Targets of activity

drm and lines regulate Bowl protein accumulation and consequent patterning in a variety of epithelial tissues

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).

Protein Interactions

Elongation of the Drosophila embryonic hindgut epithelium occurs by a process of oriented cell rearrangement requiring the genes drumstick (drm) and lines (lin). The elongating hindgut becomes subdivided into domains -- small intestine, large intestine and rectum -- each characterized by a specific pattern of gene expression dependent upon normal drm and lin function. drm encodes an 81 amino acid (10 kDa) zinc finger protein that is a member of the Odd-skipped family. drm expression is localized to the developing midgut-hindgut junction and is required to establish the small intestine, while lin is broadly expressed throughout the gut primordium and represses small intestine fate. lin is epistatic to drm, suggesting a model in which localized expression of drm blocks lin activity, thereby allowing small intestine fate to be established. Further supporting this model, ectopic expression of Drm throughout the hindgut produces a lin phenotype. Biochemical and genetic data indicate that the first conserved zinc finger of Drm is essential for its function. A pathway has thus been defined in which a spatially localized zinc finger protein antagonizes a globally expressed protein, thereby leading to specification of a domain (the small intestine) necessary for oriented cell rearrangement (Green, 2002).

In both drm and lin embryos, the hindgut is wider and shorter than that of wild-type embryos (Iwaki, 2001). Beyond this superficial similarity, however, drm and lin hindguts are quite distinct. The drm hindgut is smaller in diameter, and its epithelium consists of an undulating layer of columnar cells resembling those of the immature wild-type hindgut primordium. In contrast, the lin hindgut appears distended, consisting of a uniform layer of cuboidal cells similar in appearance to those of the wild-type small intestine. The strongly Crb-stained boundary cells, which form two parallel rows running the length of the large intestine, are duplicated in drm but are absent in lin hindguts (Iwaki, 2001; Green, 2002).

As revealed by gene expression studies, drm and lin hindguts are improperly patterned, with opposite effects on specification of the large and small intestine. Expression in the dorsal large intestine of engrailed (en) is retained in drm but absent from lin hindguts. Expression in the small intestine of unpaired (upd), encoding a ligand for the JAK-STAT pathway) is missing from drm but greatly expanded in lin hindguts. Similarly, expression in the small intestine of hedgehog (hh) is reduced in drm but greatly expanded in lin hindguts. These data indicate that, in drm embryos, the large intestine is present and the small intestine is greatly reduced or absent; in lin embryos, in contrast, the small intestine is greatly expanded and the large intestine is missing (Green, 2002).

Because their phenotypes are opposite and easily distinguishable, epistasis analysis was used to ask whether drm and lin interact genetically. By all criteria applied, the hindgut of the drm lin double mutant is remarkably like that of the lin single mutant. As in lin embryos, the drm;lin hindgut is short and distended, consists of cuboidal epithelial cells, and lacks boundary cell rows. Similarly, en expression is absent, and both upd and hh expression are expanded posteriorly. All of these observations taken together show that lin is epistatic to drm in the hindgut (Green, 2002).

Since similarities exist in the genetic regulation of foregut and hindgut development, it was asked whether lin is also epistatic to drm in the foregut. In drm embryos, the proventriculus (a multi-layered valve-like structure at the foregut-midgut junction that forms by epithelial folding) does not form, and the foregut is long and narrow. In both lin and drm;lin embryos, the proventriculus also fails to form, but the foregut is short and wide. It is concluded that lin is epistatic to drm in the foregut as well (Green, 2002).

The gene expression and phenotypic data presented in this study and previously (Iwaki, 2001) demonstrate that lin represses and drm promotes the small intestine fate. Taken together with the epistasis of lin to drm, this indicates that drm specifies small intestine by antagonizing the repressive effect of lin. To understand the molecular basis for this relief of repression, the drm gene was molecularly characterized and the interaction, both in vivo and in vitro, between Drm and Lin, was analyzed (Green, 2002).

The drm1 allele was originally identified as a spontaneously occurring lethal mutation mapping between 23F6 and 24A2. drm was mapped more precisely by using P element mobilization to generate two overlapping deletions, drmP1 and drmP2. The left and right breakpoints of these deletions were defined molecularly, thereby localizing drm to a ~60 kb interval between tim and sob. Southern blot analysis of drm1 genomic DNA revealed an insertion within the 11.9 kb BamHI fragment, characterized by inverse PCR as an I element upstream of the predicted gene CG10016. Three expressed sequence tag (EST) clones from this gene have been identified; the longest, LD26791, is 2.3 kb and contains three exons (Green, 2002).

Surprisingly, the LD26791 EST contains only one small open reading frame (ORF) with two zinc finger motifs. To address the possibility that larger proteins might arise from this gene by alternative splicing, CG10016 transcripts were characterized using several approaches. A Northern blot of embryonic RNA with probes from LD26791 identifies a single 2.5 kb transcript, similar in length to the EST clones. RT-PCR with intron-spanning primers failed to identify any splice variants. An alternative first exon was identified in a small fraction (~10%) of 5' RACE products, and several alternative poly(A) addition sites were identified by 3' RACE; none of these variants affects the ORF length. Sequence of all EST clones, RT-PCR products, 5' and 3' RACE products, and genomic DNA is consistent with the conclusion that CG10016 is an 8.5 kb transcription unit. Three exons are spliced to form a 2.5 kb mRNA encoding an 81 amino acid, 10,120 Da predicted protein (Green, 2002).

Drm is a member of the Drosophila odd-skipped (odd) family of zinc finger encoding genes that includes odd, sister of odd and bowl (sob), and bowel (bowl). These genes map close to each other, suggesting that the family has arisen by relatively recent duplication. Like bowl and sob (but not odd), drm contains a splice donor site within the R74 codon of the second zinc finger. Interestingly, this splice site has been conserved evolutionarily, since it is also present in both the mouse and human odd-skipped related (Osr) genes Osr1 and Osr2 (Green, 2002).

The Drm protein contains two zinc finger motifs (compared to four in Odd and five in both Sob and Bowl). The zinc fingers in Odd, Sob, and Bowl conform to the canonical C2H2 structure (C-X2-C-X12-H-X3-H) that is most commonly associated with a DNA-binding function, but in some cases, can have protein-binding capability. In Drm, the first zinc finger conforms to the canonical C2H2 sequence and has a high degree of similarity (~95%) to the first finger of the other Odd family members. The second zinc finger of Drm is divergent; the primary sequence conforms to the canonical C2H2 sequence up to the H73 residue, but the second His residue is replaced by a Cys, with H-X4-C spacing between the latter two zinc-coordinating residues. This residue spacing is found in other C2HC fingers with demonstrated protein-binding activity. Computer modeling with respect to the known structure of the Drosophila U-shaped (Ush) C2HC zinc finger shows that the Drm C2HC finger is theoretically capable of folding around a zinc ligand. Another distinguishing feature of Drm is the divergent linker region between its zinc fingers. The most common linker, found in over 50% of known C2H2 fingers, consists of five residues with the consensus sequence TG(E/Q)(K/R)P. The Odd, Sob and Bowl linkers all have the conserved sequence TDERP, whereas the Drm linker (KSPEIT) is different both in sequence and length. Since its C2H2 and C2HC zinc fingers are, in principle, capable of either DNA or protein binding, Drm may function by either or both of these mechanisms (Green, 2002).

Consistent with the gut defects observed in drm embryos, drm is expressed dynamically in cells that will give rise to the foregut and hindgut. In the posterior gut primordium, drm mRNA is first detected at stage 5 in a ventral crescent at 10% embryo length (EL). Cells in this region are fated to give rise to the epithelia of the posterior midgut, Malpighian tubules and hindgut. At stage 6, the posterior crescent expands dorsally to encircle the amnioproctodeal plate. By stage 7, drm is expressed in a ring within the proctodeal invagination. During germ band extension (stages 8 and 9), drm expression is seen in a region overlapping the junction of the posterior midgut and hindgut primordia. At stage 10, drm is expressed transiently in the evaginating buds of the Malpighian tubules. Between stages 11 and 13, drm mRNA is detected in the posterior midgut, the ureters of the Malpighian tubules, and the most anterior cells of the small intestine; expression in these domains persists throughout the remainder of embryogenesis (Green, 2002).

drm is also expressed in the anterior gut primordium starting at stage 5. Expression increases in this domain until stage 10 when it is internalized as the invaginating stomodeum. drm expression is then refined to a narrow ring of cells at the junction of the foregut and anterior midgut. Like odd and sob, drm is also expressed in a seven-stripe segmental pattern at stage 5; this pattern evolves into fourteen stripes that mark the anterior margin of each segment (Green, 2002).

The domains in which drm is expressed during embryogenesis include a region predicted to become small intestine. By stage 13, however, drm mRNA is not seen in the small intestine. To determine if drm is expressed early in cells fated to become small intestine, a drm-GAL4 driver was generated and it was used to drive UAS-lacZ. Owing to the perdurance of both GAL4 and ß-gal, this provides an historical summation of the drm expression pattern. By stage 13, ß-gal is detected in a much larger domain of the posterior gut in drm-GAL4:UAS-lacZ embryos than is drm mRNA in wild-type embryos. Most importantly, the presence of ß-gal in the small intestine of stage 16 embryos demonstrates that some of the drm-expressing cells in the early embryo do indeed give rise to the small intestine (Green, 2002).

If, as suggested above, spatially localized expression of drm in the anterior hindgut allows specification of the small intestine by antagonizing lin, then expression of drm throughout the hindgut should inhibit endogenous lin, thereby producing a lin-like hindgut phenotype. This was tested by driving UAS-drm using the hindgut-specific byn-GAL4 driver. The result of this gain-of-function expression of drm is a hindgut phenotype that resembles lin loss-of-function mutants: the hindgut is short and distended, consisting of a uniform layer of cuboidal cells similar to those of the wild-type small intestine, and the boundary cell rows are absent. en is not expressed in the hindgut of these embryos, but upd and hh expression is greatly expanded posteriorly. Like lin mutations, the byn-GAL4:UAS-drm combination is lethal. Overall, it appears that in both lin mutants and embryos expressing drm throughout the hindgut, the small intestine is expanded at the expense of the large intestine. It is concluded that drm functions in the hindgut by antagonizing lin (Green, 2002).

If drm acts primarily by inhibiting lin activity in the hindgut, then overexpression of lin might overcome the repressive effect of endogenous drm, thereby producing a drm-like phenotype. Consistent with this notion, overexpression of lin produces a hindgut phenotype that resembles drm loss-of-function mutants: the hindgut is short and wide, consisting of an undulating layer of columnar cells with a duplication of the boundary cell rows. As seen in drm embryos, en is expressed throughout the dorsal hindgut, upd expression is absent, and hh expression in the small intestine is reduced. Like drm mutations, the byn-GAL4:UAS-lin combination is lethal. Since weaker hindgut drivers (e.g., 14-3fkh-GAL4) and weaker UAS-lin transformants do not produce a phenotype as severe as with the strong byn-GAL4 driver or the strongest UAS-lin stocks, it is concluded that the repressive effect of drm can be titrated only by relatively high levels of lin (Green, 2002).

The epistasis and in vivo overexpression studies indicate that drm inhibits lin activity, either directly or indirectly. Since no reduction of lin expression was observed in the drm expression domains of wild-type embryos, or any changes in drm or lin expression in lin or drm mutant embryos, respectively, it is concluded that drm does not affect lin at the transcriptional level. Therefore a biochemical approach was used to ask whether Drm might interact physically with Lin. When expressed together in cultured cells, full-length Drm and full-length Lin interact with each other, as demonstrated by coimmunoprecipitation assays, indicating that the two proteins are in a complex. Since Drm and Lin interact with each other in a yeast two-hybrid assay, the Drm-Lin interaction is likely mediated by direct binding (Green, 2002).

To map the protein interaction domain, deletion and point mutation constructs of Drm were generated and their ability to coimmunoprecipitate with Lin was tested. The N-terminal portion of Drm (N-Drm) is unable to bind Lin, while the C-terminal portion of Drm, containing the two zinc finger motifs (C-Drm), retains full Lin-binding activity. Because these results map the protein-protein interaction domain to the zinc fingers, the requirement of each individual zinc finger for Lin-binding activity was tested. A mutation in the C2H2 first finger (R46C, identical to the mutation in the drm6 null allele) abolishes Lin-binding activity, while disruption of the C2HC second finger (C57G, a substitution in one of the conserved zinc-binding cysteine residues) reduces Lin-binding activity, but does not abolish it completely. To confirm these results, similar point mutations were made in the truncated C-Drm construct and their effect was tested on Lin-binding activity. Again, a mutation in the C2H2 first finger in C-Drm(R46C) abolishes Lin-binding activity while disruption of the C2HC second finger in C-Drm(C57G) reduces Lin-binding activity. It is concluded that the C2H2 zinc finger is essential for binding to Lin, while the C2HC finger contributes to binding, perhaps by stabilizing the interaction (Green, 2002).

It was next asked which portions of the Drm protein are required to block Lin function in vivo by expressing, throughout the hindgut, the same Drm deletion and point mutation constructs used in the coimmunoprecipitation studies. The activity of these mutated proteins was assayed by measuring the protein's ability to induce a lin-like hindgut phenotype when ectopically expressed with byn-GAL4 (similar to the gain-of-function phenotype induced by ectopic expression of full-length Drm). Expression of N-Drm, which lacks the zinc fingers, results in a morphologically wild-type hindgut, while expression of C-Drm, which contains only the zinc finger motifs, produces a morphologically lin-like hindgut (i.e. short and distended and lacking boundary cell rows). Because these results map the in vivo Lin-inhibiting activity to the zinc fingers, the requirement of each individual zinc finger for Lin-inhibiting activity was tested. A mutation in the C2H2 first finger (R46C) abolishes the Drm gain-of-function phenotype, while disruption of the second C2HC finger (C57G) has no effect on Drm activity. These effects are observed whether the point mutations are in full-length Drm (R46C and C57G) or in the truncated C-terminal portion of Drm (C-Drm(R46C) and C-Drm(C57G). Taken together, the results demonstrate that the first zinc finger, but not the second, is required for Lin-inhibiting activity in vivo (Green, 2002).

In summary, Drm constructs that coimmunoprecipitate with Lin are able to repress lin activity in vivo, while Drm constructs lacking Lin-binding activity in vitro are not able to repress lin activity in vivo. It is concluded that the C2H2 first finger is essential for both the Lin-binding and Lin-antagonizing functions of Drm. The C2HC second finger, while contributing to Lin binding, is not absolutely required for Lin-inhibiting activity. Thus the drm and lin antagonism observed by genetic approaches is mediated by a physical interaction between the Drm and Lin proteins (Green, 2002). <>Many questions remain about the Drm-Lin interaction and its mechanism of action. (1) It is not clear how Lin acts as a transcriptional regulator. In the hindgut, and perhaps in other tissues, Lin might act as part of a repressosome complex, binding DNA directly or associating with chromatin on the basis of an interaction with other DNA-binding proteins. Binding of Drm to a Lin-containing complex might then inhibit the repressive activity of Lin. (2) Activation of gene expression requires not only the absence (or inactivation) of repressors, but also the presence of transcriptional activators. Thus, there must be as-yet-unidentified transcriptional activators that promote expression of small intestine-specific targets once Drm has lifted the repression by Lin (Green, 2002).

Lines interacts functionally with Drumstick (Drm) and Bowl

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 brother of odd with entrails limited (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).

lines: Biological Overview | Developmental Biology | Effects of Mutation | References

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