seven in absentia


DEVELOPMENTAL BIOLOGY

Embryonic

Staining with anti-SINA antibody is localized to large cells just beneath the epidermis of late stage 12 embryos. Anti-SINA antibody also stains some epidermal cells on the ventral surface of the embryo, and at a lower level, the epidermal cells of the telson (Carthew, 1990).

In vivo function of a novel Siah protein in Drosophila

The Siah proteins, mammalian homologues of the Drosophila Sina protein, function as E3 ubiquitin ligase enzymes and target a wide range of cellular proteins for degradation. This study investigated the in vivo function of the fly protein, Sina-Homologue (SinaH), which is highly similar to Sina. Flies that completely lack SinaH are viable and in combination with a mutation in the gene, Ebi, show an extra dorsal central bristle phenotype. SinaH and Ebi can interact with each other both in vivo and in vitro suggesting that they act in the same physical complex. Flies that lack both Sina and Sina-Homologue were also created and show visible eye and bristle phenotypes, which can be explained by an inability to degrade the neuronal repressor, Tramtrack. No evidence was found for redundancy in the function of Sina and SinaH (Cooper, 2007).

The Siah E3 ubiquitin ligases have been shown to have many important functions in mammals and can target a diverse array of substrates for degradation. There are two Siah-like proteins in the Drosophila genome: Sina and the newly identified Sina-Homologue for which no mutant has existed. A defined mutation using homologous recombination to completely remove the SinaH gene was created in order to investigate the in vivo function of this potentially interesting gene. Flies that lack the SinaH gene do not show any visible phenotype in the adult. However, when homozygous, this SinaH1 allele interacts with a heterozygous Ebik16213 allele to give extra dorsal central bristles. Although the formation of extra dorsal central bristles is weak, it is a specific effect of removal of SinaH and lowering the amount of Ebi (Cooper, 2007).

As well as this genetic interaction, it was also shown that SinaH and Ebi proteins physically associate both in vitro and in vivo in S2 cells, consistent with the proteins being members of the same complex. The proteins seem to interact more strongly in vivo in the co-immunoprecipitation experiment compared with in vitro assays, suggesting that other components, which might be functionally relevant, could be present within the Ebi/SinaH complex. The lack of a visible phenotype in the SinaH mutant flies could suggest that removal of SinaH alone can be compensated by other members of the complex or it may be functionally redundant with other genes. The clear extra DC bristle phenotype when there are reduced amounts of Ebi, suggests that it is only when Ebi becomes limiting within the complex, that this compensatory mechanism is not sufficient, and the bristle phenotype is visible. This suggests that a possible role of a SinaH/Ebi containing complex in vivo is to restrict the ability to form dorsal central bristles (Cooper, 2007).

Evidence indicates that a role of the SinaH/Ebi complex is to target substrates for ubiquitination and proteasome-dependent degradation. SinaH has high homology with Sina, which can act as part of an E3 ubiquitin ligase complex containing Ebi to cause ubiquitination and degradation of Tramtrack 69. If SinaH acts in a similar manner to Sina, one explanation could be an inability to degrade Tramtrack, but this would result in fewer bristles being formed rather than additional bristles, since this neuronal repressor would inhibit SOP cell formation. This suggests that SinaH/Ebi is acting on different substrates to Sina. In humans, Siah proteins, and the homologue of Ebi (TBL1) can act together to degrade a component of the Wnt (Wg) signalling pathway, β-catenin (Armadillo). Removal of SinaH and reducing levels of Ebi might therefore cause the stabilisation of Armadillo, and the increased Wg signalling may up-regulate proneural activity in the DC cluster. Siah proteins have also been implicated in cell cycle control, and Ebi has a role in repression of the cell cycle transition between G1 and S phase. Another possibility is that increased cell division within the proneuronal cluster could result in additional SOP cell formation. However, the exact mechanism and substrates of the SinaH/Ebi complex involved in DC bristle formation is yet to be determined (Cooper, 2007).

The mouse Siah1a and Siah2 are synthetically lethal which suggests a high level of redundancy between these Siah proteins. To test if there is also redundancy between Siah proteins in flies, Both Sina and SinaH genes were removed together. Interestingly, the resultant fly displayed phenotypes very similar to flies which only lacked Sina, and could be attributed to mis/over expression of Tramtrack. Sina and SinaH therefore have distinct phenotypes suggesting that they have different roles in flies, consistent with their dissimilar expression patterns during development. Sina appears to have higher expression in the embryo and larvae whereas in this study, it was shown that SinaH mRNA is mainly expressed in later developmental stages and in males. Given that the mouse knockout of Siah1a is sterile and defective in spermatogenesis, there may still be other roles of SinaH that are yet to be uncovered and in flies, such roles can be compensated for by other E3 ubiquitin ligases or members of the complexes (Cooper, 2007).

Larval

In the eye imaginal disc, elevated staining is found in the region just anterior to the morphogenetic furrow in many if not all cells in this region. Staining is especially prominent in large rounded cells undergoing mitosis located on the apical surface of the epithelium, suggesting cell cycle regulation of SINA. Staining extends from the furrow to the posterior end of the disc. Apart from mitotic cells, staining is intense in cells that are associated with the developing ommatidia.The earliest developmental stage at which sina is expressed at high levels is at the late five-cell preclusters, three columns back from the furrow. T`he precluster is composed of the R2, R3, R4, R5 and R8 cells. Staining is intense in the R3/R4 pair, while the R2/R5 pair and R8 staining is low. The R1/R6 pair stain with SINA antibody as they associate with the cluster, followed by staining in the R7 precursor (Carthew, 1990).

In addition to the imaginal eye disc, staining with SINA antibody is observed in larval polytene tissues. There is staining of the peripodial epithelia of the eye-antennal, wing and leg discs, as well as the midgut, hindgut and fat tissues. Retinas from 40-hr-old pupae stain with SINA antibody, including the cone cells, cells R1-R8, and pigment cells. Thus, at larval stages of development, cells R2, R5 and R8 do not express high levels of SINA protein, in contrast to the high levels found expressed during pupation. Staining is also evident in each mechanosensory bristle group (Carthew, 1990).

Effects of Mutation or Deletion

In sina mutants R7 photoreceptor is missing and the normal trapezoidal arrangement of rhabdomeres is disoriented. The R8 cell is present though often in the same uncharacteristic positions as are found in sevenless mutations. R3 and R4 opsins, normally expressed in R7 cells, are reduced or eliminated. Other photoreceptor cells are also missing in some mutant ommatidia. In 40% of the ommatidia, one additional R cell is missing. The missing cells appear to be the R1-R6 class.

The mutant phenotype of sina is not restricted to the eye; other adult sensory organs are affected. Sensory bristles distributed over the body surface are frequently missing, with cases of both the cuticular bristle sheath and the socket being lost. Affected sensory organs include the microchaetes and macrochaetes of the head, notum, abdomen, and wing margin, and the stout and slender bristles of the anterior wing margin. The sina mutation also results in a 10-fold reduction in adult lifespan, and adult behavior that is generally lethargic and uncoordinated. Neither males nor females are fertile, although they produce morphologically normal sperm and eggs. No defects of the embryonic peripheral nervous system are apparent in mutants, indicating that the phenotype of mutants is restricted to late in the life cycle (Carthew, 1990).

Specification of the R7 cell fate in the developing Drosophila eye requires activation of the Sevenless (Sev) receptor tyrosine kinase, located on the surface of the R7 precursor cell, by its interaction with the Boss protein, expressed on the surface of the neighboring R8 cell. Four genes that participate in the intracellular transmission of this signal have so far been identified and molecularly characterized: Ras1, Sos, Gap1 and sina. Drosophila Raf serine/threonine kinase plays a crucial role in the R7 pathway: the response to Sev activity is dependent on raf function, and a constitutively activated Raf protein can induce R7 cell development in the absence of sev function. Raf acts downstream of Ras1 and upstream of Sina in this signal transduction cascade (Dickson, 1992).

A genetic screen was carried out for mutations that reduce the activity of sina. Nine genes may be required for normal sina activity. Three of these genes also appear to be essential for signaling by the Sevenless-Ras pathway in R7 cells, one of which corresponds to the rolled locus (rl). The rl gene is known to encode a mitogen-activated protein kinase necessary for signaling by Ras. Another is allelic to glass; it encodes a zinc finger transcription factor required for proper differentiation of all photoreceptors. Flies mutant for a third gene, peanut, show defects in cytokinesis and die at the larval-pupal transition. Three of the genes, including rolled are essential for signaling by RAS1. These results suggest that the products of these three genes may participate in a signaling pathway involving both Ras and Sina, possibly by functionally linking these two proteins (Carthew, 1994).

The first Drosophila septin gene to be discovered was peanut, isolated in a search for factors that interact with seven in absentia (sina), a gene required for the induction of neural fate in the presumptive R7 cells. peanut(pnut) behaves as a dominant enhancer of sina. Homozygous peanut mutants do not survive to adulthood but instead die shortly after pupation. peanut mutants have either severely reduced or else no imaginal discs; these are the epithelial structures that give rise to adult tissues. Such disc-less, pupal-lethal phenotypes often reflect an underlying defect in mitosis. In mitotic mutants, early divisions during embryogenesis are presumably supported by maternally supplied gene products, but later proliferation of imaginal tissues is dependent on the mutant zygotic genome. peanut mutant brains contain a large number of polyploid and multinucleate cells. Such cells have multipolar mitotic spindles and extra centrosomes (Neufeld, 1994).

Some aspect of R7 differentiation is independent of the genetic pathway(s) involving sevenless, boss and sina. An enhancer trap line, H214, was developed in which beta-galactosidase is primarily expressed in the R7 cell throughout its development. In mutations of sevenless, boss and sina, expression in H214 is initially reduced although still present in the R7 precursor and persists in the Equatorial cone cell (the alternative fate of R7 cells in mutants) into which mutant R7 cells develop. The EQC in wild type never expresses lacZ in H214. Thus the presumptive R7 cell receives positional information independent of sevenless (Mlodzik, 1992).

sevenless expression in the R7 precursor is initiated at the normal developmental time in sina mutants, but instead of shutting off by the two-cone-cell stage as in wild type, it remains high in the R7 precursor until pupation. This may reflect its adoption of an equatorial cone cell fate, since this cell maintains sevenless expression until pupation (Carthew, 1990).

Specification of the R7 photoreceptor cell in the developing Drosophila eye requires the seven in absentia (sina) gene. Ectopic expression of sina in all cells behind the morphogenetic furrow disrupts normal eye development during pupation, resulting in a severely disorganized adult eye. Thirteen independent sina transformant lines were isolated, each of which displayed eyes with abnormal exteriors, ranging from a slight roughening of the ommatidial lattice to a gross disruption of normal eye morphology. This range in phenotype is most likely due to variances in expression among the lines, since making each line homozygous results in a stronger phenotype. Eyes from strong sina overexpressing lines are notably smaller and less pigmented than wild-type, and a fusion of ommatidial surfaces results in a glazed cuticle covering the eye. Microscopic examination of sections through such eyes reveals corresponding abnormalities in the underlying retinal cells. In these lines, retinal patterning appears severely disrupted, and no normal ommatidia were identified. However, no cell types appeared to be lacking, as judged by the presence of pigment granules (pigment cells), lens structures (cone cells), rhabdomeres (photoreceptor cells) and bristles. Sections through eyes from the weaker GMR-sina lines with mild exterior phenotypes display more subtle defects, including defective ommatidial rotation, lattice disorganization, and occasional missing photoreceptors. In no case are extra R7 photoreceptor cells observed, indicating that misexpression of sina in uncommitted cells in the eye disc is insufficient to direct them into a neuronal program of development (Neufeld, 1998).

A genetic screen for dominant enhancers and suppressors of this phenotype has identified mutations in a number of genes required for normal eye development, including UbcD1, which encodes a ubiquitin conjugating enzyme; SR3-4a, a gene previously implicated in signaling downstream of Ras1, and a Drosophila homolog of the Sin3A transcriptional repressor. The genetic interaction between sina and UbcD1 presented here, as well as the demonstration of physical interaction between Sina and UBCD1, provides a molecular framework for beginning to understand how Sina may regulate the stability of proteins such as Ttk. Members of the Sin3 class of transcriptional corepressors serve as requisite components of the Mad-Max repressor complex. A Drosophila member of this family, Sin3A, interacts genetically with sina. At present, the role played by Sin3A during development is unclear. Clones of cells homozygous for Sin3A mutations could not be recovered, suggesting that this gene is required for cell proliferation or survival. Identification of mutations in Drosophila Sin3A should contribute to an understanding of this important class of transcriptional regulators (Neufeld, 1998).

During Drosophila external sensory organ development, one sensory organ precursor (SOP) arises from each proneural cluster and then undergoes asymmetrical cell divisions to produce an external sensory (es) organ made up of different types of daughter cells. phyllopod (phyl), known to be essential for R7 photoreceptor differentiation, is required in two stages of es organ development: the formation of SOP cells and cell fate specification of SOP progeny. Loss-of-function mutations in phyl result in failure of SOP formation, which leads to missing bristles in adult flies. At a later stage of es organ development, phyl mutations cause the first cell division of the SOP lineage to generate two identical daughters (IIb cells are transformed into IIa cells), leading to the fate transformation of neuron and sheath cells to hair cells and socket cells. Conversely, misexpression of phyl promotes ectopic SOP formation, and causes opposite fate transformation in SOP daughter cells. Thus, phyl functions as a genetic switch in specifying the fate of the SOP cells and their progeny. seven in absentia (sina), another gene required for R7 cell fate differentiation, is also involved in es organ development. Genetic interactions among phyl, sina and tramtrack (ttk) suggest that phyl and sina function in bristle development by antagonizing ttk activity, and ttk acts downstream of phyl. Notch (N) mutations induce formation of supernumerary SOP cells, and transformation from hair and socket cells to neurons. phyl acts epistatically to N. phyl is expressed specifically in SOP cells and other neural precursors, and its mRNA level is negatively regulated by N signaling. Thus, these analyses demonstrate that phyl acts downstream of N signaling in controlling cell fates in es organ development (Pi, 2001).

Genetic analyses show that phyl functions together with sina to promote SOP formation by antagonizing ttk activity. These results suggest that degradation of the Ttk protein is a major function of Phyl in the cell fate specification of SOP cells. Consistent with this idea, misexpression of ttk can inhibit the formation of SOP cells and suppress the ectopic bristle phenotype caused by misexpression of phyl. Several lines of evidence also indicate that Ttk functions as a repressor to inhibit SOP cell fate. (1) Ttk is expressed ubiquitously in the pupal notum except in SOP cells. (2) In embryos, overexpression of ttk inhibits the formation of es organs. (3) Injection of ttk dsRNA results in extensive increase of neurons in embryonic PNS, a phenotype observed in neurogenic mutants. All of these results suggest that ttk might play a negative role in the fate specification of SOP cells, and phyl promotes SOP fate specification by degrading Ttk (Pi, 2001).

Genetic analyses of phyl, sina and ttk are mostly consistent with the model that Phyl functions together with Sina to promote es organ development by degrading Ttk. In embryos, strong defects are detected only when both maternal and zygotic sina transcripts are removed, suggesting that maternally contributed sina transcript play an essential role in the development of embryonic es organs. Consistently, no genetic interaction between zygotic sina and phyl, and between zygotic sina and ttk was detected in embryonic PNS development. In adults, the bristle phenotypes in sina mutants are weaker than in phyl mutants. One possible reason is that the perdurance of sina gene products from maternal transcripts might supply activity for some adult bristles to develop normally. Another possibility is that phyl is able to down-regulate ttk activity in a sina-independent manner. In the Drosophila genome, a sequence (CG 13030) is located next to sina in the genome and encodes a putative protein with 50% identity and 70% similarity with Sina. It might be possible that sina functions redundantly with this gene in bristle development (Pi, 2001).

These studies of phyl/sina/ttk in es organ development and previous studies in photoreceptor differentiation indicate that the Drosophila eye and es organs depend on the same protein complex to specify their cell fate. In both cases, phyl mutations transform neural cells to non-neural cells. Both studies also show that phyl expression is tightly regulated by the upstream signaling pathways. The expression of phyl is activated by the Ras pathway in photoreceptor cells. In SOP cells, the transcription of phyl is likely activated by the proneural genes ac and sc, and is repressed by N signaling. Interestingly, it has been shown that the Egrf/Ras/Raf pathway acts antagonistically with the N pathway in SOP formation of adult macrochaetes and chordotonal organs. Whether these two pathways converge on phyl expression to regulate sensory organ formation remains to be examined (Pi, 2001).


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seven in absentia: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised:    1 August 2008

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