Mutations that influence lin-12 activity in C. elegans may identify conserved factors that regulate the activity of lin-12/Notch proteins (see Drosophila Notch). Genetic evidence indicates that sel-10 is a negative regulator of lin-12/Notch-mediated signaling in C. elegans. Sequence analysis shows that SEL-10 is a member of the CDC4 family of proteins and has a potential human ortholog. Coimmunoprecipitation data indicate that C. elegans SEL-10 complexes with LIN-12 and with murine Notch4. It is proposed that SEL-10 promotes the ubiquitin-mediated turnover of LIN-12/Notch proteins (Hubbard, 1997).
Proteolysis of LIN-12/Notch proteins might occur in response to ligand binding or occur constitutively. For a variety of cell surface receptors, ligand-induced polyubiquitination appears to be a mechanism for down-regulation. Although there is no direct evidence for ligand-induced ubiquitination of LIN-12/Notch receptors, it has been noted that LIN-12(intra), which genetically and physically interacts with SEL-10, behaves like an activated receptor. Alternatively, SEL-10 may target any form of LIN-12/Notch (activated or unactivated) for degradation. Although constitutive turnover is not strictly a mechanism for controlling receptor activity per se, it would, in effect, sensitize the system to other control mechanisms such as transcriptional regulation by generally reducing the amount of LIN-12 (Hubbard, 1997).
Constitutive turnover or ligand-induced down-regulation of LIN-12/Notch proteins may be important for cell fate decisions to occur normally. Potential roles for turnover or down-regulation can be illustrated with the AC/VU decision as an example. Initially, Z1.ppp and Z4.aaa have equal signaling and receiving potentials; ligand (LAG-2) and receptor (LIN-12) may interact, but signaling activity is below a critical threshold. SEL-10-mediated turnover or down-regulation of LIN-12 might prevent this initial signaling from causing both cells to achieve the threshold value of effector activity. Thus, one possible role for receptor turnover or down-regulation would be to limit the output from a single ligand-receptor interaction. Another potential role for receptor turnover or down-regulation is in enhancing differences in lin-12 activity between interacting cells. During the AC/VU decision, a small stochastic difference between the two cells is amplified by a feedback mechanism. The feedback mechanism appears to involve differential transcription of ligand and receptor genes: activation of LIN-12 appears to repress transcription of lag-2 and to stimulate transcription of lin-12. The feedback mechanism ensures that the cell with higher lin-12 activity becomes the VU whereas the cell with lower lin-12 activity becomes the AC. Down-regulation of LIN-12 would be necessary for differences in transcription to be manifest. In the absence of down-regulation, signaling from activated receptor would persist, masking the effects of differential transcription. Indeed, this situation is analogous to the role of ubiquitin-mediated degradation of G1 cyclins (Hubbard, 1997).
The Wnt/beta-catenin signaling pathway is responsible for the establishment of the dorsoventral axis of Xenopus embryos. The recent finding of the F-box/WD40-repeat protein Slimb in Drosophila, whose loss-of-function mutation causes ectopic activation of Wingless signaling, has led to an examination of the role of its vertebrate homolog betaTrCp in the Wnt/beta-catenin signaling and dorsal axis formation in Xenopus embryos. Co-injection of betaTrCp mRNA diminishes Xwnt8 mRNA-induced axis formation and expression of Siamois and Xnr3, suggesting that betaTrCP is a negative regulator of the Wnt/beta-catenin signaling pathway. An mRNA for a betaTrCp mutant construct (DeltaF), which lacks the F-box domain, induces an ectopic axis and expression of Siamois and Xnr3. Because this activity of DeltaF is suppressed by co-injection of DeltaF TrCP mRNA, DeltaF likely acts in a dominant negative fashion. The activity of DeltaF is diminished by C-cadherin, glycogen synthase kinase 3 and Axin, but not by a dominant negative dishevelled. These results suggest that betaTrCp can act as a negative regulator of dorsal axis formation in Xenopus embryos (Marikawa, 1998).
ebi (the term for 'shrimp' in Japanese) regulates the epidermal growth factor receptor (Egfr) signaling pathway at multiple steps in Drosophila development. Mutations in ebi and Egfr lead to similar phenotypes and show genetic interactions. However, ebi does not show genetic interactions with other RTKs (e.g., torso) or with components of the canonical Ras/MAP kinase pathway. ebi encodes an evolutionarily conserved protein with a unique amino terminus, distantly related to F-box sequences, and six tandemly arranged carboxy-terminal WD40 repeats. The existence of closely related proteins in yeast, plants, and humans suggests that ebi functions in a highly conserved biochemical pathway. Proteins with related structures regulate protein degradation. Similarly, in the developing eye, ebi promotes Egfr-dependent down-regulation of Tramtrack88, an antagonist of neuronal development (Dong, 1999).
ebi mutations have been identified in a screen for enhancers of an eye mutant called roughex, which plays a key role in regulating cell cycle progression in the developing eye. As a consequence of cell cycle defects, photoreceptor differentiation and pattern formation in the eye are disrupted. Whereas cell cycle regulators enhance and suppress the primary cell cycle phenotype, mutations in other loci, such as Star and Epidermal growth factor receptor, only modify the differentiation phenotype, and not the earlier cell cycle defects. Like Star and Egfr, ebi enhances the differentiation phenotype. These observations led to a consideration of the relationship between the Egfr signaling pathway and ebi. Evidence shows that ebi participates in Egfr signaling pathways. ebiE4, ebiE90, and ebiP7 are null, strong, and weak alleles, respectively (Dong, 1999).
That ebi functions in the Egfr pathway was initially suggested by phenotypes of a viable heteroallelic combination of ebi (i.e., ebiP7/ebiE90). These flies exhibit phenotypes similar to weak loss-of-function Egfr alleles (i.e., Egfrtop1/Egfrf2) including partial female sterility resulting from partially ventralized eggs, wing vein defects, short bristles, and abnormal eyes (i.e., rough eyes). Further evidence that ebi participates in the Egfr pathway was provided by genetic interactions between ebi and Egfr components. For instance, flies carrying two different alleles of Egfr (Egfrtop1/Egfrf2) have a weak rough-eye phenotype, which is enhanced in flies that are heterozygous for ebi. ebi and Egfr mutant embryos are also similar. Homozygous ebi null mutant embryos (ebiE4) exhibit a tail-up or U-shaped embryo with head defects. Embryos lacking both the zygotic and maternal contributions of ebi were created using ovoD and FRT/FLP-induced recombination. This results in a more severe phenotype, including the loss of ventral denticle belt structures and a tightly curled morphology indicating a marked failure in germ-band retraction. Severe head defects are also observed. In contrast to Egfr mutants, some residual ventral cuticular structures remain in embryos lacking both the zygotic and maternal contributions of ebi (Dong, 1999).
Loss of ebi also affects Egfr-dependent expression of genes in the embryo. The Egfr ligand Spitz is expressed along the ventral midline and induces expression of different target genes, including fasciclin III (fasIII) and orthodenticle (otd), in cells located in more lateral positions. In zygotic null Egfr mutants both otd and FasIII expression are lost. In wild-type stage 11/12 embryos, FasIII protein is broadly distributed in the visceral mesoderm and in a bilaterally symmetric cluster of cells flanking the midline of the ventral ectoderm. In ebi mutant embryos lacking both maternal and zygotic contribution, FasIII expression is largely abolished, although some residual patches of staining remain. Egfr-independent expression of FasIII in the anterior-most region of the embryo is unaffected in ebi mutants. In wild-type stage 10/11 embryos, otd mRNA is expressed in the preantennal head region and in the ventral-most ectoderm. In ebi mutant embryos, otd expression is markedly reduced. These data suggested that ebi may be a component in the Egfr signal transduction pathway. To assess whether ebi encoded a hitherto unidentified regulator in the Ras/MAP kinase pathway, its role in the Torso RTK pathway was assessed. Torso controls the development of the anterior and posterior termini of the embryo. Ras, Raf, MEK, and MAPK participate in both the Egfr and Torso RTK pathways. The expression of Torso target genes huckebein (hkb) and tailless (tll) in embryos entirely deficient in ebi (i.e., lacking both maternal and zygotic ebi) are indistinguishable from wild type. In summary, ebi mutant phenotypes assessed using both molecular and morphological criteria are similar to Egfr mutations. Furthermore, ebi does not function in all RTK pathways, since Torso-induced terminal development is ebi independent. These data indicate that ebi, either directly or indirectly, regulates Egfr signaling. As a step toward understanding the role of ebi in the context of a specific developmental process, the role of ebi in R7 development in the compound eye was assessed through both genetic and molecular studies (Dong, 1999).
The R7 equivalence group comprises five cells competent to become R7 neurons. They are the R7 precursor cell and the precursors to the four cone cells. Cone cell precursor cells can be induced to become R7 cells by ectopic activation of the R7 inductive pathway in these cells. Transformation of cone cells into R7 cells leads to a disorganized adult eye or a so-called rough-eye phenotype. The ability of loss-of-function ebi mutations to suppress this transformation was assessed in various genetic backgrounds. Whereas ebi dominantly suppresses R7 development induced by the activated Egfr expressed in the R7 equivalence group under the control of the sev enhancer (sev-TorDEgfr), it does not suppress R7 development induced by the activated Sev receptor (sev-TorDSev, SevS11, or activated forms of Ras, Raf, and MAPK. Hence, ebi is required for the transformation of cone cell precursors into R7 neurons by the activated Egfr (Dong, 1999).
To assess whether ebi participates in the induction of the R7 precursor cell into an R7 neuron, a genetically sensitized background in which only some 15%-20% of the R7 precursors become R7 neurons was used. The R7 inductive signal is attenuated by using a strong hypomorphic allele of sev (sevE4) and a weak gain-of-function mutation in the Ras activator, encoded by the son-of-sevenless gene, SosJC2. Aside from the loss of the majority of the R7 cells, development of the eye in this genetic background is otherwise indistinguishable from wild type. ebi is a dominant enhancer of this phenotype, as are Egfr loss-of-function mutations. These data are consistent with studies demonstrating a requirement for both the Egfr and Sev receptor in R7 induction. Hence, ebi is required for induction of the R7 precursor cell into an R7 neuron and for transformation of cone cell precursors into R7 in response to ectopic activation of Egfr. Ttk88 down-regulation is required for R7 induction of the R7 precursor cell. This is supported by the finding that Ttk88 mutations are dominant suppressors of the SevE4;SosJC2/+ phenotype (Dong, 1999).
To assess the role of ebi on R7 development in an otherwise wild-type background, attempts were made to generate homozygous null mutant clones. Such clones could not be generated using X-ray and heat shock Flp-induced mitotic recombination. Hence, like Egfr, ebi is required for cell proliferation and/or survival during the proliferative phase of disc development. To increase the efficiency of Flp-induced mitotic recombination, a Flp source driven by the eyeless (ey) promoter was used. The ey promoter drives expression from the earliest cell divisions in the eye primordium until the last cell division of precursor cells in the third instar. This results in the production of multiple mutant clones throughout development. Mutant clones in the eye disc have been recognized by the loss of Ebi immunoreactivity. Rather small clones have been observed: clusters within these clones contain differentiating R cells. Each cluster contains a single R8 cell (i.e., stained with antibody to the Boss protein), and early clusters appear normal. Although clusters containing eight neurons form, disorganized clusters containing fewer differentiated neurons are also observed (Dong, 1999).
Adult ommatidia containing homozygous mutant cells are frequently highly disorganized and show a marked reduction in R cells. Mutant R cells, including R7 cells, are seen in adult mosaic ommatidia; some 80% of these cells show an altered cellular morphology. Hence, although ebi is required for R7 development in a genetically sensitized background, R7 neurons can develop in an ebi mutant. Although the formal possibility that these R7 neurons develop because of perdurance of Ebi protein in the R7 precursor cell cannot be ruled out, these data strongly suggest that R7 cells can form in an ebi-independent fashion, though less efficiently than in wild type. These data are consistent with ebi subserving a redundant function in R7 development. To gain clues to the molecular pathways regulated by ebi, the gene was cloned and sequenced (Dong, 1999). This transcription unit encodes a protein of 700 amino acids with a carboxy-terminal segment containing six WD40 repeats. The ebiE4 and ebiE90 alleles result in missense mutations. In ebiE4 the methionine encoded by codon 1 is changed to an isoleucine, and in ebiE90 a highly conserved cysteine, located at amino acid 510 between WD40 repeats 3 and 4, is changed to a tyrosine (Dong, 1999).
ebi-related human cDNA sequences and genomic sequences from S. cerevisiae and Arabidopsis thaliana, have been identified in the database. Because the initial human expressed sequence tag was not complete, additional cDNAs were isolated from adult human spleen cDNA library and sequenced. Both an amino-terminal 89-amino-acid segment and the carboxy-terminal WD40 repeats of fly ebi correspond remarkably well to these regions in the mammalian, plant, and yeast genes; the amino-terminal 89 amino acids and the WD40 repeat region share 81%, 34%, and 51% identity with the human, yeast, and plant sequences, respectively. In addition to these conserved regions the fly protein is predicted to contain an insertion of 160 amino acids between the amino terminus and the WD40 repeats (Dong, 1999).
The bipartite structure of Ebi is reminiscent of three proteins involved in protein degradation: Cdc4 from S. cerevisiae; Slimb from Drosophila melanogaster, and Sel-10 from C. elegans. All three proteins contain an amino-terminal F-box and carboxy-terminal WD40 repeats; these proteins have been shown (Cdc4) or proposed (Slimb and Sel-10) to target proteins for degradation by linking them to a ubiquitin-conjugase complex. Although the amino-terminal domain of Ebi is divergent from the Cdc4 F box (as is Slimb), it shares weak sequence and structural homology. The amino-terminal half of the F box is more highly related to ebi than the carboxy-terminal region. The periodic spacing of hydrophobic residues in both Ebi and F-box sequences is consistent with these regions being able to assume an alpha-helical amphipathic conformation. Three residues in the amino-terminal region of the Cdc4 F box have been shown to be required for binding to Skp1 (a component in the E3 complex). These amino acids are conserved in Ebi, and correspond to residues P45, I52, and L57 in the Ebi sequence (Dong, 1999).
Ebi is widely expressed in nuclei of the embryo and larvae. Immune staining is largely, if not exclusively, nuclear. Double staining of salivary gland nuclei with anti-Myc antibodies to detect Myc-tagged Ebi and the DNA stain DAPI demonstrates that Ebi was not associated with chromatin but, rather, is distributed in a reticular pattern throughout the nucleoplasm. The similarity of Ebi to F-box/WD40 repeat-containing proteins and its nuclear localization suggests that Ebi may regulate Egfr signaling through degradation of nuclear proteins. Recent studies have revealed an important role for both Egfr and degradation of a specific transcription factor Tramtrack88, for R7 development. The structural similarity between Ebi and F-box/WD40-repeat proteins involved in protein degradation prompted an exploration of the relationship between ebi and Ttk88 protein levels in the developing eye. Ttk88 is expressed at very low levels in undifferentiated cells in the developing eye disc and at high levels in developing cone cell nuclei; it is not expressed in developing photoreceptor cells. Transformation of cone cells into R7 by misexpression of phyl under the sev promoter leads to Ttk88 degradation. Ectopic R7 induction by TorDEgfr driven by the sev promoter also leads to marked degradation of Ttk88. sev-TorDEgfr-induced Ttk88 degradation is dominantly suppressed by ebi. Similarly, ebi dominantly suppresses the pGMR-phyl-induced decrease in Ttk88, as well as the pGMR-phyl-induced eye phenotype (Dong, 1999).
The role of ebi in regulating Ttk88 levels in an otherwise wild-type eye disc was examined. Analysis of Ttk88 levels on the small mutant clones generated with ey-Flp reveals no obvious differences. To explore this issue further, reduction in ebi was achieved by expressing the dominant-negative form of ebi in all cells posterior to the morphogenetic furrow in an ebi heterozygous background. Dominant-negative ebi contains the amino-terminal half of the protein from amino acids 1-334 expressed under the control of the pGMR promoter. In wild-type eye discs, Ttk88 staining is not observed in a focal plane in which photoreceptor cell nuclei are located. In contrast, in mutant discs, an average of 36 ± 6 Ttk88-positive nuclei are observed in this region. Most Ttk88-positive nuclei are found 8-10 rows posterior to the morphogenetic furrow. This increase in Ttk88-positive cells also parallels a concomitant decrease in the number of cells stained with the pan-neuronal stain, anti-Elav. In wild-type eye discs, all ommatidia 8-10 rows posterior to the morphogenetic furrow have at least seven Elav-positive cells (R1-R6 and R8). However, in mutant discs, many ommatidia in this region contained less than seven stained cells. Interestingly, a considerably smaller fraction of ommatidia in rows 11-13 contain less than eight Elav-positive R cells; in wild-type discs, all clusters contain eight Elav-positive cells in this region. Hence, a reduction in ebi activity delays neuronal development and this is correlated with persistent nuclear expression of the Ttk88 protein. In summary, both ebi and Egfr promote Ttk88 down-regulation, thereby promoting neuronal development. Further work is required to assess the relationship between ebi and ttk in Egfr signaling in other developmental contexts (Dong, 1999).
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