seven in absentia

Targets of Activity

prospero expression is regulated by the Sevenless-Ras1 pathway acting through Yan and Pointed. sina null mutants express prospero in the R7 equivalence group comparable to wild type. prospero expression in sevenless-sina double mutants resembles expression in a sevenless mutant, indicating that sevenless is epistatic to (or, dominate in effect over) sina. Cells transformed into cone progenitors by removing phyllopod function express prospero at a level comparable to wild type. Cone cells express pros at a relatively low level, but when derived from R7 transformation in a sevenless-phyllopod mutant they express pros at an elevated level. This elevated level requires sina function. It is concluded that sina is not required for prospero function, and that sina and phyl do not upregulate pros transcription singlehandedly in R7 cells, because the Pointed-Yan pathway makes sina and phyl redundant. Nevertheless, it is suggested that sina and phyl are involved in a secondary, redundant pathway for prospero activation (Kauffmann, 1996).

Protein Interactions

SINA protein can be shown to form a complex with PHYL. The amino acids 108 to 130 of PHYL are critical for this interaction (Kauffmann, 1996).

Genetic interactions of yan with downstream components of the Sevenless pathway have been studied. A reduced activity of tramtrack results in enhancement of the mutant yan phenotype. ttk mutations produce extra R7 cells even in sina homozygotes while the yan mutation does not. This results indicates that TTK represses R7 induction downstream of the sites where YAN and SINA function (Yamamoto, 1996).

A collection of transposable-element-induced mutations have been screened for those which are dominant modifiers of the extra R7 phenotype of a hypomorphic yan mutation. The members of one of the identified complementation groups correspond to disruptions of the tramtrack gene. As heterozygotes, ttk alleles increase the percentage of R7 cells in yan mutant eyes. Just as yan mutations increase ectopic R7 cell formation, homozygous ttk mutant eye clones also contain supernumerary R7 cells. However, in contrast to yan, the formation of these cells in ttk mutant eye tissue is not necessarily dependent on the activity of the sina gene. Furthermore, although yan mutations are dominant in interactions with mutations in the Ras1, Draf, Dsor1, and rolled genes to influence R7 cell development, ttk mutations only interact with yan and rl gene mutations to affect this signaling pathway. These data suggest that yan and ttk both function to repress inappropriate R7 cell development but that their mechanisms of action differ. In particular, TTK activity appears to be autonomously required to regulate a sina-independent mechanism of R7 determination (Lai, 1996).

Tramtrack RNA is alternatively spliced, giving rise to two forms. One is a protein of 69 kDa that binds the fushi tarazu promoter. A second is a protein of 88 kDa with an alternative set of zinc fingers, having a DNA binding specificity distinct from that of the first protein. Tramtrack (Ttk88) expression represses neuronal fate determination in the developing Drosophila eye. Ttk88 was ectopically expressed in R3, R4 and R7 photoreceptor precursors and the four cone cell precursors using a Sevenless enhancer. The resultant transgenic flies have three missing photoreceptor precursors per ommatidium. Ectopic expression of Ttk88 in all cells posterior to the morphogenetic furrow results in flies devoid of photoreceptors (Tang, 1997).

Phyllopod acts to antagonize this repression by a mechanism that requires Seven In Absentia and is associated with decreased Ttk88 protein levels, but not reduced ttk88 gene transcription or mRNA stability. Sina, Phyl, and Ttk88 physically interact. Phyl interacts with Sina and Phyl interacts strongly with Ttk88 (but not with TTK69). Sina and TTK88 show a weak interaction. Sina interacts genetically and physically with UBCD1, a component of the ubiquitin-dependent protein degradation pathway (Treier, 1992). These results suggest a model in which activation of the Sevenless receptor tyrosine kinase induces Phyl expression, which then acts with Sina to target the transcriptional repressor Ttk88 for degradation, thereby promoting R7 cell fate specification (Tang, 1997).

Musashi (Msi), the Drosophila neural RNA-binding protein, plays a part in eye development. Msi expression is observed in the nuclei of all photoreceptor cells (R1-R8). Although a msi loss-of-function mutation results in only weak abnormalities in photoreceptor differentiation, the msi eye phenotype is significantly enhanced in a seven in absentia (sina) background. sina is known to be involved in the degradation of the Tramtrack (Ttk) protein, leading to the specification of the R7 fate. Msi also functions to regulate Ttk expression. The sina msi mutants show significantly high ectopic expression of Ttk69 and failure in the determination of the R1, R6, and R7 fates. Other photoreceptor cells also fail to differentiate, with abnormalities occurring late in the differentiation process. These results suggest that Msi and Sina function redundantly to downregulate Ttk in developing photoreceptor cells (Hirota, 1999).

To investigate the functions of Msi during eye development, the eye phenotype of the msi1/msi1mutants was examined. msi1/msi1eyes contain abnormal ommatidia, with deformed rhabdomeres and/or irregular orientation, at a low frequency (3.05%). Staining developing msi1/msi1-eye discs with antibodies against several neuronal markers reveals that the number of photoreceptor cells is not affected, suggesting that msi is involved in late processes of photoreceptor cells differentiation, including the formation of rhabdomeres. However, the penetrance of the msi1/msi1phenotype is so low that it was difficult to investigate the Msi function. Also examined were the genetic interactions of msi with ttk, a possible target gene of Msi, and sina, a factor involved in the degradation of Ttk. Strong genetic interactions occur between msi and sina mutations in eye development. In wild-type flies, the compound eye has a regular array of ommatidia, each of which contains eight photoreceptor cells (R1-R8). At the R7 level, the rhabdomeres of seven photoreceptor cells (R1-R7) are arranged in a characteristic asymmetrical trapezoid. Since Sina is essential for the differentiation of R7, R7 is missing in 90% of the ommatidia of sina2/sina3 mutants (in which little if any functional gene products are produced) and the external morphology of the eyes shows a slight roughness. Notably, the eye phenotype of the double homozygous mutants of msi and sina (sina2msi1/sina3msi1) show synergistic, but not additive, enhancement. The external morphology of the double homozygous mutants shows strikingly disturbed ommatidial arrays. Most of the rhabdomeres are severely deformed and no ommatidia contain more than five rhabdomeres, suggesting that msi and sina have important and distinct roles in the differentiation of photoreceptor cells. To confirm the function of Msi, rescue experiments of sina msi double mutants by Msi were performed. A genomic region containing the wild-type msi gene (referred to as P[msi1+]) was introduced into the sina msi mutant flies by P element-mediated germline transformation. The P[msi1+] fragment significantly rescues the defects in the sina msi eyes. The external morphology of the eye and the formation of rhabdomeres are nearly normal. This result indicates that the sina msi eye phenotype is partially due to the loss of msi function. Since Msi contains two RNA recognition motifs (RRMs), RRM-A and RRM-B, the RNA-binding activity of Msi is likely to be essential for its rescuing activity. To test this possibility, a transgenic rescue experiment was performed in which mutant Msi proteins whose RNA-binding activities were designed to have been abolished were expressed. The RRM domains contain a consensus sequence that is composed of two highly conserved short segments, referred to as RNP1 (ribonucleoprotein octamer consensus) and RNP2. Since the aromatic side chains of RNP1 are known to be crucial for RNA binding, mutations that change phenylalanine to alanine in three places in the RNP1 were induced into both of the two RRMs of Msi (referred to as P[msiA*B*]). The P[msiA*B*] fragment does not rescue the sina msi eye phenotype. Additionally, P[msi1+] fully rescues the weak defects in the msi1eyes described above, while P[msiA*B*] had no effect. Taken together, it is concluded that the RNA-binding ability of Msi is involved in normal eye development (Hirota, 1999).

To examine how the sina and msi mutations causes the severe eye defects described above, the neuronal differentiation of this double mutant was examined by staining with anti-Elav antibody. All the ommatidia posterior to the eighth row from the MF contain eight photoreceptor cells in wild-type eye discs. In the same region, 90% of the ommatidia of sina2/sina3 lack R7. In the same region, all the ommatidia of sina2msi1/sina3msi1contain only five cells, consistent with the appearance of the phenotype in adult eyes. In the sina2msi1/sina3msi1eye discs, a nearly normal pattern of Elav staining was observed in developing ommatidia up to the five-cell precluster stage, which is composed of R2, R3, R4, R5, and R8. Subsequently, however, R1, R6, and R7 are never added to the ommatidia. These results suggest that Msi and Sina have redundant functions in the cell fate determination of R1, R6, and R7. Furthermore, in the posterior region of the eye discs, the spatial arrangement of the five cells in each ommatidium changes and overlaps abnormally. In the most posterior region of the eye discs, many ommatidia with reduced numbers of Elav-positive cells are observed. These results indicate that R2, R3, R4, R5, and R8, which had once expressed Elav, gradually have their spatial arrangement in the ommatidia disrupted, and that some of the photoreceptor cells fail to maintain Elav expression, resulting in a strikingly deformed eye (Hirota, 1999).

To confirm the requirement of Sina and Msi for the cell fate determination of R1 and R6, the sina msi eye discs were stained with antibody against Bar, an R1/R6-specific marker. Bar is expressed in R1 and R6 in wild-type eye discs. In 40% of the ommatidia of sina2/sina3 eye discs, the number of Bar-positive cells is reduced to one or zero, suggesting that Sina has some roles in the cell fate determination of R1 and R6, where Sina is known to be expressed. Consistent with anti-Elav staining, Bar-positive cells completely disappear in the sina2msi1/sina3msi1eye discs. In combination with sina1, a hypomorphic allele, instead of sina2, one or two Bar-positive cells per ommatidium are detected in 50% of the sina2msi1/sina3msi1ommatidia, confirming that Msi and Sina have redundant functions required for the cell fate determination of R1 and R6 (Hirota, 1999).

Sina has been suggested to be involved in the degradation of Ttk, a general inhibitor of neuronal differentiation. Since sina msi double mutants show defects in neuronal differentiation, the possibility that the expression pattern of Ttk69 is different in these animals was tested by examining the Ttk expression pattern in eye discs stained with anti-Ttk69 antibody. In wild-type eye discs, Ttk69 is detected in four cone cells per ommatidium. The expression pattern of Ttk69 in the msi1/msi1eye discs is indistinguishable from that of wild-type. In sina2/sina3 eye discs, five cells were labeled in 5% of the ommatidia, suggesting that degradation of Ttk69 is reduced. Notably, 50% of the ommatidia in the sina2msi1/sina3msi1eye discs contain additional Ttk69-expressing cells, indicating that the average number of cells per ommatidium that express Ttk69 ectopically is larger in sina2msi1/sina3msi1 than in sina2/sina3 eye discs. Since both photoreceptor and cone cells are deformed at later stages of development the cell types ectopically expressing Ttk69 could not be identified. This result suggests that the Ttk69 expression is negatively regulated by both Msi and Sina. Consistent with this idea, the morphology of sina msi double mutants is significantly recovered in a heterozygous background for ttkosn (sina2msi1ttk osn/sina3msi1), a mutation that disrupts expression of both the Ttk69 and Ttk88 proteins. The external morphology of the eye and the formation of the rhabdomeres are nearly normal. Thirty percent of the sina2msi1ttkosn/sina3msi1ommatidia show the normal number and arrangement of photoreceptor cells and are indistinguishable from wild-type ommatidia. These results suggest that the severe eye phenotype of the sina2msi1/sina3msi1 double mutant may result from an elevation in Ttk expression levels (Hirota, 1999).

These results demonstrate that Msi and Sina redundantly function as factors required for the downregulation of Ttk69; however, the mechanism remains unknown. A recent study, exploring the target RNA for Msi, indicates that TTK69 mRNA contains multiple sites that could potentially be recognized by Msi. Therefore, it is likely that Msi binds to the TTK69 mRNA to inhibit its translation or reduce its stability. In contrast, Sina has been shown to function with Phyl to target Ttk protein for degradation. Thus, Msi and Sina are likely to function to down-regulate Ttk at different levels in independent manners. If one functions via the other, the phenotype of the sina msi double null mutants would be identical to the phenotype of single null mutants for the gene that functions downstream of the other. Instead, the sina msi mutants show a synergistically enhanced eye phenotype that is much more severe than that of the single mutants. Furthermore, the finding that a half-reduction in the gene dosage of ttk suppresses the sina msi double null mutants indicates that ttk is downstream of msi and sina. Taken together, the expression of Ttk is likely to be regulated posttranscriptionally by factors including Msi, and posttranslationally by Sina, Phyl, and Ebi (a WD repeats protein). The negative regulation of Ttk by Msi and Sina is required for both the early processes of R1, R6, and R7 differentiation and the late processes of the differentiation of other photoreceptor cells in eye development. Further studies will extend knowledge of how the posttranscriptional regulation of gene expression by Msi controls ommatidial development (Hirota, 1999).

Three lines of evidence were found that link Ebi to Sina and Phyllopod-dependent degradation of Ttk88. The first line of evidence comes from transient transfection experiments. Sina and Phyl are able to target Ttk88 for ubiquitin-dependent degradation when expressed in transient transfection experiments in S2 cells. S2 cells contain high levels of endogenous Ebi. To interfere with the activity of this protein, an N-terminal fragment (EbiN) was expressed that has been used (Dong, 1999) as a dominant-negative mutant to interfere with Ttk88 degradation in the eye disk. Cells were co-transfected with pIZT vectors constitutively expressing either Cat or EbiN from the OpIE2 promotor, in the presence of metallothionine-inducible vectors containing Phyl, Sina and Ttk88-myc. Following transfection and copper induction, cells were metabolically labeled with [³⁵S]methionine and then chased with cold methionine for various time points to monitor Ttk88 degradation. In the presence of Sina and Phyl, Ttk88 is degraded with a half life of ~25 min, a process that can be blocked by the proteosome inhibitor MG132. Ttk88 degradation is blocked in cells expressing the EbiN dominant-negative form of pIZT, whereas a control pIZT vector that constitutively expressed Cat gave no effect (Boulton, 2000).

The second line of evidence stems from an in vitro degradation assay. To investigate whether Ebi is linked to Ttk88 degradation, attempts were made to reconstitute Ttk88 degradation in vitro. These studies show that Sina and Phyl are needed for Ttk degradation, mirroring their requirement in vivo. The results of the in vitro degradation assay suggested that Ebi might physically associate with Ttk88, Sina and Phyl. Ebi was found to interact strongly with Sina and Phyl, and weakly with Ttk88, when these proteins were expressed and assayed individually. When Sina, Phyl and Ttk88 are co-expressed in the same lysate, Ebi is able to pull down Ttk88 with a much higher affinity, compared with Ttk88 when expressed and bound alone. This suggests that the Ebi-Ttk interaction is indirect and may require Sina and Phyl to facilitate association. Since the ß-propeller structure formed by WD-repeat domains provides a surface for many protein-protein interactions, it is likely that this domain in Ebi provides a scaffold for Sina, Phyl and Ttk88 association (Boulton, 2000).

If stabilization of Ttk88 is responsible for the cell cycle phenotypes associated with Ebi, a consistent pattern of genetic interactions between GMR-p21 or GMR-E2F-DP-p35 and mutations in the Egfr pathway would be expected that function to regulate Ttk88 levels. However, unlike mutations in Ebi, loss-of-function alleles of egfr, gap1, raf1, ras1, mapk, yan, sina, phyl and ttk have no strong effect on either the GMR-p21 or the GMR-E2F-DP-p35 phenotype. Mutations in the Ets-domain transcription factor Pointed (pnt) enhance the GMR-E2F-DP-p35 phenotype but fail to modify the GMR-p21 phenotype. While it is possible that Ebi is the only dosage-sensitive component of the Egfr pathway whose levels affect cell proliferation, these results suggest that Ebi might have an activity that is independent of Ttk88 degradation (Boulton, 2000).

To test directly whether stabilization of Ttk88 is likely to be responsible for the changes in cell cycle control caused by ebi mutant alleles, the effects of elevating the levels of Ttk88 were tested. Ectopic expression of Ttk88 was induced by heat shock for 30 min at 39°C in embryos carrying hs-Ttk88. Following recovery, embryos were either pulsed with BrdU or aged for immunohistochemistry. The ectopic expression of Ttk88 from a heat shock-regulated transgene is sufficient to disrupt neuronal differentiation in stage 13-14 embryos. Unlike the ebi mutant embryos, BrdU incorporation demonstrates that the block to differentiation in hs-Ttk88 embryos does not result in a failure to exit the cell cycle in the PNS and CNS. Furthermore, expression of Ttk88 from a GMR transgene inhibits differentiation in the eye disc. However, the GMR-Ttk88 transgene fails to suppress the GMR-p21 eye phenotype and is unable to restore S phases in the GMR-p21 eye disc. In contrast, halving the dosage of Ebi or expressing cyclin E from the GMR promoter is sufficient to restore the second mitotic wave of S phases. It is concluded that increasing Ttk88 protein to a level where differentiation is perturbed in either the embryo or the eye disc is insufficient to promote S phase entry in either of the situations where ebi mutations give this effect. It is inferred that Ebi must have a second function, independent of Ttk88 degradation, that is important for regulating cell cycle exit (Boulton, 2000).

To investigate whether Ebi is linked to Ttk88, attempts were made to reconstitute Ttk88 degradation in vitro. Initial experiments showed that Ttk88 is not degraded when co-expressed with Sina and Phyl in rabbit reticulocyte lysate. In an attempt to stimulate the degradation activity, GST (glutathione S-transferase) or GST-Ebi beads were incubated together with ubiquitin, an ATP regeneration system and Sina-Phyl-Ttk co-expressed in a reticulocyte lysate. Neither GST nor GST-Ebi is able to promote Ttk88 degradation in this setting. It was speculated that Ebi may function as a bridge between the Sina-Phyl-Ttk88 complex and an activity required for ubiquitylation and subsequent degradation of Ttk88. In order to provide this missing activity, GST or GST-Ebi beads were preincubated with S2 extracts (now referred to as loaded beads) prior to performing the degradation reaction. Loaded GST-Ebi beads are unable to degrade Ttk88 when expressed alone. However, when Sina and Phyl are co-translated with Ttk88, loaded GST-Ebi beads are able to promote degradation of TtK88 by a mechanism that is blocked by the proteosome inhibitor, LLnL, whereas loaded GST beads cannot. The need for Sina and Phyl for Ttk degradation mirrors their requirement in vivo. In vitro-translated E2F, dDP or cyclin E is not degraded in any of the experiments described. In order to map the region of Ebi that associates with the activity required for Ttk88 degradation, loaded GST-EbiN and GST-EbiC beads were pre-incubated and then the degradation assay was performed. Interestingly, neither half of Ebi alone is capable of targeting Ttk88 for degradation. These data suggest that the full-length Ebi protein may act to bring the Sina-Phyl-Ttk88 complex and a ubiquitylation activity into close proximity (Boulton, 2000).

The results of the in vitro degradation assay suggest that Ebi might physically associate with Ttk88, Sina and Phyl. This was tested using the in vitro translated Sina, Phyl and Ttk88, and the GST-Ebi fusion proteins described above. GST-Ebi was found to interact strongly with Sina and Phyl, and weakly with Ttk88, when these proteins were expressed and assayed individually. The GST control shows no association. Interestingly, when Sina, Phyl and Ttk88 are co-expressed in the same lysate, GST-Ebi is able to pull down Ttk88 with a much higher affinity compared with when Ttk88 is expressed and bound alone. This suggests that the Ebi-Ttk interaction is indirect and may require Sina and Phyl to facilitate association. To define the region of Ebi required for Sina, Phyl and Ttk association, GST-EbiN (N-terminal domain) and GST-EbiC (C-terminal WD-repeat domain) fusion proteins were tested for binding. GST-EbiN does not associate with any of the proteins, whereas GST-EbiC is able to bind to all three proteins, although with a slightly reduced affinity when compared with the full-length protein. Since the ß-propeller structure formed by WD-repeat domains provides a surface for many protein-protein interactions, it is possible that this domain in Ebi provides a scaffold for Sina, Phyl and Ttk88 association. To provide further evidence for these interactions, pIZT-Ebi with pIZT-V5His-Sina or pIZT-V5His-Phyl, or all three constructs were transiently co-transfected into S2 cells. Sina and Phyl (His₆- and V5-tagged) were purified from lysates from the various transfected populations by Ni-NTA agarose chromatography. The beads were then subjected to Western blotting using a monoclonal antibody to Ebi. Ebi was found to co-precipitate with Sina and Phyl but was not observed in the untransfected control (Boulton, 2000).

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