phyllopod

Transcriptional Regulation

R7 photoreceptor fate in the Drosophila eye is induced by the activation of the Sevenless receptor tyrosine kinase and the RAS/MAP kinase signal transduction pathway. Expression of a constitutively activated Jun isoform in ommatidial precursor cells is sufficient to induce R7 fate independent of upstream signals normally required for photoreceptor determination. Jun interacts with the ETS domain protein Pointed to promote R7 formation. This interaction is cooperative when both proteins are targeted to the same promoter and is antagonized by another ETS domain protein, Yan, a negative regulator of R7 development. Furthermore, phyllopod, a putative transcriptional target of RAS pathway activation during R7 induction, behaves as a suppressor of activated Jun. Taken together, these data suggest that Jun and Pointed act on common target genes to promote neuronal differentiation in the Drosophila eye, and that phyllopod might be such a common target (Treier, 1995).

Drosophila muscles originate from the fusion of two types of myoblasts -- founder cells (FCs) and fusion-competent myoblasts (FCMs). To better understand muscle diversity and morphogenesis, a large-scale gene expression analysis was performed to identify genes differentially expressed in FCs and FCMs. Embryos derived from Toll^10b mutants were employed to obtain primarily muscle-forming mesoderm, and activated forms of Ras or Notch were expressed to induce FC or FCM fate, respectively. The transcripts present in embryos of each genotype were compared by hybridization to cDNA microarrays. Among the 83 genes differentially expressed, genes known to be enriched in FCs or FCMs, such as heartless or hibris, previously characterized genes with unknown roles in muscle development, and predicted genes of unknown function, were found. These studies of newly identified genes revealed new patterns of gene expression restricted to one of the two types of myoblasts, and also striking muscle phenotypes. Whereas genes such as phyllopod play a crucial role during specification of particular muscles, others such as tartan are necessary for normal muscle morphogenesis (Artero, 2003).

The Toll^10b mutation gives rise to embryos composed primarily of somatic mesoderm. In these embryos FCs and FCMs are readily detected, and they respond to the Ras and Notch signaling pathways in the same way as their wild-type counterparts. Advantage was taken of this fact to enrich Toll^10b mutant embryos for FCs or FCMs, which allowed a concentration on the transcription in these two specific cell types within the context of the entire embryo. Genes known to be expressed and regulated in FCs or FCMs emerged from the screen in the proper categories. Not all known FC/FCM genes were detected in the screen for several reasons: the high stringency set for interpretation of the array data; the presence of only about one-third of the genome on the arrays; the loss of Dpp in the Toll^10b background, and the specific window of myogenesis (5- to 9-hours) that was the focus of this investigation. However, a plethora of potential new muscle regulators were uncovered, including known genes with no previously recognized function in the mesoderm (such as phyl and asteroid), and genes predicted from the Drosophila genome sequence but not previously analyzed (Artero, 2003).

Notch and Ras signaling pathways interact during muscle progenitor segregation. The results suggest that phyl and polychaetoid (pyd) may be additional links between the two signaling pathways in FCs. phyl and pyd both interact genetically with Notch and Delta. The transcription of phyl, which promotes neural differentiation, is negatively regulated by Notch signaling during specification of SOPs and their progeny. This study shows a similar regulation in muscle cells, where Notch signaling represses phyl expression and Ras signaling increases phyl expression. Likewise, in the nervous system, the segregation of SOPs requires pyd, a Ras target gene, to negatively regulate ac-sc complex expression. Similarly, Pyd may restrict the muscle progenitor fate to a single cell, perhaps by regulating lethal of scute transcription. Thus, Pyd would collaborate with Notch signaling to restrict muscle progenitor fate to one cell (Artero, 2003).

FCMs appear to integrate Ras and Notch signaling differently. Two genes whose transcripts were enriched under activated Notch conditions, parcas and asteroid (ast), have been implicated in Ras signaling in other tissues, directly (ast) or indirectly (parcas). These data are suggestive of a role for Ras signaling in the FCMs, in addition to its role in FC specification. In addition, Notch signaling to FCMs may prime cells for subsequent Ras signaling during muscle morphogenesis, much as occurs in FCs where Ras signaling primes the cell for subsequent Notch signaling during asymmetric division of the muscle progenitor (Artero, 2003).

Embryos that lack or ectopically express phyl have morphological defects in specific muscles, for example, in LL1 and DO4 in response to diminished phyl function, and in DT1 and LT4 in response to increased phyl function. The morphological defects in the loss-of-function embryos appear to be due to a failure to specify particular FCs, a conclusion that is based upon missing or abnormal production of the FC marker Kr. In eye development and SOP specification, Phyl directs degradation of the transcriptional repressor Tramtrack. In a subset of the primordial muscle cells, Phyl may work similarly, targeting Tramtrack for degradation. The presence of Tramtrack would contribute to the specific identity program of the muscle. Since Tramtrack is expressed in the mesoderm, this possibility is likely. Alternatively, Phyl may be required for targeted degradation of some other protein in a subset of FCs. The molecular partner for Phyl during muscle differentiation is unknown, although preliminary data suggest that sina is also expressed in somatic mesoderm and thus may be its partner. These studies have identified a new role for Phyl in muscle progenitor specification and suggest the importance of targeted ubiquitination for proper muscle patterning (Artero, 2003).

Protein Interactions

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).

The transcription repressor Tramtrack (Ttk) is found in cone cells but not photoreceptor cells of the Drosophila eye. Down-regulation of Ttk expression occurs in photoreceptor cells and is required for their fate determination. Down-regulation requires the presence of Phyllopod, which is induced by the ras pathway, and Seven In Absentia (Sina). Loss of either gene causes accumulation of Ttk in photoreceptor cells, and Ttk does not accumulate in cone cells if both Phyl and Sina are present. Reduction of Ttk levels by Phyl depends on sina function. Reduction of Ttk expression induced by ectopic phyl does not occur in a sina mutant background. Down-regulation of Ttk expression by phyl does not occur at the transcriptional level. That is, the level of ttk expression as detected using a ttk expression vector is uneffected by alteration in phyl function. Sina and Phyl promote ubiquitination and rapid degradation of Ttk by the proteasome pathway in cell culture. Both Sina and Phyl bind to the N-terminal domain of Ttk. Deletion of amino acids 1-286 of Ttk88 abolishes the interaction with Sina, whereas a polypeptide containing amino acids 1-116 of Ttk88 results in a strong interaction. Interestingly, this region corresponds precisely to the BTB domain shared by both Ttk69 an Ttk88. These results argue that photoreceptor differentiation is regulated by the RAS pathway through targeted proteolysis of the Ttk repressor (Li, 1997).

prospero(pros) gene becomes transcriptionally activated at a low level in all Sevenless-competent cells prior to Sevenless signaling, and this requires the activities of Ras1 and two Ras1/MAP kinase-response ETS transcription factors, Yan and Pointed. Activation of pros transcription in all cells within the R7 equivalence group requires the down-regulation of Yan activity through phosphorylation by MAPK in R7 and cone cell precursors. Loss of pointed results in a reduction in the number of pros expressing cells. Two other nuclear factors, Seven in absentia and Phyllopod are required for R7 determination, but are not absolutely for pros expression. However, the presence of phyl in cells is sufficient to induce them to express elevated levels of pros. phyl requires sina activity to stimulate pros expression. Sina protein can be shown to form a complex with Phyl (Kauffmann, 1996)

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).

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.