerite development in the thorax. Without spen and teashirt functions, Antennapedia loses its ability to specify thoracic identity in the epidermis. Spen is the only known homeotic protein with RNP binding motifs: this indicates that splicing, transport, or other RNA regulatory steps are involved in the diversification of segmental morphology. Other studies have identified spen as a gene that acts downstream of Raf to suppress Raf signaling in a manner similar to the ETS transcription factor Aop/Yan. This raises the intriguing possibility that the Spen RNP protein might integrate signals from both the Raf and Hox pathways (Wiellette, 1999).
Loss of zygotic spen function results in embryonic lethality that is associated with a loss of the anterior portion of the H-piece, and with a kinked median tooth. The anterior parts of the H-piece are derived from the ventral maxillary segment, and are dependent on Dfd function. Head development is profoundly disrupted in maternal/zygotic spen mutants. These mutant embryos have non-involuted heads that are missing many head sclerites, including the base of the mouth hooks, median tooth, anterior regions of the lateralgraten, and the dorsal bridge. The ventral arms and vertical plates are also strongly reduced. Many of these spen-dependent sclerites are also dependent on head Hox genes such as labial (lab), and Sex combs reduced (Scr); gap/homeotic genes such as empty spiracles (ems) and orthodenticle (otd), and other head patterning genes. In the maternal/ zygotic mutants, the trunk region of the embryos appears to be largely unaffected. The denticle belts and posterior structures such as filzkorper appear normally shaped, although these structures, as well as the remnants of head skeleton (and ecopic head-like sclerites, see below), are somewhat less pigmented/ sclerotized than normal. The overall low level of sclerotization may be due to embryonic death in maternal/zygotic mutants before the cuticle is fully developed (Wiellette, 1999).
Approximately 50% of the embryos that are zygotic or maternal/zygotic spen mutants also develop sclerites in the thoracic segments. These sclerites appear most frequently in the second and third thoracic segments (T2 and T3), and overlap the anterior/posterior compartment boundary. The expressivity of this phenotype ranges widely. The weakest phenotypes exhibit a row of small chunks of ectopic sclerotic material, usually limited to T2 and T3. Strong phenotypes show broad bands of sclerotic material in all thoracic segments, often accompanied by sclerotic patches in lateral regions of the abdominal segments. No matter how extensive, the sclerites do not form in the fields of denticle belts, nor do they change the overall size of a segment. In small patches, the ectopic material is variably sclerotized, scar-like, and not recognizable as any other embryonic structure (Wiellette, 1999).
When manifest in broad patches, the sclerotic material is brown and striated, reminiscent of the brown, striated appearance of the ventral arms, vertical plates and dorsal arms of the head skeleton. It is concluded that one role of spen is to suppress the production of head-type sclerotization from ventral thoracic cells, and it is needed to a lesser extent for this function in lateral abdominal cells (Wiellette, 1999).
This spen minus homeotic phenotype is not specific to alleles isolated on the basis of Dfd interaction. spen was also isolated in a screen for suppressors and enhancers of an activated Raf construct. Dickson (1996) isolated three alleles of E(Raf)2A. These alleles do not complement the lethality of spen alleles that generate homeotic phenotypes, and the E(Raf)2A alleles also exhibit ectopic head-like sclerites, as do 11 additional alleles of spen that enhance the rough eye phenotype generated by ectopic expression of Cyclin E in the eye (Wiellette, 1999 and references therein).
Production of ectopic head-like sclerotic material in the trunk indicates that loss of spen function might result in de-repression of head patterning genes. The cuticular phenotypes were examined of embryos doubly mutant for spen and Distal-less, cnc, lab, Dfd, buttonhead, otd and ems, all of which have been shown to be required for determining head-specific pathways. All of the double mutant combinations show the same degree of thoracic sclerotization as spen single mutants. The transcription patterns of Dll, cnc, lab, Dfd, ems, otd and apontic were examined in spen mutant embryos and found to be indistinguishable from wild type. Therefore it is concluded that spen does not repress ectopic head-like cuticle by repressing the expression or function of these known head-determining genes. Finally, ectopic expression of Dfd and lab in spen mutant embryos does not alter the degree or character of the ectopic sclerites in the thorax (Wiellette, 1999).
Null mutations in Antp result in a transformation of T2 and T3 towards T1 in the embryonic body plan. In addition, Antp mutant embryos develop ectopic head-like sclerites in the dorsal thorax (between T1 and T2), similar in kind but not in position to the ectopic sclerite phenotype seen in spen mutants. To test whether spen and Antp function in an additive or synergistic manner in the repression of head-like sclerites in the thorax, spen-; Antp- cuticle phenotypes were examined. Embryos mutant for both spen and Antp have more sclerotic material in dorsal T2 than do Antp mutants alone. In addition, the ectopic head-like sclerites in the ventral thorax of spen-;Antp- mutants are more sclerotized and extensive than in spen mutants alone. The sclerotic material in spen-;Antp- mutants frequently appears in two distinct bands, one in the center of the segment similar to the position in spen mutants, and at another position in the posterior of T1 and T2. These posterior ectopic sclerites do not develop in T3. The enhanced formation of head-like sclerites in spen-;Antp- mutants suggests that spen and Antp function in a common or interacting pathway(s) in subregions of T1 and T2 (Wiellette, 1999).
The synergistic effect of Antp and spen might be due to a regulatory effect of Antp on spen transcription pattern, or to Spen effects on Antp transcript pattern or translation. However, Antp transcript and protein expression patterns are unchanged in spen mutant embryos, and spen transcript expression is unchanged in Antp mutant embryos. Therefore, spen and Antp appear to be acting in parallel, presumably due to direct or indirect regulation of common downstream genes (Wiellette, 1999).
If spen and Antp regulate common targets, then induction of high levels of exogenous Antp expression might result in suppression of the spen mutant phentoype. The ability of excess Antp protein to suppress the spen mutant phenotype was examined. Overexpression of Antp under heat shock promoter control (hsAntp) causes a transformation of head regions to thoracic identity, but leaves T2 and T3 nearly unchanged. When Antp is overexpressed in a spen mutant background, the ectopic head-like sclerites are strongly suppressed. The number of hsAntp; spen- embryos that exhibit any detectable ectopic sclerites is less than half the expected number compared to spen- mutant siblings from the same cross, or compared to spen-; hsAntp embryos that were not subjected to heat shock. In addition, the sclerites which do occasionally appear in heat shocked hsAntp; spen- embryos are smaller than those in their spen- siblings. The ability of excess Antp to suppress the spen- homeotic transformation indicates that the two genes interact to repress ectopic head-like sclerites (Wiellette, 1999).
In the head region, where spen is required for the development of sclerites that also require Dfd, Scr and other head genes, it is also possible that spen might work in parallel to Hox pathways. This is the case for Dfd and Scr, since mutations in these genes have no effect on spen transcript expression pattern, and conversely, spen mutants have no effect on Dfd or Scr transcript or protein expression patterns. Attempts were made to test whether the overexpression of Dfd could rescue the H-piece defect in spen mutant embryos, but the morphology of the head skeleton was so disrupted by heat shock induced ectopic Dfd protein that it was not possible to conclude whether the anterior H-piece was restored (Wiellette, 1999).
Another Drosophila gene involved in distinguishing head from body is teashirt (tsh). Tsh protein is expressed only in the labial segment and trunk region of embryos, where it is required to repress head identity and to promote thoracic and abdominal segment identities. tsh transcription levels in the thorax are maintained by Antp, but a variety of genetic interaction tests have shown that Antp and Tsh have independent functions in repressing head development. Is spen integrated into the Antp;tsh pathways by regulation of the tsh or Antp expression patterns? Experiments show that (1) expression pattern of Tsh protein is unchanged in spen mutant embryos; (2) protein expression patterns of Scr or Antp in spen-;tsh- double mutant embryos are unchanged from the pattern seen in tsh mutants alone, and (3) the spen transcript pattern is normal in tsh mutants. Therefore, Spen suppression of head-like sclerites is not exerted by a regulatory effect on the Tsh protein expression pattern, nor by Tsh effects on the spen transcript pattern, nor through combinatorial effects of spen and tsh on Scr or Antp protein abundance (Wiellette, 1999).
The phenotype of tsh - , spen- mutant embryos suggests that the two genes act to promote thoracic development. In tsh mutant embryos, the T1 denticle belt is absent and although the remaining denticle belts appear to have the appropriate segmental identities, the denticles themselves are disorganized and smaller than in wild type. In contrast, tsh-;spen- double mutants completely lack denticle belts in the thorax. This may be due to the the death of cells in the denticle field in the thorax of the double mutants, or to the inability of Antp protein, still expressed in the remaining cells, to promote the development of thorax-specific structures (Wiellette, 1999).
As to whether tsh and spen collaborate in repressing head-like sclerites, it was found that the tsh-, spen- double mutants still have bits of sclerite in the 'thorax' of the double mutants, so this phenotype is not enhanced. However, the effects of Tsh overexpression on the ectopic head-like sclerites was also examined in spen mutants. In wild-type embryos, overexpression of Tsh protein throughout the embryo results in transformation of head regions toward thoracic identity, as well as poorly differentiated denticle belts, especially in the thorax. In the thorax of spen mutant embryos that also overexpress Tsh, the ectopic ventral head-like sclerites are strongly suppressed. Taken together, these results suggest that spen, tsh and Antp function in a combinatorial manner to repress the development of head-like sclerites and promote the development of thoracic identity (Wiellette, 1999).
The activity of the E2F transcription factor is regulated in part by pRB, the protein product of the retinoblastoma tumor suppressor gene. Studies of tumor cells show that the p16(ink4a)/cdk4/cyclin D/pRB pathway is mutated in most forms of cancer, suggesting that the deregulation of E2F, and hence the cell cycle, is a common event in tumorigenesis. Extragenic mutations that enhance or suppress E2F activity are likely to alter cell-cycle control and may play a role in tumorigenesis. An E2F overexpression phenotype in the Drosophila eye was used to screen for modifiers of E2F activity. Coexpression of dE2F and its heterodimeric partner dDP in the fly eye induces S phases and cell death. Thirty-three enhancer mutations of this phenotype were isolated by EMS and X-ray mutagenesis and by screening a deficiency library collection. The majority of these mutations sorted into six complementation groups, five of which have been identified as alleles of brahma, moira, osa, pointed, and polycephalon (poc: split ends). osa, brm, and mor encode proteins with homology to SWI1, SWI2, and SWI3, respectively, suggesting that the activity of a SWI/SNF chromatin-remodeling complex has an important impact on E2F-dependent phenotypes. Mutations in poc also suppress phenotypes caused by p21(CIP1) expression, indicating an important role for polycephalon in cell-cycle control (Staehling-Hampton, 1999).
In higher eukaryotes, cyclin E is thought to control the progression from G1 into S phase of the cell cycle by associating as a regulatory subunit with cdk2. To identify genes interacting with cyclin E, a screen was carried out in Drosophila for mutations that act as dominant modifiers of an eye phenotype caused by a Sevenless-CycE transgene that directs ectopic Cyclin E expression in postmitotic cells of the eye imaginal disc and causes a rough eye phenotype in adult flies. The majority of the EMS-induced mutations that were identified fall into four complementation groups corresponding to the genes split ends, dacapo, dE2F1, and Cdk2 (Cdc2c). The Cdk2 mutations in combination with mutant Cdk2 transgenes have allowed the regulatory significance of potential phosphorylation sites in Cdk2 (Thr 18 and Tyr 19) to be addressed. The corresponding sites in the closely related Cdk1 (Thr 14 and Tyr 15) are of crucial importance for regulation of the G2/M transition by myt1 and wee1 kinases and cdc25 phosphatases. In contrast, the results presented here demonstrate that the equivalent sites in Cdk2 play no essential role (Lane, 2000).
kinase suppressor of ras (ksr) encodes a putative protein kinase that by genetic criteria appears to function downstream of RAS in multiple receptor tyrosine kinase (RTK) pathways. While biochemical evidence suggests that the role of Ksr is closely linked to the signal transduction mechanism of the MAPK cascade, the precise molecular function of Ksr remains unresolved. To further elucidate the role of Ksr and to identify proteins that may be required for Ksr function, a dominant modifier screen was conducted in Drosophila based on a Ksr-dependent phenotype. Overexpression of the Ksr kinase domain in a subset of cells during Drosophila eye development blocks photoreceptor cell differentiation and results in the external roughening of the adult eye. Therefore, mutations in genes functioning with Ksr might modify the Ksr-dependent phenotype. Approximately 185,000 mutagenized progeny were screened for dominant modifiers of the Ksr-dependent rough eye phenotype. A total of 15 complementation groups of Enhancers and four complementation groups of Suppressors were derived. Ten of these complementation groups correspond to mutations in known components of the Ras1 pathway, demonstrating the ability of the screen to specifically identify loci critical for Ras1 signaling and further confirming a role for Ksr in Ras1 signaling. Mutations in genes encoding known components of the Ras pathway were isolated in a screen for the 14-3-3epsilon, Dsor1/mek, rolled/mapk, pointed, yan, and ksr loci. Furthermore, due to the ability of dominant-negative KSR (KDN) to block RAS/MAPK-mediated signaling, mutations in genes expected to function upstream of ksr were also isolated. These included mutations in the Egfr, Star, Sos, and Ras1 loci. In addition, 4 additional complementation groups were identfied. One of them corresponds to the kismet locus, which encodes a putative chromatin remodeling factor (Therrien, 2000).
Since alleles of EK2-9 failed to complement two independent P elements recently shown to disrupt the RRM-motif protein locus split ends (spen), it is concluded that EK2-9 is allelic to spen. This gene encodes at least three large (~5500 amino acids) and closely related protein isoforms. The Spen proteins contain three RRMs at the N terminus and a conserved region of unknown function at the C terminus. The presence of three RRMs suggests that the Spen proteins mediate their effect via a RNA binding-dependent mechanism such as RNA processing or transport. Interestingly, mutant alleles of spen have been isolated in several independent genetic screens in Drosophila. They were initially recovered in a screen for mutations affecting peripheral nervous system development. Subsequently, they were isolated in two related screens that, like the KDN screen, were designed to identify novel components of the Ras1 pathway. Mutations in spen were isolated as dominant enhancers of a Raf-induced [E(Raf) 2A complementation group] and a Yan-induced rough eye phenotype. The fact that three separate screens targeting Ras1 signaling (KSR-, Raf-, and Yan-dependent) recovered mutant alleles of spen suggests that this locus is relevant for Ras1-mediated signal transduction. Examination of the genetic interactions, however, indicates that the role of spen in Ras1 signaling may not be straightforward. For example, the ability of the E(Raf)2A/spen alleles to dominantly enhance an activated Raf-dependent phenotype suggests that spen is a negative regulator of the pathway. However, alleles of spen also enhance RafHM7 lethality at 18°, and homozygous mutant clones in the eye are often missing R7 and outer photoreceptor cells, although extra photoreceptor cells are occasionally found. These results are more consistent with a positive role for spen during Ras1 signaling and would agree with the fact that spen mutations have been recovered as enhancers of sE-KDN and gmr-yanact. Alleles of spen were also recovered as enhancers of a loss-of-function phenotype for the Hox gene Deformed (Dfd). In that context, the genetic analysis of spen suggests that it functions in parallel to Dfd for the specification of head cuticular structures. Finally, mutations in spen have been identified as dominant enhancers of an E2F/Dp-induced rough eye phenotype and as dominant suppressors of p21CIP1-induced phenotypes. The fact that S-phase entry is stimulated by the overexpression of E2F and Dp proteins in the eye but inhibited by p21CIP1 overexpression suggests that spen may be involved in the negative regulation of cell cycle progression (Therrien, 2000).
Currently it is unknown how spen activity links Ras1-dependent cell differentiation, Hox-dependent segment specification, and E2F-dependent cell cycle control. Nonetheless, their common requirement for spen function suggests that they are interconnected. In agreement with this idea, other loci have been found in common in the screens mentioned above, as well as in other related screens. One of these loci corresponds to the kismet (kis) gene. In addition to the EK2-4/kis alleles identified in this KDN screen, mutations in kis were recovered as dominant enhancers in the Dfd screen and as dominant suppressors in a Polycomb (Pc) loss-of-function screen. Alleles of kis have also been identified as dominant suppressors of the synthetic lethality generated by the coexpression of activated Sevenless (SevS11) and Ras1V12 (Therrien, 2000).
Wingless directs many developmental processes in Drosophila by regulating expression of specific target genes through a conserved signaling pathway. Although many nuclear factors have been implicated in mediating Wingless-induced transcription, the mechanism of how Wingless regulates different targets in different tissues remains poorly understood. The split ends gene is shown to be required for Wingless signaling in the eye, wing and leg imaginal discs. Expression of a dominant-negative version of Split ends resulted in more dramatic reductions in Wingless signaling than split ends-null alleles, suggesting that it may have a redundant partner. However, removal of split ends or expression of the dominant-negative has no effect on several Wingless signaling readouts in the embryo. The expression pattern of Split ends cannot explain this tissue-specific requirement, because the protein is predominantly nuclear and present throughout embryogenesis and larval tissues. Consistent with its nuclear location, the Split ends dominant-negative acts downstream of Armadillo stabilization. These data indicate that Split ends is an important positive regulator of Wingless signaling in larval tissues. However, it has no detectable role in the embryonic Wingless pathway, suggesting that it is a tissue or promoter-specific factor (Lin, 2003).
In this study, a total of seven distinct readouts of Wg signaling were examined in imaginal discs. They are: inhibition of interommatidial bristle formation; MF initiation/progression; repression of Wg and DFz2, and activation of Sens expression at the presumptive wing margin; inhibition of dpp expression in the dorsal leg; and reduction of eye size. Wg regulation of six of these readouts is significantly blocked by partial or complete removal of spen and/or the expression of spenDN. These results provide a strong genetic argument that spen is required for Wg signaling in these tissues (Lin, 2003).
Interpreting spen phenotypes is complicated by the fact that spen has been implicated in several other pathways. Can these functions explain the apparent loss of Wg signaling phenotypes observed? spen has been found to act with Deformed to suppress head identity in the embryonic trunk and spen genetically interacts with cell cycle mutants. It is thought unlikely that these spen functions can account for the phenotypes observed. However, Spen has also been shown to be involved with the Ras and Notch signaling pathways, which do affect the readouts employed for studying Wg signaling. Therefore, it is possible that some of the spen phenotypes documented in this study are due to disruption of these signaling cascades, though it is argued that this is unlikely (Lin, 2003).
spen mutations affect some Ras targets in a way that suggests it acts positively in Ras signaling. This may be the explanation for the non-autonomous derepression of Ac expression adjacent to spen clones in P[sev-wg] eyes, since Dl expression is activated by the EGF/Ras pathway in the eye. Ras signaling plays a positive role in MF progression and elevated Ras signaling can suppress a Wg or Arm induced small eye phenotype. Therefore, a reduction in Ras signaling caused by loss of spen cannot explain the observations. Ras signaling has no effect on wing margin formation and acts downstream of Wg/Dpp crossregulation in the leg, again arguing that the role of Spen in Ras signaling cannot account for the apparent Wg signaling defects observed (Lin, 2003).
Expression of Suppressor of Hairless [Su(H)], a transcription factor required for Notch signaling, is significantly reduced in spen mutant embryos. Can a reduction of Notch signaling explain the results? Notch signaling is required for interommatidial bristle inhibition so this could explain the requirement of spen for Wg-dependent Ac inhibition. However, Notch is absolutely required for Wg expression at the DV stripe in the wing and plays a positive role in MF progression. Thus, reducing Notch activity by loss of spen or spenDN cannot explain the wider Wg stripe and suppression of the dppblk MF defect observed (Lin, 2003).
Though no evidence for elevated Notch signaling in spen mutants has been reported in Drosophila, a recent report has suggested that SHARP, a human Spen homolog, functions as a transcriptional co-repressor for RBP-Jkappa/CBL, the ortholog of Su(H). In addition, the fly homolog of human SMRT, which binds to SHARP, has been shown to act as a negative regulator of Notch signaling. This could mean that loss of spen activity in flies results in higher expression of Notch/Su(H) targets, owing to derepression. Although this could conceivably contribute to the MF and wing phenotypes that were found, such derepression could not account for the suppression of Wg-dependent reduction of eye size and bristle inhibition or the derepression of dpp expression in the leg. In summary, the only explanation consistent with all the spen (or spenDN) imaginal disc phenotypes is a loss of Wg signaling (Lin, 2003).
In contrast to the data in the imaginal discs, no evidence was found for the involvement of spen in Wg signaling in the embryo, either by removing spen gene activity or expressing spenDN. Thus, it appears that Spen may be a tissue-specific regulator of Wg signaling. Spen is a predominately nuclear protein expressed ubiquitously in embryos and imaginal discs. It could be that a Spen co-factor is not expressed in embryos, or that Spen is post-translationally modified in a tissue-specific way. Alternatively, the specificity could lie in the promoters of the targets that were tested. This appears to be the case in the wing, where Wg and Sens regulation by Wg signaling is spen dependent, while that of Fz2 is not (Lin, 2003).
The negative results obtained in the embryo cannot be viewed as definitive. Embryos that lack maternal and zygotic spen activity could be normal for Wg signaling because of redundancy. Likewise, even though expression of spenDN in the imaginal discs causes strong Wg loss of function phenotypes, and causes spen-like phenotypes under mild expression conditions in the embryo, it is possible that adequate amounts of spenDN were not supplied in embryonic assays (Lin, 2003).
Experiments with loss of function spen alleles indicate that spen is not absolutely required for Wg signaling in the wing and eye. Although reduction of spen activity suppresses a dppblk MF defect, which can be explained by a reduction in Wg signaling, complete removal of spen does not cause an ectopic MF. Because removal of Wg signaling is known to induce an ectopic MF, this indicates that sufficient Wg signaling still occurs in the spen clones. In the wing, spen clones affect Wg readouts, but with incomplete penetrance: this again indicates a partial reduction in Wg signaling in the absence of spen (Lin, 2003).
Experiments with spenDN suggest that the partial loss of Wg signaling in spen mutants may be due to redundancy. Expressing spenDN causes more severe phenotypes and much higher penetrance in disruption of Sens and expansion of Wg in the wing than complete removal of spen. A likely explanation is that the SpenDN protein also inhibits the function of another gene that has roles in the Wg pathway redundant to spen (Lin, 2003).
Although many genes exist in the fly genome that encode proteins containing RRMs, only one other besides Spen is predicted to encode a protein with both RRMs and a SPOC domain. This factor has been called short Spen-like protein (SSLP or DmSSp) and is referred to as CG2910 in the annotated genome. No genetic or molecular characterization of SSLP has been reported and its possible redundancy with spen is currently being examined (Lin, 2003).
Where does Spen act in the Wg pathway? Epitasis experiments in the eye indicate that SpenDN blocks Wg signaling downstream of Arm stabilization. Thus, Spen could act in Arm nuclear import, or in mediating TCF/Arm transcriptional regulation. Consistent with a role in Wg target gene transcription, Spen is predominantly nuclear in imaginal tissues. In addition, the mouse and human homologs of Spen have been implicated as transcription factors (Lin, 2003).
Studies on the vertebrate homologs of Spen have provided functions for the RRM and SPOC domain that these proteins share with Spen. Spen has three predicted RRMs near its N terminus. The role of RRMs in specific RNA binding is well established and the RRM domains in the human Spen homolog SHARP has been shown to bind to the steroid receptor RNA co-activator SRA. By contrast, the RRM domain of the mouse Spen homolog, MINT, has been shown to bind to specific double-stranded DNA, including the proximal promoter of the osteocalcin gene. SHARP also binds to the nuclear receptor co-repressor SMRT and acts as a transcription corepressor by recruiting histone deacetylases (HDACs) through its SPOC domain. A similar co-repressor function for SHARP with the DNA-binding protein RBP-Jkappa/CBL has also been reported. Finally, MINT was also found to interact with Msx2, a known transcriptional repressor. These studies on the vertebrate homologs suggest that Spen may bind DNA or RNA at its N terminus, and may regulate the Wg pathway as a transcription corepressor (Lin, 2003 and references therein).
Why is spen required for only a subset of Wg targets? Based on studies with its vertebrate homologs, could spen only regulate the Wg targets that are transcriptionally repressed by TCF/Arm? Wg-dependent transcriptional inhibition through TCF has been shown for the stripe gene in the embryo and has been suggested for bristle inhibition in the eye. However, no direct targets of Wg signaling in the imaginal discs have been determined and attempts to determine whether stripe repression in the embryo requires spen have been inconclusive. It is interesting to note that two embryonic targets tested which were spen independant, eve and slp1, are both directly activated by TCF/Arm. Identification of spen-dependent direct targets of Wg signaling will be necessary to explore this model (Lin, 2003 and references therein).
Two factors have previously been reported that are tissue/promoter-specific regulators of Wg signaling. tsh has been shown to be required for Wg-mediated inhibition of denticle formation in the ventral embryonic epidermis and lines, which is needed for Wg signaling only in the dorsal epidermis. A third factor, Spen, has been reported, that is only needed for imaginal disc regulation of Wg targets. The existence of these specific factors begs the question: what is the difference between the various Wg targets that requires such specificity (Lin, 2003)?
The Drosophila split ends (spen) gene encodes a large nuclear protein containing three RNP-type RNA binding motifs, and a conserved transcriptional co-repressor-interacting domain at the C-terminus. Genetic analyses indicate that spen interacts with pathways that regulate the function of Hox proteins, the response to various signaling cascades and cell cycle control. Although spen mutants affect only a small subset of morphological structures in embryos, it has been difficult to find a common theme in spen mutant structural alterations, or in the interactions of spen with known signaling pathways. By generating clones of spen mutant cells in wing imaginal discs, it has been show that spen function is required for the correct formation and positioning of veins and mechanosensory bristles both on the anterior wing margin and on the notum, and for the maintenance of planar polarity. Wing vein phenotypic alterations are enhanced by mutations in the crinkled (ck) gene, encoding a non-conventional myosin, and correlate with an abnormal spatial expression of Delta, an early marker of vein formation in third instar wing imaginal discs. Positioning defects were also evident in the organization of the embryonic peripheral nervous system, accompanied by abnormal E-Cadherin expression in the epidermis. The data indicate that the role of spen is necessary to maintain the correct positioning of cells within a pre-specified domain throughout development. Its requirement for epithelial planar polarity, its interaction with ck, and the abnormal E-Cadherin expression associated with spen mutations suggest that spen exerts its function by interacting with basic cellular mechanisms required to maintain multicellular organization. This role for spen may explain why mutations in this gene interact with the outcome of multiple signaling pathways (Mace, 2004).
How could Spen instruct cells to maintain a specific position, without affecting their fate directly? A plausible explanation is that it could affect cell adhesion. In fact, in spen mutant embryos, the expression of E-cadherin was up-regulated at sites of high epithelial morphogenetic activity, generating a phenotype similar to the E-cadherin mutant embryos, as it has been observed in other studies. It is plausible that the increase in E-cadherin expression is the result of a wound response to a defect in epithelial integrity, caused by spen mutations. A defect in cell adhesion and/or cytoskeletal rearrangements could also explain specific aspects of the spen embryonic phenotype. The holes that result in abnormal cuticle deposition in the embryonic epidermis are due to a failure of epidermal epithelial integrity. These cells subsequently undergo a wound response at the end of embryogenesis. Additionally, some of the phenotypes resulting from the loss of spen are indeed similar to those seen in mutants for the gene encoding Daschous, a cadherin involved in cell adhesion. However, blistering of the wings, a phenotype that is often found in cell adhesion mutants, was never observed in any of the spen mutant clones (Mace, 2004).
A role for Spen in cell adhesion and/or cytoskeletal rearrangements could also be inferred through its genetic interaction with crinkled (Myosin VIIA), and the planar cell polarity phenotype observed in mutant cells for spen in the wing blade. Myosin VIIA is associated with the cadherin-catenins complex and participates in the creation of a tension force between the actin cytoskeleton and adherens junctions, which is predicted to strengthen cell-cell adhesion. Furthermore, ck acts downstream of Drosophila Rho-associated kinase (Drok), which links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton. Myosin VIIA mutations have been described in vertebrates, including those causing the Usher syndrome in humans, the shaker-1 mutation in mice, and the mariner mutation in zebrafish. Interestingly, these mutations, among other symptoms, cause splaying and abnormal distribution of sensory hair cells in the inner ear, leading to deafness in mice and humans, and mechanosensory defects in zebrafish. It seems plausible that spen may regulate the expression or function of components affecting the outcome of pathways involved in cytoskeletal rearrangements and epithelial planar polarity and, hence, affect cell positioning. However, a direct requirement for spen function in the Ck or Drok pathways is unlikely, since mutations in these genes result in different phenotypes from those observed in spen mutants (Mace, 2004).
An influential role for spen in mechanisms of intercellular adhesion and/or cytoskeletal rearrangements may also be relevant to understanding its suggestive role in human cancer. The search of public human sequence resources reveals one spen ortholog (SHARP), and three putative Short Spen-like Protein (SSLP) orthologs in the human genome. At least one of these genes (OTT/RBM15) is involved in a recurrent translocation detected in acute megakaryocitic leukemia, and a potentially aberrant transcript for another human SSLP ortholog at 3p21 has been identified in cDNA isolated from human cancer cells. Despite the presence of common domains, the functional relationship between large and small Spen-related polypeptides is still unknown. It is plausible that in Drosophila, SSLP might rescue some of the functions of spen during early embryonic development, as evidenced by the incomplete penetrance of phenotypes seen in spen maternal and zygotic mutant embryos. Complementation at this level has been suggested by others to explain the incomplete penetrance of spen mutations in wing discs, although it should be noted that the region required for Spen to interact with transcription factors such as Msx-2 or nuclear receptors, is apparently missing in SSLP proteins. Therefore, the potential redundancy of Spen and SSLP will have to be determined (Mace, 2004).
This study has shown that the function of the spen gene is essential for all stages of development. The experimental evidence indicates that Spen participates in processes that regulate planar cell polarity and may influence cytoskeletal organization, and its loss results in specific phenotypes that can not be solely explained by defects in a specific signaling pathway. In order to unify this observations with those previously reported by others, it is proposed that the function of Spen is necessary for the maintenance of correct cell positioning during growth, ensuring that structures that are determined early during development are correctly positioned in the adult. Since cells are determined early during development to become part of a specific structure, their position has to be maintained during growth according to a pre-established pattern. If cells were unable to maintain their position, phenotypes similar to those obtained in spen mutant clones would be expected. Structures would be misplaced, and in some cases would be absent if the cells that were predetermined to adopt a specific fate fall within 'forbidden' positions. This mechanistic model could explain why spen interacts genetically with signaling pathways that require and/or specify precise spatial organization during metazoan development (Mace, 2004).
The novel family of SPOC domain proteins is comprised of broadly conserved nuclear factors that fall into two subclasses, termed large and small, based on protein size. Members of the large subgroup, which includes Drosophila SPEN and human SHARP, have been characterized as transcriptional corepressors acting downstream of a variety of essential cell signaling pathways, while those of the small subclass have remained largely unstudied. Since SPEN has been implicated in Drosophila eye development, and the small SPOC protein (Spenito) Nito is also expressed in the developing eye, this context was used to perform a structure/function analysis of Nito and to examine the relationship between the two SPOC family subclasses. The results demonstrate that the phenotypes obtained from overexpressing Nito share striking similarity to those associated with loss of spen. Dosage sensitive genetic interactions further support a model of functional antagonism between Nito and SPEN during Drosophila eye development. These results suggest that large and small SPOC family proteins may have opposing functions in certain developmental contexts (Jemc, 2006).
spen encodes the founding member of a family of proteins characterized by three N-terminal RNA recognition motifs (RRMs) and a novel C-terminal domain, called the SPEN Paralog Ortholog Conserved domain or SPOC domain. SPEN orthologues have been identified in worms, flies, mosquito, mouse, human, and other vertebrates, and more recent studies have identified proteins in plants and yeast carrying the SPOC domain in conjunction with other functional motifs. The RRMs suggest a role for SPOC family proteins in RNA or DNA binding and in the case of SPEN are necessary for nuclear localization, while the SPOC domain of SPEN and its human and mouse orthologs SHARP (SMRT/HDAC1 Associated Repressor Protein) and MINT (Msx2-interacting nuclear target protein) has been implicated in transcriptional regulation and repression (Kuroda, 2003; Oswald, 2002; Shi, 2001; Yang, 2005). SPOC family proteins can be further divided into two subclasses based on their size. In contrast to large SPOC family proteins almost nothing is known about the functions of small SPOC proteins. Thus far, only the human small SPOC family member One Twenty Two (OTT)/ RNA-Binding Motif protein-15 (RBM15) has been studied. Specifically, chromosomal translocations identified in cases of acute megakaryocytic leukemia revealed a fusion with MAL (Megakaryocytic Acute Leukemia)/MKL1 (Megakaryoblastic Leukemia-1) that results in a chimeric protein that includes almost the entire coding region of both genes, with 4 RBM15/OTT at the N-terminus and MAL/MKL1 at the C-terminus (Ma, 2001; Mercher, 2001). Recent evidence suggests that the RBM15-MKL1 fusion may contribute to leukomogenesis through an increased ability to activate serum response factor (SRF) target genes (Jemc, 2006 and references therein).
Sequence conservation defines two distinct SPOC family subclasses: SPOC family proteins fall into two apparent subclasses based on their size. To determine whether such a distinction might be functionally significant, sequence alignments of the conserved Cterminal SPOC motif were performed to compare the level of sequence conservation in the SPOC family in general and subclass members in particular. Analysis revealed only 27% identity and 50% overall similarity between the SPOC domains of SPEN and Nito, the Drosophila representatives of the large and small subfamilies, respectively; however, upon comparison of the SPOC domains of these proteins with those of their respective subclass family members, a higher level of conservation was revealed. Drosophila SPEN and human SHARP exhibit 58% sequence identity and 79% overall sequence similarity, while Drosophila Nito and human RBM15/OTT share 47% sequence identity and 62% overall sequence similarity. Comparable results were obtained by comparing the RRM motifs. These results reveal a higher level of sequence conservation within SPOC family subclasses relative to the family in general, raising the possibility that subclasses may have adopted divergent functions (Jemc, 2006).
Overexpression of nito perturbs adult eye morphology: To better understand the relationship between large and small SPOC proteins, it was determined if spen and nito function synergistically or antagonistically in vivo. Because the large SPOC family member spen is required for Drosophila eye development and the fly eye provides a uniquely powerful system in which to explore functional relationships between signaling molecules, this analyses focused on the eye. RT-PCR confirmed that nito, like spen, is expressed in the developing eye disc (Jemc, 2006).
Because no nito mutants are currently available, an in vivo structure-function analysis was undertaken to investigate nito function during eye development. While the phenotypes resulting from overexpression of a gene must be interpreted with caution, such overexpression models frequently result in sensitized genetic systems that can provide powerful tools for investigating in vivo relationships between signaling molecules. Myc-tagged full-length Nito (Nito-FL), Nito lacking the N-terminus (NitoDN; an exogenous nuclear localization sequence was added to ensure proper nuclear targeting) and Nito lacking the C-terminus (NitoDC) were cloned downstream of a UAS promoter and the transgenes were expressed in flies using eye specific GAL4 drivers. Three different sevenless-Gal4 (sev-Gal4) drivers, which promote expression in photoreceptors R1, R3, R4, R6, R7, the cone cells and the 'mystery' cells, which are poorly understood interommatidial cells that are never recruited to the ommatidia and ultimately apoptose, were utilized in this study: sevstrong couples the sev enhancer to the hsp70 promoter, resulting in the highest levels of expression; sevmedium contains both the sev enhancer and sev promoter, and expresses at an intermediate level; sevweak contains the same regulator sequences as sevmedium, but expresses at lower levels, presumably as a consequence of position effect of the transgene. To avoid unnecessary confusion, these will be referred to collectively as sev-Gal4 (Jemc, 2006).
Sev-Gal4 driven overexpression of Nito-FL and NitoDC yielded dosage dependent adult rough eye phenotypes, while overexpression of NitoDN was indistinguishable from wild type. Western blots confirmed the expression of all transgenes, and immunohistochemistry showed nuclear localization of Nito in all cases, indicating that the lack of a NitoDN phenotype is not due to the absence or mislocalization of protein. While overexpression of Nito-FL and NitoDC both perturb eye development, the resulting phenotypes are distinct. This data is not unexpected given previous results for the large SPOC family protein, SPEN, in which overexpression of SPENDC functions as a dominant negative with respect to spen (Jemc, 2006).
It was therefore speculated that NitoDC functions analogously as a dominant negative relative to nito whereas Nito-FL expression simply augments the pool of full-length Nito. Specifically, it was observed that overexpression of Nito-FL results in roughening of the posterior part of the eye and an overall decrease in eye size, whereas overexpression of NitoDC more uniformly perturbs the external morphology of the eye (Jemc, 2006).
In order to distinguish the Nito-FL and NitoDC rough eye phenotypes at the cellular level, adult eyes were sectioned and examined for defects. In wildtype ommatidia, photoreceptors are arranged in a trapezoidal array with seven of the eight photoreceptors visible in one plane of view. The regular trapezoidal arrangement of photoreceptors is disturbed in both overexpression systems. When Nito-FL is overexpressed, a decrease in the number of photoreceptors per ommatidia, elongated rhabdomeres, as well as a general disorganization of the ommatidia are seen. These observations suggest that the rough eye phenotype is due to a loss of photoreceptors and possible defects in the accessory cells, which normally provide support for the rhabdomeres in the ommatidia. This phenotype is strikingly reminiscent of that seen in sections of spen mutant eye clones, raising the possibility that overexpressed nito may function antagonistically with respect to spen in the developing eye (Jemc, 2006).
Eyes overexpressing NitoDC also appear disorganized compared to wildtype, although in contrast to Nito-FL ommatidia, photoreceptor number is not strongly affected. Rather, the most prevalent defect appears to be ommatidial fusions suggesting that cone and pigment cells, rather than photoreceptors are most affected. Given that the Gal4 driver used for these experiments is expressed primarily in a subset of photoreceptors, the cone cells and interommatidial mystery cells, the accessory cell defects observed upon nito overexpression may be due in part to indirect effects on pigment cells. Thus, Nito-FL ommatidia have defects in photoreceptor number and ommatidial morphology, while NitoDC ommatidia have defects in accessory cells required for the spacing of ommatidia (Jemc, 2006).
To further investigate the defects caused by overexpressing nito, the effects of increasing nito expression in early eye development were examined. First, recruitment of the photoreceptor neurons into ommatidia was examined by looking at expression of the panneural marker ELAV in the larval precursor to the eye, the eye imaginal disc. Consistent with the differences observed in the adult phenotypes, the larval phenotypes associated with sev-Gal4 driven expression of Nito-FL and NitoDC are also distinct. In eye discs overexpressing Nito-FL, initial recruitment of photoreceptors appears normal. However, approximately seven rows posterior to the furrow, there is a decrease in the number of photoreceptors per ommatidia. Thus while Nito-FL expression does not perturb initial photoreceptor recruitment, subsequent development and/or survival are compromised, resulting in the reduced number of photoreceptors observed in the adult eye. The loss of photoreceptors upon overexpression of Nito-FL is also similar to spen mutant clones, which have reduced numbers of photoreceptors in mutant ommatidia in the developing imaginal disc, consistent with the observations made in adult eye sections. In contrast to Nito-FL and spen mutant clones, and consistent with the ommatidial fusions observed in adult eye sections, overexpression of NitoDC causes loss of spacing between ommatidia in the larval eye disc, while recruitment of photoreceptors is not affected (Jemc, 2006).
To examine the possibility that the phenotypes associated with overexpression of Nito-FL and NitoDC were due primarily to cell death, eye discs were stained with the apoptotic marker acridine orange. In the wildtype eye disc very little cell death is observed. In Nito-FL eye discs, a stripe of cell death occurs in the posterior part of the differentiating eye disc, consistent with the loss of photoreceptors observed in the ELAV-probed eye disc and similar to the elevated cell death phenotype observed in spen mutant clones. However coexpression of the apoptotic inhibitor p35 or introduction of the H99 Deficiency that removes the proapoptotic genes hid, reaper and grim, did not suppress the Nito-FL rough eye phenotypes, suggesting that increased apoptotic cell death is unlikely to be the primary factor contributing to the Nito-FL associated eye defects. In discs overexpressing NitoDC, increased cell death is observed more anteriorly relative to that for Nito-FL, consistent with the ommatidial spacing defects observed in the ELAV-probed disc (Jemc, 2006).
The potential for functional antagonism between SPEN and Nito was suggested by the similarity of phenotypes observed in adult eye sections overexpressing Nito-FL and in spen mutant eye clones. To further investigate this potential antagonism, a series of dose-sensitive genetic interactions between spen and nito was performed (Jemc, 2006).
Initially, the effects of reducing spen levels in the Nito-FL overexpression background was examined. If Nito-FL antagonizes SPEN function, as suggested by the phenotypic analysis, further reducing spen should exacerbate the Nito-FL overexpression phenotype. An important requirement for such an experiment is the need for dose-sensitive Nito-FL phenotypes. Two observations suggest Nito-FL provides a dose-sensitive phenotype ideal for studying genetic interactions: (1) expression of independent transgenic lines with the same sev-Gal4 driver results in a range of phenotypes, and (2) expression of a given Nito-FL transgene with sevweak results in a mild rough eye phenotype whereas expression of the same line at a higher level produces a more severe phenotype. Consistent with the hypothesis of an antagonistic relationship between spen and nito, it was found that heterozygosity for a null spen allele enhanced the rough eye phenotype associated with Nito-FL expression, as demonstrated by an increased number of ommatidia lacking photoreceptors. Next, the consequences of increasing or decreasing nito levels in the background of a dominant negative spen transgene (spenDN), which encodes the C-terminal 936 amino acids of spen), and also produces dose sensitive phenotypes, that were examined. Because both transgenes are capable of perturbing eye development on their own, in order to distinguish between additive and synergistic interactions a Nito-FL transgenic line was used that when expressed with sevweak exhibits only very mild perturbations of the adult eye (Jemc, 2006).
As expected, given the Nito structure-function analysis, Nito-FL causes an enhancement of the spenDN rough eye phenotype, an increase in necroses in the eye and a complete loss of organization. Thus, overexpression of nito and overexpression of spenDN appear to act in the same direction, suggesting opposing functions for Nito and Spen. Since loss-of-function mutations in nito have not been isolated, a nito transgenic dsRNA construct was generated to investigate the consequences of reducing endogenous nito expression levels with respect to spen function. RT-PCR from Drosophila eye discs confirmed that this construct mediates partial knockdown of nito expression. In vivo, while dsRNA-mediated knockdown of nito expression does not perturb eye morphology on its own, nito-RNAi partially rescues the rough eye phenotype resulting from overexpression of spenDN, again suggesting antagonism between nito and spen. Eye sections show fewer missing ommatidia in nito-RNAi, spenDN adult eyes relative to those overexpressing spen DN alone, as well as fewer missing photoreceptors in ommatidia lacking the full complement of photoreceptors and more normal rhabdomere morphology. Together, these dose-sensitive genetic interactions argue for mutual antagonism between the large SPOC family member spen and the small SPOC family representative nito during Drosophila eye development (Jemc, 2006).
It remains to be determined if the antagonistic relationship between nito and spen is maintained in developmental contexts outside of the eye. Previous work examining the role of SPEN in Wingless signaling suggested the presence of a redundant partner for SPEN, for which Nito would be a good candidate, given their sequence conservation. In situ hybridization for nito and spen suggests they are also both ubiquitously expressed throughout embryonic development, and considering the broad range of embryonic phenotypes attributed to spen mutants, exploration of context specific interactions between spen and nito in the embryo will likely improve understanding of the relationships between these two related proteins. It is predicted that certain developmental events will require synergism between nito and spen, whereas others, as was demonstrate in the eye, will require antagonism (Jemc, 2006).
At the cellular level, spen is implicated as a positive component of Wingless and RTK/RAS signaling, and large SPOC family proteins SHARP and MINT are implicated as negative regulators of Notch signaling (Kuroda, 2003; Oswald, 2002). Given the ability of nito to antagonize spen function in the developing eye, it seems reasonable to speculate that Nito also acts as a downstream regulator/effector of some or all of these pathways. Furthermore, the antagonism between nito and spen may provide a mechanism for differential regulation of output from these pathways. Mechanistically, how might one envision the mutual antagonism between Spen and Nito? Large SPOC proteins have been previously shown to serve as transcriptional corepressors. Thus one attractive possibility is that small SPOC proteins might serve as transcriptional activators. In this model, by virtue of their conserved RRM and SPOC motifs, small and large SPOC proteins might compete for access to common binding partners. The resulting complexes, depending on whether they contain Spen or Nito, would then either repress or activate transcription. In a slight variation of the model, one could propose that SPOC proteins might be able to either repress or activate transcription, and so depending on context, would either act synergistically or antagonistically. Unfortunately, Drosophila cultured cells do not provide an appropriate environment in which to assay the activity of SPOC proteins so it was not possiable to test this model with respect to Spen and Nito. However, using mammalian COS cells, it was observed that while the SPOC motif of SHARP represses transcription, the SPOC motif of RBM15, the human Nito ortholog, strongly activates transcription. Thus, perhaps the antagonistic relationship between Spen and Nito that is reported in this study in the context of Drosophila eye development reflects a conserved antagonistic relationship between large and small SPOC proteins that is manifested at the level of transcriptional output (Jemc, 2006).
In conclusion, an antagonistic relationship has been demonstrated between the large and small SPOC family proteins in the developmental context of the Drosophila eye. The finding that SPOC family proteins function as downstream effectors of a variety of signaling pathways suggests they may act to fine-tune transcriptional output downstream of these cascades. Thus, it will be extremely interesting to determine whether the antagonistic relationship have observed between Nito and Spen in the eye is a general property of small and large SPOC proteins, or if it is unique to Drosophila eye development. Determination of transcriptional targets and cofactors will be required to understand how SPOC family proteins function to regulate and integrate information from these signaling pathways (Jemc, 2006).
The Notch and Epidermal Growth Factor Receptor (EGFR) signaling pathways interact cooperatively and antagonistically to regulate many aspects of Drosophila development, including the eye. How output from these two signaling networks is fine-tuned to achieve the precise balance needed for specific inductive interactions and patterning events remains an open and important question. The gene split ends (spen) functions within or parallel to the EGFR pathway during midline glial cell development in the embryonic central nervous system. This study shows that the cellular defects caused by loss of spen function in the developing eye imaginal disc place spen as both an antagonist of the Notch pathway and a positive contributor to EGFR signaling during retinal cell differentiation. Specifically, loss of spen results in broadened expression of Scabrous, ectopic activation of Notch signaling, and a corresponding reduction in Atonal expression at the morphogenetic furrow. Consistent with Spen's role in antagonizing Notch signaling, reduction of spen levels is sufficient to suppress Notch-dependent phenotypes. At least in part due to loss of Spen-dependent down-regulation of Notch signaling, loss of spen also dampens EGFR signaling as evidenced by reduced activity of MAP kinase (MAPK). This reduced MAPK activity in turn leads to a failure to limit expression of the EGFR pathway antagonist and the ETS-domain transcriptional repressor Yan and to a corresponding loss of cell fate specification in spen mutant ommatidia. It is proposed that Spen plays a role in modulating output from the Notch and EGFR pathways to ensure appropriate patterning during eye development (Doroquez, 2007).
This study demonstrates that loss of spen perturbs the normal balance between the EGFR and Notch pathways as evidenced by the patterning disruptions and aberrant expression of multiple pathway components. These findings raise the question of whether Spen functions primarily in the Notch pathway, primarily in the EGFR pathway, or as a critical component of both. Although definite resolution is difficult given the extensive and intricate feedback regulation within and between these two signaling networks, a model is proposed in which Spen-mediated antagonism of the Notch pathway regulates the signaling flow through the EGFR pathway to achieve proper retinal cell fate specification (Doroquez, 2007).
Loss of spen results in hyperactivation of the Notch pathway as evidenced by elevated levels of both Notch and its transcriptional targets, the E(spl)-bHLHs. Therefore, a normal function of Spen in the developing eye is to limit the activity of Notch. Consistent with this model, heterozygous reduction of spen was shown to be sufficient to suppress the heterozygous Notch wing margin phenotype. However, loss of spen does not lead to the anti-neurogenic phenotypes typically associated with overexpression/overactivation of canonical members of the Notch pathway, suggesting that although Notch signaling output is elevated, the increase is below the threshold needed to achieve such phenotypes. Consistent with this interpretation, recruitment of the initial R8 photoreceptor neuron, a process influenced at multiple stages by Notch signaling, occurs normally in the absence of spen (Doroquez, 2007).
Where might Spen interface with the Notch signaling pathway? The striking increase in Sca expression in spen mutant clones at the MF is consistent with Spen regulating Notch activation by limiting the expression of sca either through transcriptional repression or by destabilizing the transcript. This suggests that in the Drosophila eye Spen may have an upstream role in the Notch pathway in contrast to the downstream role described for Spen mammalian orthologs. In contrast, because of extensive feedback regulation in Notch signaling, it is plausible that Spen interfaces with the network at a more downstream point. For example, ectopic expression of Notchintra was shown to promote Sca expression, which in turn activates Notch signaling. Additionally, although no such role was detected with respect to yan, it is possible that Spen limits Notchintra/Su(H)-mediated transactivation at the level of transcriptional repression of other Notch pathway targets, including the E(spl)-bHLHs, as is the case for the mammalian Spen orthologs. This latter mechanism might also be relevant posterior the MF, where Notch signaling remains elevated as judged by increased levels of both Notch and the E(spl)-bHLHs in spen mutant clones, but where Sca is no longer expressed (Doroquez, 2007).
Although pinpointing where Spen interfaces with the Notch signaling pathway remains a challenge, the simplest interpretation of the data is that at the MF, Spen either directly or indirectly regulates Sca expression to restrict Notch pathway output. Posterior to the MF, as discussed below, mutual antagonism between the Notch and EGFR pathways may stabilize the initial signaling imbalance independent of Sca, leading to sustained up-regulation of Notch and down-regulation of EGFR output in spen mutant tissue (Doroquez, 2007).
What might the consequences of a moderate increase in Notch pathway output be? Given the extensive functional antagonism that has been reported between the Notch and EGFR pathways in the eye, a likely outcome is that the increased Notch signaling in spen mutant tissue would dampen EGFR pathway output. Supporting the idea that spen plays a positive role with respect to EGFR signaling, the cell fate specification defects observed in spen mutant clones are highly reminiscent of phenotypes associated with hypomorphic mutants in positive components of the EGFR pathway. Thus, the defective specification of neuronal and non-neuronal cell types and the perturbed R8 spacing adjacent to the MF all suggest reduced, but not ablated, EGFR pathway function in spen clones (Doroquez, 2007).
Lending further support to a model in which elevated Notch signaling in spen clones dampens EGFR pathway output, both Ato and dpERK expression at the MF are reduced. Because previous work has shown that Ato is required for activation of the EGFR pathway at the MF, one possibility is that Spen stabilizes dpERK levels at the MF by antagonizing Notch-mediated lateral inhibition to ensure appropriate Ato expression. Another plausible mechanism for Spen-mediated regulation of dpERK activity would be downstream of or in parallel to Ras. In this scenario, Spen might mediate transcriptional repression of an inhibitor such as a MAPK phosphatase. However, qRT-PCR analysis in imaginal discs predominantly mutant for spen do not indicate a role for Spen in regulating the expression of two characterized Drosophila MAPK phosphatases - dMKP3 and PTP-ER. Thus, validation of such a mechanism will require identification of other MAPK phosphatases or pathway inhibitors that might be regulated by Spen (Doroquez, 2007).
It should be noted that the results of this analysis of spen function in the eye appear contradictory to those from a prior study that suggested spen antagonizes EGFR output and promotes Notch signaling during embryonic neural development (Kuang, 2000). Specifically, elevated EGFR signaling was reported in spen maternal/zygotic null embryos, as evidenced by increased numbers of midline glial cells and loss of Yan expression. However, these results could not be reproduced (F. Chen and I. Rebay, unpublished data reported in Doroquez, 2007). On the contrary, analysis of spen function during midline glial cell development in the embryonic central nervous system was entirely consistent with a role for spen as a positive contributor to EGFR signaling. Thus, at least with respect to EGFR signaling, it is believed that Spen serves an analogous role in multiple developing tissues (Doroquez, 2007).
With respect to Notch signaling, Kuang reported a strong reduction in E(spl)-bHLH expression throughout the embryo but no change in Notch levels, exactly opposite to the current findings in the eye. Additional work will be needed to determine whether and how spen interfaces with the Notch pathway during embryogenesis, and whether distinct or identical mechanisms operate in retinal versus embryonic neural development (Doroquez, 2007).
It is not yet clear whether spen's role in Notch-EGFR interactions posterior to the MF is identical to its role in events occurring at the MF. The failure to down-regulate Yan and the resulting cell fate specification defects show that EGFR signaling posterior to the MF is compromised in spen mutant tissue. Given that Yan up-regulation in spen clones does not result from loss of Spen-mediated transcriptional repression, but rather reflects loss of post-translational control, two models for Spen function seem likely. First, if the inability to detect changes in dpERK protein levels posterior to the MF in situ accurately indicates unaltered dpERK levels, then the ability of dpERK to phosphorylate Yan must be compromised in spen mutants. Alternatively, dpERK levels may be sufficiently reduced to increase Yan stability, but the change may be below the immunohistochemical detection threshold (Doroquez, 2007).
In terms of the signals that impinge on dpERK, whereas Notch signaling and Ato expression are critical for proper dpERK expression in the MF, reiterative EGFR signaling takes over posterior to the MF to maintain dpERK activity. Thus, it is possible that a Spen-dependent, Notch-independent mechanism may regulate EGFR output posterior to the MF. Alternatively, because Notch, E(spl) and Yan expression are all elevated in spen mutant tissue both in and posterior to the MF, Spen-mediated antagonism of Notch signaling may be relevant to EGFR regulation in both contexts. An extension of this idea that results in perhaps the most appealing model is that the initial increase in Notch output at the MF dampens EGFR signaling, which in turn leads to elevated Notch signaling in more posterior regions resulting in reduced EGFR output. In this way, the initial signaling imbalance created by loss of spen at the MF could be maintained over the entire eye disc through mutual antagonism and feedback regulation between the Notch and EGFR pathways (Doroquez, 2007).
In summary, this study analyzed the requirement for spen in regulating the EGFR and Notch pathways during Drosophila eye development, and it is proposed that increased Notch pathway activity upon loss of spen may be sufficient to dampen EGFR signaling, but not to disrupt other downstream effects of Notch signaling. Therefore, because the effects of spen loss appear to be at a threshold below the production of bona fide Notch-related phenotypes, it is suggested that Spen plays a subtle role in the regulation of the Notch pathway or functions redundantly alongside other components. An equally likely hypothesis is that Spen regulates the Notch and EGFR pathways separately and that the phenotypes reported reflect a composite of independent disruptions to both signaling networks (Doroquez, 2007).
Although much of the literature focuses on a primary role for Spen family proteins as co-repressors, recent findings suggest members of this family may also regulate non-coding RNA sequestration, mRNA export, RNA splicing, and proteolysis. Therefore, future identification of the precise molecular mechanisms by which Spen interfaces with the EGFR and Notch pathways may reveal novel modes of interaction between these two critical and conserved signaling networks (Doroquez, 2007).
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date revised: 20 December 2006
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