Given the profound effect of bowl mutants on tarsal segmentation and similarities with Notch phenotypes, it was expected that bowl would be expressed at the sites where Notch is active in the tarsus. Therefore, the expression of bowl was compared with E(spl)mß, a known target of Notch signalling in the leg, using an E(spl)mß-lacZ transgene and an antibody that recognizes Bowl. Although Bowl and ß-galactosidase are clearly co-expressed at some positions, including the t5/pretarsus boundary and the tibia/t1 boundary, Bowl was not detected at sites of Notch activity within the tarsus. Indeed, the distribution of Bowl and Odd appears to be identical and neither is detected at tarsomere boundaries (Rauskolb, 1999). Both are present at all the proximal joints (coxa/femur, femur/tibia, tibia/t1) and at a distal site, the t5/pretarsal boundary (the latter has not previously been documented as a site of Notch activity, although it clearly expresses E(spl)mß and gives rise to an articulated joint). In summary, therefore, Bowl and Odd are present at a subset of the segmental boundaries where Notch is active in the developing leg. These correspond to the boundaries between 'true' segments and not to those between tarsomeres (de Celis Ibeas, 2003).
Expression of the Notch ligands is a key step in regulating Notch activity in the developing leg. To investigate the relationship between Bowl and Notch activity, the timing and distribution of Bowl expression was compared with that of Serrate and Delta, which both regulate Notch activity in the leg disc. By monitoring expression from early third instar, it was found that the evolution of Bowl/odd-lacZ expression closely parallels that of the Notch ligands. The only significant discrepancy appears late in the third instar, when Serrate and Delta are detected at intertarsomere boundaries but Bowl and odd-lacZ are not. Before that stage, Bowl/Odd expression occurs distal to each domain of Delta that is established. For example, the central t5/pretarsal ring of Bowl appears at and correlates with the appearance of Delta in the tarsus and a transient expression of Serrate on the distal, pretarsal, side (de Celis Ibeas, 2003).
Whether Bowl accumulation at segment boundaries depends on Notch activity was tested more directly, by generating clones of Notch mutant cells in the disc epithelium. In all cases in which Notch clones crossed between t5 and the pretarsus, the ring of nuclear Bowl protein at the boundary was interrupted. The effects at the t1/tibia and tibia/femur boundaries were less clear cut, with some clones showing absence or reductions in Bowl, whereas others retained apparently wild-type levels. Many of the last group were small clones (seven cells or less). In converse experiments, expression of a constitutively activated form of Notch (Notchicd) resulted in ectopic Bowl accumulation at a subset of locations in the disc. These broadly correspond to the areas where Bowl is normally detected. Taken together, these data indicate that Bowl is responsive to Notch regulation but that the regulation is limited to a specific time window and/or position. Similar results have been obtained with odd, which is only responsive to Notch in selected regions (de Celis Ibeas, 2003).
The operation of the Drm/Lines/Bowl regulatory pathway was examined in the context of the epidermal organizer. Across the dorsal embryonic epidermis, Hedgehog and Wingless are the key pattern-organizing signals. Hedgehog specifies cell fate in half the PS (the 1°-3° cell fates), while Wingless specifies the remaining cell fate (the 4° cell fate) in the complementary half. To investigate whether Hedgehog and Wingless engage the Drm/Lines/Bowl regulatory pathway, drm gene expression and Bowl protein accumulation were examined under conditions of loss or excess of Hedgehog or Wingless signaling. Expression of drm was found to be decreased in hedgehog mutants, and expanded posteriorly in embryos expressing the secreted form of Hedgehog in Engrailed/Hedgehog-expressing cells. Two points are noteworthy here: (1) while Hedgehog can directly control drm expression posterior to the Hedgehog domain, control within the Hedgehog domain is likely indirect since these cells cannot themselves respond to Hedgehog signaling; (2) the fact that excess Hedgehog does not induce drm expression in anterior cells suggests that Wingless signaling represses drm expression in this region. Consistent with this prospect, it was found that drm expression is ectopically activated in wingless mutants and repressed upon ectopic activation of the Wingless pathway. It was also found that changes in drm expression due to manipulations of Hedgehog and Wingless signaling largely led to the expected changes in Bowl protein accumulation. For instance, broadened drm expression caused by excess Hedgehog leads to a broadened Bowl domain, while the ectopic stripe of drm expression in wingless mutants also leads to increased Bowl accumulation, although Bowl accumulates rather more broadly than the narrow drm stripe would suggest. These changes in Bowl accumulation correlate nicely with the patterning changes observed with inactivation or activation of Hedgehog or Wingless signaling. It is concluded that the asymmetric response of drm to Hedgehog underlies the pattern of epidermal cell differentiation since drm promotes the accumulation of Bowl in drm-expressing cells and consequent cellular responses elicited by Bowl. Note that Bowl accumulates in two rows of cells but apparently is required for patterning across a broader region. This observation implies that Bowl controls expression of a new signal that further elaborates epidermal pattern (Hatini, 2005).
Whether drum and lines regulate Bowl abundance in various epithelia was tested along with whether the restricted accumulation of Bowl in these epithelia controls distinct developmental fates, as it does across the embryonic epidermis. Initially, the regulation of Bowl accumulation was investigated in the gut. Genetically, bowl is required both in the foregut, where it distinguishes proventriculus from anterior gut, and in hindgut, where it distinguishes small from large intestine. Indeed, Bowl protein accumulates in two narrow domains in the gut: the primordia for the proventriculus and for the small intestine. In addition, these domains coincide with the sites of drm expression, and in drm mutants, Bowl protein was barely detectable across these domains. Conversely, in lines, as well as drm lines double mutants, Bowl accumulates ubiquitously across the foregut and hindgut primordia. Thus, in the gut just as in the embryonic epidermis, the restricted accumulation of Bowl appears to control distinct developmental fates (Hatini, 2005).
Next the analysis was extended to the leg imaginal disc epithelia, where bowl has been shown to regulate distal leg identities and leg-joint morphogenesis. It was found that the Bowl protein is detected at a set of five rings within the leg imaginal discs, and drm mRNA is detected at a set of five similar rings, supporting the idea that the Drm/Lines/Bowl regulatory pathway also operates in this tissue. To determine whether lines controls Bowl accumulation in the leg also, Bowl accumulation was examined in clones of cells mutant for lines. A cell-autonomous increase in Bowl protein accumulation was found in these clones. This ectopic Bowl accumulation disrupts the normal pattern of gene expression in the leg, as it leads to cell-autonomous reduction of bric-a-brac expression, a target gene repressed by Bowl. These regulatory interactions likely extend to several other imaginal disc epithelia, since a strong correlation was observed in the areas where Bowl is detected at high levels and the domains of drm expression in the wing and eye-antennal disc (Hatini, 2005).
Hedgehog and Wingless signaling in the Drosophila embryonic epidermis represents one paradigm for organizer function. In patterning this epidermis, Hedgehog and Wingless act asymmetrically, and consequently otherwise equivalent cells on either side of the organizer follow distinct developmental fates. To better understand the downstream mechanisms involved, mutations that disrupt dorsal epidermal pattern were investigated. The gene lines contributes to this process. The Lines protein interacts functionally with the zinc-finger proteins Drumstick (Drm) and Bowl. Competitive protein-protein interactions between Lines and Bowl and between Drm and Lines regulate the steady-state accumulation of Bowl, the downstream effector of this pathway. Lines binds directly to Bowl and decreases Bowl abundance. Conversely, Drm allows Bowl accumulation in drm-expressing cells by inhibiting Lines. This is accomplished both by outcompeting Bowl in binding to Lines and by redistributing Lines to the cytoplasm, thereby segregating Lines away from nuclearly localized Bowl. Hedgehog and Wingless affect these functional interactions by regulating drm expression. Hedgehog promotes Bowl protein accumulation by promoting drm expression, while Wingless inhibits Bowl accumulation by repressing drm expression anterior to the source of Hedgehog production. Thus, Drm, Lines, and Bowl are components of a molecular regulatory pathway that links antagonistic and asymmetric Hedgehog and Wingless signaling inputs to epidermal cell differentiation. Finally, it is shown that Drm and Lines also regulate Bowl accumulation and consequent patterning in the epithelia of the foregut, hindgut, and imaginal discs. Thus, in all these developmental contexts, including the embryonic epidermis, the novel molecular regulatory pathway defined here is deployed in order to elaborate pattern across a field of cells (Hatini, 2005).
The Drosophila embryonic epidermis is composed of a series of parasegments (PS). lines is required in the epithelium of the dorsal epidermis to specify one of the four (1°-4°) cell fates present across each PS, such that in lines mutants the 4° fate is missing and all the cells adopt only the 1°-3° fates. If lines operates in the context of the drm/lines/bowl regulatory pathway to control epidermal patterning, drm and bowl should have phenotypes opposite to lines, as they do in the gut. To test this hypothesis, the cuticle phenotype of drm and bowl mutants was examined either alone or in combination with lines. Indeed, it was found that the drm and bowl mutant phenotypes are the opposite of the lines phenotype. In both mutants, the 1°-3° fates are replaced with 4°. In addition, gain-of-function phenotypes for lines and drm parallels those observed in the gut -- while lines gain-of-function phenocopies a drm mutant, drm gain-of-function phenocopies a lines mutant. Therefore, similar to lines, drm and bowl control cell fate decisions across the dorsal embryonic epidermis. In all three mutants, cells make abnormal fate decisions early during development: these are reflected later during development in specific abnormalities in the cuticle pattern. Finally, the epistatic relationships between lines and bowl and between drm and lines are the same as those observed in the gut: lines bowl double mutants look like bowl single mutants, while drm lines mutants look like lines. These results imply that the three genes act in a linear relief-of-repression pathway to pattern the dorsal embryonic epidermis -- lines inhibits bowl across the PS allowing specification of the 4° cell fate, while drm inhibits lines in a subset of cells, allowing bowl to specify the 1°-3° cell fates. Consistent with this model, expression of lines and bowl mRNA is ubiquitous, whereas expression of drm mRNA is localized (Hatini, 2005).
Whether direct molecular interactions underlie these genetically defined inhibitory interactions was investigated. Drm and Bowl are members of the conserved Odd-skipped family of zinc-finger proteins. The bowl gene encodes a protein containing five C2H2 fingers. drm encodes an 81-amino-acid peptide containing a single C2H2 finger most similar to the first zinc finger of Bowl. lines encodes a pioneer protein, conserved in mammals, with no motifs that would suggest a biochemical function. Lines has been shown to bind to the N-terminal C2H2 finger of Drm. This finger shares a high degree of homology with the N-terminal finger of Bowl, suggesting that Lines inhibits Bowl by binding to this finger. Using protein-protein interaction assays, combined with deletion and point mutation analyses, this hypothesis was investigated. Yeast two-hybrid and coimmunoprecipitation (IP) assays suggest direct interactions between Bowl and Lines. The zinc-finger domain (ZFD) was sufficient for the interaction with Lines. Within this domain, a mutation in the first finger (R258C) abolishes interaction with Lines, while a mutation in the second finger (C268G) has little or no effect. Because the N-terminal zinc fingers of Bowl and Drm are each essential for binding to Lines, one likely mechanism for Drm to antagonize Lines is to disrupt, by competition, the Lines-Bowl interaction. This hypothesis was tested by cotransfecting Lines and Bowl into Schneider line 2 cells (S2), with increasing amounts of Drm. It was found that in the absence of Drm, Lines coimmunoprecipitates with Bowl. However, cotransfection with increasing amounts of Drm decreases the amount of Lines associated with Bowl, and does so in a dose-dependent manner, supporting the hypothesis (Hatini, 2005).
In principle, the physical interactions between Lines and Bowl and between Drm and Lines could influence either the activity or the abundance of Bowl, the key downstream effector of this pathway. To determine whether these interactions affect Bowl abundance in vivo, the distribution of Bowl protein was investigated in wild-type embryos. While Bowl mRNA is expressed uniformly, Bowl protein accumulates in the nuclei of only two cell rows in each PS, the posteriormost Engrailed cells and a row of cells just posterior to this. These two cell rows flank the segment border. In addition, the formal genetics suggest particular roles for lines and drm is this regulation. In agreement, in drm mutants, the normal discrete accumulation of Bowl protein accumulation is decreased dramatically in these two cell rows. Conversely, in lines mutants, Bowl protein accumulates ubiquitously across the PS, even when drm function is also removed. These effects on Bowl accumulation are cell-autonomous; the localized expression of Drm in drm mutants results in the increased accumulation of Bowl only in cells that express Drm, while localized expression of Lines (En-Gal4/UAS-Lines) in lines mutants results in the decreased accumulation of Bowl only in cells that express Lines. Finally, to confirm that the Lines-Bowl protein-protein interaction is necessary for the regulation of Bowl accumulation in vivo, the distribution of wild-type Bowl was compared to that of Bowl(R258C), which is compromised for binding to Lines. These proteins were expressed across the embryonic epidermis using Ptc-Gal4, a driver expressed across most but not all cells of the PS. Epitope-tagged wild-type Bowl was found to accumulate to the greatest degree in cells that normally express drm. This is roughly a single-cell-wide stripe since the domains of Ptc-gal4 and drm overlap in only the posterior drm-expressing cells. In contrast, an epitope-tagged form of Bowl(R258C), compromised for binding to Lines, accumulates in all cells in which it is expressed. It is thus concluded that changes in the nuclear abundance of Bowl across the embryonic epidermis are dependent on regulated physical interaction between Lines and Bowl (Hatini, 2005).
Changes in the intensity of the Bowl immunofluorescent signals could reflect either changes in the steady-state level or subcellular distribution of the Bowl protein. These possibilities were distinguished by immunoblotting embryonic extracts from different genotypes. Lower levels of Bowl were detected in drm mutants compared to wild type, and approximately fivefold higher levels of Bowl were detected in lines mutants, drm lines double mutants, or in embryos overexpressing drm. Thus, these data confirm that drm and lines control the steady-state level of Bowl protein. It is concluded that the Lines protein regulates Bowl protein accumulation post-translationally by physically binding to Bowl, consistent with Lines activity leading either directly or indirectly to the degradation of Bowl protein. Drm may inhibit the degradation of Bowl by antagonizing lines in the narrow domain of cells that express drm (Hatini, 2005).
Next, whether Drm antagonizes other aspects of Lines function was investigated. Across a PS, the Lines protein exhibits distinct subcellular localization that correlates with its genetic requirement. An epitope-tagged version of Lines, when expressed either broadly using Arm-GAL4 or more discretely using Ptc-Gal4, accumulates in the nuclei of cells where lines is required genetically, but is either less focused to nuclei or quite cytoplasmically enriched within a narrow domain where lines is not required genetically. The cytoplasmic enrichment of Lines occurs in a region that flanks the segment border, which is where drm is transcribed and Bowl protein accumulates. Since the subcellular distribution of Lines is independent of bowl function, whether it was controlled by drm was tested.The reduced nuclear accumulation of Lines in cells flanking the segment border suggests that Drm disrupts the Lines-Bowl interaction by segregating Lines away from nuclearly localized Bowl. This was investigated by cotransfecting cells with constant amounts of Lines and Bowl together with increasing amounts of Drm. Consistent with the hypothesis, Lines and Bowl localize to the nucleus in the absence of transfected drm. However, Lines redistributes to the cytoplasm with increasing amounts of cotransfected drm. To determine whether this interaction occurs in vivo as well, the subcellular distribution of Lines was examined in drm mutants or when drm was ectopically expressed. In wild type, the epitope-tagged form of Lines is cytoplasmic posteriorly adjacent to the segment border, and nuclear in remaining cells that express Ptc-Gal4. In drm mutants, the epitope-tagged form of Lines is nuclear in all cells in which it is expressed by Ptc-Gal4, while in embryos coexpressing lines and drm, Lines is cytoplasmic in all cells expressing the two proteins. To confirm that the interaction between Drm and Lines is functionally significant, the biological activities were investigated of a mutant derivative of Drm, Drm(R46C), which failed to bind to Lines in co-IP assays and failed to elicit gain-of-function phenotypes in the gut in ectopic expression assays. Ectopic expression of Drm(R46C) failed to transform the cuticle pattern, failed to redistribute Lines to the cytoplasm, and failed to increase the steady-state accumulation of Bowl. Thus, each of the newly discovered in vivo activities of the Drm protein defined in this study require the interaction between Drm and Lines. It is concluded that, in those cells requiring Bowl activity for patterning, Drm is expressed and inhibits Lines through a dominant interfering mechanism. The Drm peptide disrupts the Lines-Bowl interaction, alters the subcellular distribution of Lines, and thereby allows the nuclear accumulation and consequent action of Bowl. Drm localizes Lines to the cytoplasm either by stimulating nuclear export or by inhibiting nuclear import of Lines. Although these findings do not distinguish between these two possible mechanisms, it is suspected that Drm disrupts the Lines-Bowl interaction in nuclei, and subsequently stimulates nuclear export of Lines, and in this manner eliminates residual activity of Lines in the nucleus (Hatini, 2005).
The most important biological implication of these findings is that the Drm/Lines/Bowl pathway can be engaged by a variety of positional cues, depending on context, to elaborate pattern across a field of cells. While Hedgehog and Wingless engage this regulatory pathway in the embryonic epidermis, these signals are not involved in the developing gut epithelia, and the relevant positional cues remain unknown. In the leg imaginal disc, it has been suggested that the Notch signaling pathway regulates drm expression and Bowl accumulation. The Notch pathway may engage lines and bowl in order to control the identity of distal leg identities and the morphogenesis of leg joints. The regulation of bric-a-brac expression by lines nicely substantiates this idea, since bric-a-brac itself specifies distal leg identities. Taken together with the results presented here, it is proposed that the drm gene can integrate distinct signaling inputs depending on the specific tissue invloved (Hatini, 2005).
Across the dorsal embryonic epidermis, the regulation of drm gene expression can explain how the Drm/Lines/Bowl pathway links the antagonistic inputs of Hedgehog and Wingless signaling to subsequent steps in epidermal differentiation. Indeed, changes in drm expression account nicely for the transformation of the epidermal pattern observed in conditional hedgehog and wingless mutants. Loss of drm expression, as seen in hedgehog mutants, leads to the establishment of the 4° cell type in place of the 1°2°3° portion of the pattern, resulting in a 4°-4° pattern. In contrast, symmetric drm expression, as seen in wingless mutants, leads to the establishment of mirror-symmetric 3°2°1° fates in place of the 4°, resulting in a 1°2°3°-3°2°1° pattern. The asymmetric induction of drm expression is then used to modulate Lines and Bowl function. This is reflected by the asymmetry of Lines subcellular distribution and Bowl accumulation relative to the source of Hedgehog production. Although Bowl accumulates in only two cell rows in each PS, it has a remarkable influence on a broader field of cells that spans approximately six cell rows. Bowl may therefore organize the pattern indirectly by regulating expression of a new signal (Hatini, 2005).
Pattern across each PS in the ventral embryonic epidermis is not organized by a single morphogen but by a combination of distinct signals, with each signal acting fairly locally. Early during development, the expression of Hedgehog and Wingless is established by reciprocal induction across the parasegment border. At a later stage, Hedgehog induces expression of rhomboid only on the segment border side within the anterior compartment. rhomboid controls the production of secreted Spitz, a TGFalpha homolog that activates the EGF-R pathway. In addition, Hedgehog and Wingless appear to act at a distance to restrict Serrate expression to the middle of the anterior compartment. Finally, cell differentiation is controlled by Hedgehog, Wingless, Spitz, and Serrate, each controlling a subset of cell fates. For example, Hedgehog, Spitz, and Wingless each induce expression of the gene stripe by short-range inductive signaling, leading to tendon differentiation at three discrete positions across each abdominal PS. While rhomboid and consequent EGF-R activation are crucial for ventral patterning, no role was detected for rhomboid in dorsal cuticle patterning. The current findings suggest that the Drm/Lines/Bowl pathway organizes the pattern in response to Hedgehog signaling dorsally and thus substitutes for rhomboid. Although drm responds to Hedgehog asymmetrically, there is an important distinction between the regulation of drm expression and the regulation of other Hedgehog targets such stripe and rhomboid. While previously known Hedgehog targets are induced only in anterior compartment cells, the drm gene is induced in both anterior and posterior compartments, on either side of the segment border. The induction of drm expression in the posterior compartment is likely not due to Hedgehog directly, because Hedgehog-producing cells are refractory to Hedgehog signaling. There is likely a reciprocal induction between anterior and posterior compartment cells with Hedgehog inducing drm expression in the anterior compartment, and a new signal inducing drm in the posterior compartment. Understanding the logic underlying this regulation will require identifying the signal(s) downstream of Bowl that lead to broad patterning. Given that the Drm/Lines/Bowl regulatory pathway is conserved and operates reiteratively in development, such signals are likely to be used in patterning of other epithelial tissues (Hatini, 2005).
Drosophila Groucho, like its vertebrate Transducin-like Enhancer-of-split homologues, is a corepressor that silences gene expression in numerous developmental settings. Groucho itself does not bind DNA but is recruited to target promoters by associating with a large number of DNA-binding negative transcriptional regulators. These repressors tether Groucho via short conserved polypeptide sequences, of which two have been defined: (1) WRPW and related tetrapeptide motifs have been well characterized in several repressors; (2) a motif termed Engrailed homology 1 (eh1) has been found predominantly in homeodomain-containing transcription factors. A yeast two-hybrid screen is described that uncovered physical interactions between Groucho and transcription factors, containing eh1 motifs, with different types of DNA-binding domains. One of these, the zinc finger protein Odd-skipped, requires its eh1-like sequence for repressing specific target genes in segmentation (Goldstein, 2005).
The eh1 Gro recruitment domain was originally defined as a heptapeptide motif that is conserved in members of the En family of homeodomain proteins and their vertebrate homologues. More recently, eh1-dependent binding to Gro has also been demonstrated in vitro for various other Drosophila and mammalian proteins, nearly all of which contain homeodomains. Given that Bowl and Odd, two non-homeodomain ZnF transcription factors, contain this motif and interact with Gro, the possibility was explored that eh1 motifs are prevalent among additional non-homeodomain transcription factor families. Indeed, an unbiased yeast screen for Gro-interacting proteins selected two additional transcriptional regulators that contain eh1-like motifs, namely, Sloppy-paired (Slp; Forkhead related) and Dorsocross (Doc; T box). Alignment of the eh1-like sequences of Bowl, Odd, Slp, and Doc with those of En and Gsc revealed three conserved amino acids: phenylalanine-x-isoleucine-x-x-isoleucine (Phe-x-Ile-x-x-Ile, where x is any amino acid). Subsequent database searches for presumptive Drosophila transcription factors containing this minimal peptide sequence identified a wide range of potential negative regulators belonging to different superfamilies as classified by their distinct DNA-binding domain types. Remarkably, eh1-related motifs have been preserved in many human homologues of these fly proteins, indicating that the ability to bind Gro/TLE has been evolutionarily conserved in human transcriptional regulators and that this sequence may have been widely adopted throughout the proteome as a Gro recruitment domain (Goldstein, 2005).
Several representatives, corresponding to different transcription factor families, were tested for the ability to bind Gro in biochemical assays. Where possible, full-length expressed sequence tags encoding these proteins were obtained; otherwise, single exons containing the eh1-like sequence were PCR amplified from genomic DNA. Each polypeptide was assessed for the ability to pull down radiolabeled Gro in vitro. GST-tagged Slp and Doc (amino acids 254 to 391) readily retain Gro, as do Eyes absent (Eya) and the homeodomain proteins Ventral nervous system defective (Vnd, 1 to 465), Bagpipe (Bap, 1 to 129), BarH1, and Empty spiracles (Ems, 1 to 360), as well as the orphan nuclear hormone receptor DHR96. To confirm that these interactions rely on intact eh1-related sequences, the eh1 motif of one of these, BarH1, was mutated by substituting glutamic acid for Phe at position 1, finding that its binding to Gro is reduced by >60% (Goldstein, 2005).
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