InteractiveFly: GeneBrief

bowel: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - brother of odd with entrails limited

Synonyms - bowel

Cytological map position - 24C3--4

Function - transcription factor

Keywords - foregut, hindgut, leg

Symbol - bowl

FlyBase ID: FBgn0004893

Genetic map position - 2-16

Classification - Odd family zinc finger

Cellular location - nuclear

NCBI link: Entrez Gene

bowl orthologs: Biolitmine
Recent literature
Mojica-Vazquez, L. H., Benetah, M. H., Baanannou, A., Bernat-Fabre, S., Deplancke, B., Cribbs, D. L., Bourbon, H. M. and Boube, M. (2017). Tissue-specific enhancer repression through molecular integration of cell signaling inputs. PLoS Genet 13(4): e1006718. PubMed ID: 28394894
The bric-a-brac2 (bab2) gene is required for distal leg segmentation. Previous work has shown that the Distal-less (Dll) homeodomain and Rotund (Rn) zinc-finger activating transcription factors control limb-specific bab2 expression by binding directly a single critical leg/antennal enhancer (LAE) within the bric-a-brac locus. This study shows that the EGFR-responsive C15 homeodomain and the Notch-regulated Bowl zinc-finger transcription factors also interact directly with the LAE enhancer as a repressive duo. The appendage patterning gene bab2 is the first identified direct target of the Bowl repressor, an Odd-skipped/Osr family member. Moreover, C15 was shown to act on LAE activity independently of its regular partner, the Aristaless homeoprotein. Instead, C15 interacts physically with the Dll activator through contacts between their homeodomain and binds competitively with Dll to adjacent cognate sites on LAE, adding potential new layers of regulation by C15. Lastly, C15 and Bowl activities were shown to regulate also rn expression. These findings shed light on how the concerted action of two transcriptional repressors, in response to cell signaling inputs, shapes and refines gene expression along the limb proximo-distal axis in a timely manner.
To, V., Kim, H. J., Jang, W., Sreejith, P. and Kim, C. (2021). Lin28 and Imp are Required for Stability of Bowl Transcripts in Hub Cells of the Drosophila Testis. Dev Reprod 25(4): 313-319. PubMed ID: 35141457
Hub cells comprise a niche for germline stem cells and cyst stem cells in the Drosophila testis. Hub cells arise from common somatic gonadal precursors in embryos, but the mechanism of their specification is still poorly understood. This study found that RNA binding proteins Lin28 and Imp mediate transcript stability of Bowl, a known hub specification factor; Bowl transcripts were reduced in the testis of Lin28 and Imp mutants, and also when RNA-mediated interference against Lin28 or Imp was expressed in hub cells. In tissue culture Luciferase assays involving the Bowl 3'UTR, stability of Luc reporter transcripts depended on the Bowl 3'UTR and required Lin28 and Imp. These findings suggest that proper Bowl function during hub cell specification requires Lin28 and Imp in the testis hub cells.

In the Drosophila leg, activation of Notch leads to the establishment of the joints that subdivide the appendage into segments. Mutations in bowl result in phenotypes similar to Notch, causing fusion and truncations of tarsal segments (tarsomeres). Like its close relative Odd-skipped, Bowl is produced in response to Notch signalling at a subset of segment boundaries. However, despite the fact that bowl mutant clones result in fusion of tarsomeres, Bowl protein is only found at the t1/tibial and t5/pretarsal boundaries, not at tarsomere joints. One hypothesis to reconcile these data is that bowl has a role at an earlier stage in tarsal development. Therefore, the effects were investigated of bowl mutations on the expression of leg 'gap' genes that confer regional identity on the developing leg. Several of these genes have altered expression in bowl mutant cells. For example, bric-a-brac2 is normally expressed in the central part of the tarsus domain but expands into distal and proximal regions in bowl clones. Conversely, ectopic bowl leads to a reduction in bric-a-brac2, with a concomitant expansion of proximal (t1) and distal (t5) tarsomere fates. The bowl gene is therefore required for the elaboration of pattern in the tarsus and its effects suggest a progressive model for the determination of P/D identities. This mechanism might be important in the diversification of arthropod limbs, because it explains how segmented tarsomeres could have arisen from an ancestral limb with an unsegmented tarsus (de Celis Ibeas, 2003).

A second study has independently shown that the odd-skipped gene family as a key group of genes that function downstream of the Notch receptor to promote morphological changes associated with joint formation during leg development. odd, sob, drm, and bowl are expressed in a segmental pattern in the developing leg, and their expression is regulated by Notch signaling. Ectopic expression of odd, sob, or drm can induce invaginations in the leg disc epithelium and morphological changes in the adult leg that are characteristic of endogenous invaginating joint cells. These effects are not due to an alteration in the expression of other genes of the developing joint. While odd or drm mutant clones do not affect leg segmentation, and thus appear to act redundantly, bowl mutant clones do perturb leg development. Specifically, bowl mutant clones result in a failure of joint formation from the distal tibia to tarsal segment 5, while more proximal clones cause melanotic protrusions from the leg cuticle. Together, these results indicate that the odd-skipped family of genes mediates Notch function during leg development by promoting a specific aspect of joint formation, an epithelial invagination. Since the odd-skipped family genes are involved in regulating cellular morphogenesis during both embryonic segmentation and hindgut development, it is suggested that they may be required in multiple developmental contexts to induce epithelial cellular changes (Hao, 2003).

Animal limbs develop as outgrowths from the main body axis that acquire proximal/distal (P/D) patterning to form a series of specialized skeletal structures. These structures are articulated and so one key consequence of P/D patterning is the establishment of joints between each skeletal element. In the Drosophila leg, the P/D axis is established through the combined activities of Wingless (Wg) and Decapentaplegic (Dpp), which intersect in the centre of the limb primordium. Wg and Dpp together induce the expression of Distal-less (Dll), a homeodomain protein required for the development of all distal leg structures, and Dachshund (Dac), a nuclear protein required for intermediate leg segments (femur and tibia). By the beginning of the third larval instar, the leg primordium is therefore subdivided into at least three regions. Subsequent patterning involves interactions between the factors expressed in these early territories. For example, several genes are required for the development of the tarsus, including rotund and bric-a-brac. Expression of these genes is promoted by Dll and restricted proximally by the combined activities of Dac and distally by a gradient of epidermal-growth-factor-receptor signalling. By the stage that a series of P/D regions have been established, further patterning appears to be independent of the initial inducers Wg and Dpp. However, it is not clear how these P/D regions are elaborated (for example, to give diversity to the distal tarsal structures) (de Celis Ibeas, 2003 and references therein).

A final stage in translating the P/D patterning into the definitive segmented structure of the insect adult leg is the formation of the inter-segmental joints. The leg consists of six true segments or podites (coxa, trochanter, femur, tibia, tarsus and pretarsus), which are independently moveable by muscles. In Drosophila, the tarsus is further subdivided into five tarsomeres (t1-t5), which have distinct characteristics but lack independent musculature. Development of both 'true' joints and inter-tarsomere joints requires Notch activity, shown by the loss of joints and fused segments in Notch mutant cells, and by the ectopic joints that are formed when extra sites of Notch activity are engineered. Consistent with its pivotal role in specifying joint development, Notch activity is detected at all segment/subsegment boundaries at the end of larval development, using transcription of the Enhancer of split target genes as a measure. However, expression of Notch ligands is first observed at a subset of locations at a much earlier stage shortly after the initial 'regional' domains of gene expression are established. There are two explanations for this. One is that the specification of joints occurs sequentially, with some joints being determined early and others (e.g., tarsomere joints) much later. Alternatively, Notch activity might have both earlier roles in P/D regionalization and patterning and later roles that build on these earlier events to establish the segmental boundaries and joints at the correct locations (de Celis Ibeas, 2003).

To investigate further the mechanisms involved in P/D limb development, genes were sought whose expression is dependent on Notch activity. Analysis of these genes could allow discovery whether they have roles in the initial P/D patterning as well as in the subsequent establishment of joints. The zinc-finger protein encoded by the gene bowl is detected at a subset of sites of Notch activity and its expression is dependent on Notch. The bowl gene is closely related to the segmentation gene odd-skipped, and is required for development of the embryonic hindgut. Analysis of bowl and odd-skipped function in the developing leg indicates that these genes are involved in the elaboration of pattern in the tarsus, leading to the proposal that Notch is important for patterning as well as for joint formation. The effects of Bowl on tarsal development suggest that P/D tarsal identities are determined progressively and might also explain how different numbers of tarsomeres could have arisen from an ancestral limb that is thought to have contained an unsegmented tarsus (de Celis Ibeas, 2003).

Thus the genes bowl and odd are involved in a novel aspect of this process that elaborates the pattern within the tarsus to generate the correct number and structural diversity of the tarsomeres. Mutations in bowl or odd cause cells at the proximal and distal positions in the tarsal region to acquire fates of more centrally placed cells, giving rise to truncated or fused tarsomeres. Conversely, ectopic Bowl leads to a transformation of central fates to more proximal or distal fates, again causing distortions and truncations of the tarsus. The changes in fate are manifest in the expression patterns of genes such as bab1 and bab2, which are normally present at the highest levels in t3/t4 tarsomeres and at lower levels in t2 and t5. Absence of bowl leads to elevated Bab2 levels in t2 or t5 and to expression in proximal regions (t1), where bab2 is normally silent. One notable feature of Bab1/Bab2 expression is that it is modulated into rings of higher and lower expression. This modulation is also partially lost in bowl mutant clones (and in Dac mutants), arguing that bowl is intimately associated with the elaboration of patterning (de Celis Ibeas, 2003).

Previous studies have shown that bab1/bab2 expression is promoted by Dll and that its proximal and distal limits are dependent on Dac proximally and on epidermal-growth-factor-receptor signalling distally. It is proposed that these activities not only define the initial domain of bab1/bab2 expression but also indirectly regulate the production of Bowl and Odd through their effects on Notch-ligand expression. Bowl is then necessary to fine tune bab2 expression so that its levels are low or absent in the extremities of the tarsus, allowing these to adopt t1 and t5 characteristics. If one of the factors responsible for positively regulating bab1/bab2 expression was present transiently, its decay would also contribute to the gradation in Bab2 expression and could explain why Bab2 is not turned on in the t1 cells that have lost Bowl at late stages (de Celis Ibeas, 2003).

The effects of Bowl and Odd on tarsal development were initially difficult to reconcile with their expression. In late stages of limb development (late L3/early pupal), the proteins are present only at sites of Notch activity outside the tarsus, not within the tarsus, even though the most obvious phenotypes are tarsomere fusions. All of the sites of expression are precursors for the 'true' joints (those with tendon attachments and direct muscle control), suggesting that Bowl/Odd could have a primary role in the establishment of joints and that the regulation of tarsal patterning has been acquired secondarily. It is proposed that effects on patterning occur because the proximal and distal parts of the tarsus are formed by cells that synthesize Bowl/Odd at an earlier stage and that the levels of Bowl/Odd determine the extent of tarsal gene expression. When the tarsus is first defined by the expression of bab, Bowl/Odd directly flank this domain. As the tarsus expands, Bowl and Odd are only retained at the boundary and are lost from the intervening cells; as a consequence, bab2 is derepressed. In this way, cells closest to the initial domain of Bab2 expression would contain Bowl/Odd for the least time and therefore have higher levels of Bab2 than those closer to the tibial boundary. A similar relationship between expression and phenotype has been seen with drumstick (drm; a gene related to bowl and odd that is required for hindgut morphogenesis). At late embryonic stages, drm expression is detected only in the most anterior cells of the small intestine, even though it influences cell behaviour along the whole length of the intestine. By tracing earlier phases of expression, it has been shown that drm is transiently expressed more broadly and gradually becomes restricted to the anterior hindgut boundary (Green, 2002), which is similar to what was observed for odd-lacZ expression in the leg. It is possible that these similarities in drm, odd and bowl regulation reflect a common underlying mechanism conserved between hindgut and leg morphogenesis (de Celis Ibeas, 2003).

Notch activation appears to be one key factor in promoting the accumulation of Bowl and Odd at the tarsal boundaries, but some data indicate that other factors are required and that the regulation might be indirect. (1) Bowl and Odd can only be induced at a subset of the locations where Notch is active, so Notch alone is not sufficient. (2) Although all Notch clones at the t5/tibia boundary result in a loss of Bowl protein, not all clones at the more proximal boundaries have a phenotype. Because the smaller clones tend to have the least effect on Bowl, Notch appears to initiate but not to maintain Bowl expression at these locations. (3) Although regulation of odd can be fully explained by its effects on transcription, Bowl might be subject to post-transcriptional regulation. When expression of bowl mRNA is driven through the leg (using GAL4 drivers), only low levels of Bowl protein are detected, at best, within the tarsus, suggesting that the translation or the stability of the protein are regulated. Candidates to participate in Odd and Bowl regulation include Spineless and Lines, a protein that acts antagonistically to Bowl and Drm in hindgut morphogenesis. Although the combined actions of Notch and these factors might explain the initial expression of Bowl and Odd, the mechanism that maintains their expression specifically at the boundaries of the tarsus is unclear. This aspect of regulation is crucial for the diversification of the tarsomeres and, if this model is correct, would be linked to proliferation. It is predicted that tarsal cells should show a bias in their patterns of proliferation, as is the case in more proximal regions of the leg, and that the progeny of Bowl-expressing cells should occupy the t1/t2 and t5 tarsal segments. It has not yet been possible to specifically monitor the proliferation pattern and fate of Bowl-expressing cells to test these predictions (de Celis Ibeas, 2003).

One extrapolation from the proposed model for tarsal development in Drosophila is that the basal or ancestral state consisted of a single tarsal segment, specified by uniform levels of Bab and directly flanked by sites of Bowl expression prefiguring the tarsal/tibial and tarsal/pretarsal joints. This is in agreement with the phylogenetic evidence, which points towards the ancestral arthropod limb having an unsegmented tarsus (as remains the case for many modern arthropods, including some insects). Furthermore, there is considerable variation in the extent of tarsal subdivision, with most insects having between two and five tarsomeres (some arachnids have further subdivisions). These differences in pattern could be explained by differences in either the duration or the rate of proliferation during the crucial phase when bowl/odd influence bab2 patterning. Although mutations in Notch or bowl/odd affect the extent of tarsal proliferation, as do mutations in spineless and bab2, none of these activities alone is sufficient to cause an increased length of the tarsus (although ectopic Notch activity can give ectopic outgrowth). Further investigation of how these factors combine to coordinate tarsal patterning and proliferation should help in the unraveling of the mechanism underlying the diversification of arthropod limb structure. Furthermore, as modifications of bab2 expression are correlated with diversification of pigmentation and trichome patterns in Drosophila species, the possibility that bab2 expression is intrinsic to diversification of tarsal patterning suggests that changes in the regulation of a single gene could contribute to the diversification of many different morphological traits (de Celis Ibeas, 2003).


Transcriptional Regulation

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).

Hedgehog promotes Bowl protein accumulation by promoting drm expression, while Wingless antagonizes Hedgehog function and Bowl accumulation by repressing drm expression

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).

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

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).

Groucho interaction with Engrailed homology 1 (eh1) proteins

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).


Transcripts of the bowel gene initially accumulate at the cellular blastoderm stage in three domains that are more terminally located than the odd and sob stripes; these include a strong cap at the posterior pole (from 0 to 11% EL), a relatively weak and nonuniform domain at the anterior pole (84%-100% EL), and a broad transverse stripe (approximately 6 cells wide) that lies just anterior to the presumptive cephalic furrow (extending from 76% EL dorsally to 67% EL ventrally). While some cells in these regions may also express odd and sob (e.g., the anterior cap of odd partially overlaps the anterior bowl domain, as do the anteriormost secondary stripes of odd/sob and the bowl cephalic stripe), the overall patterns have little in common. Therefore, the initial regulation of bowl is clearly distinct from that of sob and odd. However, at early gastrulation, the terminal expression of bowl is supplemented by transverse stripes in the trunk primordium. As with odd and sob, these initially appear in a pair-rule pattern of seven stripes, but rapidly evolve to a segment polarity-like pattern as the number of stripes doubles. The intensity of these stripes is very low compared to the terminal domains and the single cephalic stripe, suggesting that bowl may retain only vestigial regulatory elements in common with odd and/or sob (Hart, 1996).

The embryonic expression patterns of the Drosophila odd-family genes can be placed into two classes, one exemplified by drm, and the other by bowl. The three family members that map closest to one another, drm, sob, and odd, display striking similarities in patterns (but not levels) of mRNA expression. Expression of these genes begins in seven pair-rule-like stripes at the cellular blastoderm stage; these then resolve into 14 stripes (Green, 2002 and Hart, 1996). drm, sob, and odd are also expressed in the invaginating stomodeum and proctodeum at their respective junctions with the midgut primordia. By stage 13, drm, sob, and odd are expressed in the keyhole region that will form the proventriculus, and in a wide ring at the most posterior portion of the midgut (Green, 2002 and Hart, 1996). Their expression continues in these regions through the remainder of embryogenesis (Johansen, 2003b and references therein).

bowl expression is overall quite different from that of drm, sob, and odd; in contrast to the spatially localized expression of drm, sob, and odd, the expression of bowl is relatively uniform throughout the anlagen, primordia, and epithelia of the foregut and hindgut (Wang, 1996). By stage 13, expression of bowl in the hindgut is greatly reduced, but is maintained strongly throughout the foregut, where it remains high until stage 17 (Johansen, 2003b).

odd expression during leg development has previously been defined by an embryonic lethal enhancer trap insertion in the odd locus, oddrK111 (Rauskolb, 1999). To confirm that this expression faithfully represents the endogenous gene expression pattern, in situ hybridization to leg discs using an odd probe was performed. Moreover, since the odd-skipped family genes are segmentally expressed during embryogenesis, attempts were made to determine their expression during leg development and how this expression compares with that of odd. During development, the leg disc ultimately gives rise to a cylindrical adult leg, and thus the expression of genes in a segmental pattern is observed as a reiterated pattern of concentric rings in the leg imaginal disc. All four odd-skipped family genes are expressed during leg development in segmentally repeated patterns; the expression profiles of odd, sob, and drm are identical, while that of bowl is distinct (Hao, 2003).

odd, sob, and drm are expressed in a narrow band of cells at the distal edge of each presumptive leg segment, except in tarsal segments 1-4. Robust expression of drm is consistently observed, while both odd and sob are expressed at lower levels. Endogenous expression of odd is identical to that previously observed for oddrK111. Comparison of drm and sob expression to that of oddrK111-driven ß-galactosidase expression has confirmed that all three genes are expressed in the same cells. Expression of drm was further characterized at different stages of leg development. In early third instar discs, expression is first observed in a proximal ring, corresponding to the presumptive coxa. As development proceeds, progressively more distal rings of drm expression are added, until the full complement of five segmental rings is observed by the end of larval development. This progressive increase in the number of rings of drm expression is similar to the previously observed (Rauskolb, 2001) progressive increase in Notch ligand expression in the leg (Hao, 2003).

Notch activation occurs in a narrow stripe of cells within each presumptive leg segment, just distal to cells expressing the ligands Serrate and Delta. The expression of oddrK111 (and thus presumably also odd, sob, and drm) was compared with that of Serrate and Delta to determine whether odd-driven ß-galactosidase expression is observed in cells in which Notch is activated. Indeed, oddrK111 expression was observed in cells directly adjacent, and distal, to ligand-expressing cells. Thus, odd (and drm and sob, as well) might be direct targets of Notch activation. In support of this notion, ectopic activation of Notch within the leg induces the expression of oddrK111, drm, and sob (Rauskolb, 1999). Further, expression was reduced in cells along the anterior-posterior (A-P) axis of the leg when Notch activity was inhibited in a stripe of anterior cells at the A-P compartment boundary by expression of the extracellular domain of Notch, which acts as a dominant-negative receptor (Hao, 2003).

bowl is also segmentally expressed in the developing leg, although its expression is not as restricted as that observed for the other three genes. bowl is expressed in a broad stripe within each presumptive leg segment, including tarsal segments 1-4, in a pattern that appears overlapping with, but also broader than, that of odd, sob, and drm. The bowlk08617 enhancer trap is also expressed in developing leg discs, in both larval third instar and pupal discs, in a pattern similar to that observed for the endogenous bowl transcripts (Hao, 2003).

Taken together, these results indicate that the odd-skipped family genes are candidate effectors of the influence of Notch on Drosophila leg segmentation. However, given that odd, sob, and drm are expressed identically, they may act redundantly in this process. bowl, in contrast, may act independently, especially within the distal leg, as it is expressed more broadly and is the only gene family member expressed within tarsal segments 1-4 (Hao, 2003).

An antennal-specific role for bowl in repressing supernumerary appendage development in Drosophila

In Drosophila, antennae and legs are serially homologous appendages, and yet they develop into organs of very different structure and function. This implies that different genetic mechanisms operate onto a common developmental ground state to produce antennae and legs. Still few such mechanisms have been uncovered. During leg development, bowl, a member of the odd-skipped gene family, has been shown to participate in the formation of the leg segmental joints. This study reports that, in the antennal disc, bowl has a dramatically different role: bowl is expressed in the ventral antennal disc to prevent inappropriate expression of wg early during development. The removal of bowl function leads to the activation of wg in the dpp-expressing domain. This ectopic expression of wg, together with dpp, results in a new proximo–distal axis that promotes non-autonomous antennal duplications. The role of bowl in suppressing a supernumerary PD axis is maintained even when the antennal disc is homeotically transformed into a leg-like appendage. Therefore, bowl is part of a genetic program that suppresses the formation of supernumerary appendages specifically in the fly’s head (Brás-Pereira, 2008).

In Drosophila, antennae, mouthparts, legs and genitalia are considered to be serially homologous ventral appendages. This means that despite their very different structure and function, they are thought to develop from a common developmental ground state. It is the segment-specific selector gene expression that, acting upon this ground state, defines their specific morphologies. Of these ventral appendages, the development of the leg is best understood. The leg primordium is set aside as a cluster of epidermal cells, composed of a distal population, that expresses Distal-less (Dll) and a proximal one, expressing homothorax (hth), teashirt and escargot. This early genetic subdivision corresponds to the proximo–distal (PD) telopodite–coxopodite subdivision of the insect appendages. hedgehog (hh), expressed by posterior cells within the leg primordium, triggers the expression of the decapentaplegic (dpp) and wingless (wg) signaling molecules in anterior cells which, through mutual repression, become expressed in a dorsal and a ventral wedge, respectively. wg and dpp expressions only coincide at the center of the leg disc and it is this confluence of maximal signaling that defines the distal tip of the future leg and triggers growth. The larval development of the leg primordium –called leg imaginal disc – progresses by the successive definition of intermediate domains of gene expression that specify the segments of the leg (coxa, trocanter, femur, tibia and tarsus) are defined. During late larval life, leg development becomes wg/dpp-independent, and the distal disc tip becomes a source of EGFR signaling, which is responsible of the further segmentation of the tarsus into the five tarsomeres and the terminal claw. Growth and segmentation of the leg also depends on Notch signaling. Activation of Notch by its ligands Delta (Dl) and Serrate (Ser) is necessary for the disc to grow, and the overlapped expression of Dl and Ser in concentric rings defines the position of the joints of the leg segments as the cells immediately distal to these rings (Brás-Pereira, 2008 and references therein).

The odd-skipped family of genes, odd-skipped (odd), drumstick (drm) and sister of odd and bowl (sob) are among the Notch targets in legs. These genes are expressed in concentric rings at the prospective leg joints, just distal to the Dl/Ser ring domains. A fourth member of the family, brother of odd with entrails limited (bowl), has a more widespread expression pattern. Genetic data indicate that bowl is required for the segmentation of the leg, and that the localized co-expression of the other family members allows (probably in a redundant fashion) the activation of bowl at the prospective joints. Further molecular and genetic experiments show that, at least during embryogenesis, the product of the gene lines blocks bowl function by directly binding to Bowl and preventing its nuclear accumulation. Drm and likely Odd are able to competitively displace Lines from Bowl, thus allowing Bowl to become nuclear and functional (Brás-Pereira, 2008 and references therein).

The distinct antennal development is promoted by the distal maintenance of hth expression in the antennal disc, resulting in the co-expression of hth and Dll. This co-expression selects the antennal fate. Compared to the leg, the antenna is a much shorter appendage, with four segments (antennal (a) segments 1–3, plus a distal arista), and functions in olfaction, through the specialization of its a3 segment. The antennal disc does not develop as an independent disc, like the leg one, but forms part of the eye–antennal disc complex. This disc comprises cells derived from several embryonic head segments and the unsegmented acron. All the cells of the eye–antennal disc complex express the Pax6 genes eyeless (ey) and twin-of-eyeless during first larval stage (L1), but during L2, only the posterior two-thirds of the complex express Pax6 genes, while the anterior third expresses cut (ct). The L2 ct and Pax6 domains correspond to the antennal and eye discs, respectively. The smaller size and fewer segments of the adult antenna when compared to the leg correlate with a different expression of the Dl and Ser ligands in antennal and leg discs. Accordingly, the antennal disc has only two odd-expressing rings, instead of the six present in leg discs. The different control of growth and segmentation in the antenna indicates that there must be mechanisms operating differently in antennal and leg discs (Brás-Pereira, 2008 and references therein).

The fact that bowl has been placed downstream of Notch signal in the elaboration of distal leg patterning prompted a test of whether bowl had any function during antennal development, and if it did, whether it was similar to its role during leg segmentation. The results indicate that, during antennal disc development, bowl has a dramatically different role: bowl is expressed at early stages in the ventral antennal disc, where it prevents inappropriate expression of wg. If bowl is removed, the activation of wg results in non-autonomous antennal duplications. bowl is still required to prevent PD axis duplication in homeotically transformed antennal discs, which indicates that there are genetic differences between head and thorax discs that are selector gene independent (Brás-Pereira, 2008). .

During the development of the antennal disc, bowl has two phases of expression: an early expression in the ventral disc, required to maintain wg repressed, and a later one in concentric rings. Both phases have antennal-specific properties. The early bowl expression and function is unique to the antenna. Its expression in rings associated to prospective joints, which recapitulates the ring expression in leg discs, does not seem required for joint formation in the antenna, contrary to what has been described in the legs. In addition, bowl is still required to repress a ventral supernumerary PD axis even if the antenna has been homeotically transformed into a leg-like appendage by overexpression of the leg selector Antp. All these results indicate that the development of the head structures deriving from the antennal disc depends not only on the activity of selector genes, but also on a cephalic-specific genetic program. Supporting this claim, it was found that the expression of eyg, an antennal-specific marker, is maintained in homeotically transformed antennal discs (Brás-Pereira, 2008).

These cephalic vs. thoracic differences might reflect the very different developmental histories of antennal and leg discs. While each leg disc primordium is formed from cells derived from just two adjacent parasegments (or one embryonic segment), the antennal disc is part of a composite disc, the eye–antennal disc, which forms by the fusion of imaginal primordia derived from several embryonic head segments [the labial, antennal, intercalary, mandibular and maxillary segments plus the unsegmented acron. The coalescence of all these primordia in a single imaginal disc might have required the repression of some domains of gene expression carried along by precursor cells. In this sense bowl might have been recruited to block wg expression in the ventral cells of the antennal disc during the early stages of its development. The lack of bowl at this stage would release wg expression which, in turn and with dpp, would trigger the development of a new appendage (Brás-Pereira, 2008).

The repressive function of bowl might extend to other parts of the eye–antennal eye disc. bowl minus clones in the ventral region of the stem that connects the antennal and eye disc lobes develop autonomously into eye tissue. In contrast to the antennal suppressing function, bowl is required autonomously to repress eye development. This autonomy indicates that either the signals normally operating to spread retinal differentiation in the normal eye are not produced in these ectopic retinal patches, or that the wild type tissue is refractory to these signals. At present, no choice can be made between these two hypotheses. It was noticed, however, that the overexpression of the bowl inhibitor Lines driven by the dpp-GAL4 driver leads to two phenotypic outcomes: antennal duplication or ectopic ventral eyelet. Interestingly, only in one case out of more than 20 discs analyzed these two phenotypes co-occurred. This suggests that the cells in the sensitive region adopt collectively only one two fates, antenna or eye, and that deciding upon one excludes the other. Since wg normally acts by limiting the eye field, eye fate might be blocked in those ventral bowl minusclones derepressing wg. In addition, it is noted that this ct, ey-expressing region is particularly prone to develop into eye upon genetic perturbations. For example, it is this region that is preferentially transformed into eye when hth function is removed or when tsh is ectopically expressed. Perhaps, the unique fact that this region co-expresses antennal and eye determinants makes its fate more ambiguous. In the absence of bowl, hth might tilt the equilibrium towards head capsule or antennal development, while the opposite fate – eye – would be adopted in the presence of tsh and ey. It will be interesting to determine whether functional relationships between bowl and these factors exist to determine specific fates within the eye disc (Brás-Pereira, 2008).

Mechanistically, bowl function seems to lie downstream of hh and dpp. In bowl minus cells associated with an antennal duplication, hh is still expressed and the Hh-coreceptor patched is normally up-regulated in anterior cells abutting the hh-expressing domain, which indicates correct hh-signaling. Accordingly, wg derepression in bowl minus cells occurs closest to the P cells, as expected for a hh target gene. In the embryo, bowl has also been placed downstream of hh during the process of epidermal differentiation (Brás-Pereira, 2008).

In the antenna, as in the leg disc, the dpp and wg signaling pathways repress each other to establish two opposing wedges of dpp and wg expression. In bowl minus clones, though, dpp expression, monitored by a lacZ-expressing reporter, is not turned off, despite the induction of wg expression ventrally. Although this might be due to the perdurance of the LacZ product, bowl minus cells accumulate normal levels of phosphorylated-Mad. This indicates that bowl-mutant cells transduce the dpp signal. Therefore, these results suggest that bowl is required for the mutual repression of wg and dpp in the ventral portion of the antennal disc. Nevertheless, bowl is not sufficient to repress wg in the antenna. Simple explanations for this fact have been ruled out, such as low levels of the induced Bowl protein, or its retention in the cytoplasm. This insufficiency is not due to the inhibition by Lines, because even in the presence of Drm, which prevents Lines from binding to Bowl, this latter is still unable to repress wg. Although further work is required to identify which other factor or factors collaborate with bowl during ventral antennal disc development, the simplest explanation would be that Bowl acts in concert with a factor induced by dpp. This is because bowl cannot block the ectopic wg expression in ventral antennal cells devoid of dpp signal. Nevertheless, when bowl expression is forced in the leg disc using the ptc-GAL4 driver, wg is repressed by bowl cell-autonomously in the most distal region of the disc, but not in the more proximal domain. This result strengthens the idea that bowl acts as a wg repressor. Such repression takes place in the distal part of its domain, closest to the dpp source, which also supports the claim that bowl requires the dpp signaling to repress wg (Brás-Pereira, 2008).

This study has shown that bowl is expressed in the ventral antennal disc, the realm of the dpp pathway, and that dpp signaling can activate bowl transcription in this disc. These results suggest that high levels of dpp induce bowl which, in turn, is required to prevent inappropriate expression of wg in the antennal disc together with the dpp pathway. Two are the likely sources of Dpp: the wedge of dpp that can be visualized using the dpp-disc enhancer reporters in the antenna, and a ventral disc expression that is controlled by a separate enhancer. This enhancer drives dpp expression in the prospective ventral head region, close to the region where bowl is transcribed in early discs (Brás-Pereira, 2008).

bowl and the related genes odd and drm show a late pattern of expression in rings, similar to the one deployed in leg discs. But contrary to their requirement for leg segmentation, bowl seems to be dispensable for antennal segmentation. A similar situation has been described for the gene dachshund (dac). dac is expressed in the medial segment of both leg and antennal discs, but while loss of dac in the leg leads to the loss of intermediate adult leg structures, the antenna develops normally. These results might reflect the fact that, although antennal and leg discs have specific developmental programs, the mechanisms for generating the PD axis are shared by both appendages. This mechanism would call a similar battery of genes, even if only a subset of them is effectively used for the development of each appendage. In fact, ectopic activation of the Notch pathway by overexpression of the ligand Delta induces ectopic expression of drm in the antenna. This indicates that, similarly to what happens in the leg discs, ring expression of Odd-family genes in the antenna might also be under Notch control. In this sense, in the antenna the segmentation function might have been taken over by other(s) member(s) of the Odd family, expressed as well in the future joints (Brás-Pereira, 2008).

In summary, the results show that the zinc-finger encoding gene bowl is part of a cephalic-specific program that represses appendage formation in the ventral eye–antennal disc. Here, bowl is required to repress wg, downstream of dpp, to prevent the generation of supernumerary antennae. These extra appendages might arise from some silenced primordium in the proximal part of the antenna, which would be normally fated to become part of the head capsule. In addition, bowl also silences the development into eye of another cell population of the prospective head that presents mixed expression of antenna and eye selector genes. The repressive action of bowl that is described here might have been essential for the coalescence of cells deriving from several different embryonic cephalic segments into a single imaginal disc, as well as for the formation of the head structures of adult cyclorraphan flies, such as Drosophila (Brás-Pereira, 2008).


Bowl is required downstream of Notch for elaboration of distal limb patterning

In the developing leg, Notch mutant cells result in fused leg segments, owing to the absence of joints, and in severely reduced growth. Accumulation of pigmented tissue also occurs at joints in proximal regions. Mutations in bowl result in similar phenotypes; the mutant cells are associated with fusions and truncations of tarsal segments as well as with melanotic patches at the proximal joints. Because the gene is essential at earlier stages in development (bowl mutant embryos do not hatch; Wang, 1996), it is difficult to determine the consequence of completely eliminating bowl in the leg. However, when the mutant clones cover most of the distal part of the leg, the limb is severely truncated, with little or no segmentation/joint tissue evident. Conversely, clones that are restricted to the central part of the tarsus often fail to result in a detectable phenotype (de Celis Ibeas, 2003).

The bowl gene encodes a zinc-finger transcription factor and is closely related to odd-skipped, a gene that has already been implicated in leg segmentation (Wang, 1996; Rauskolb, 1999). Phenotypes produced by odd mutant cells were examined and defects similar to bowl were found -- segmental fusions and truncations in the tarsal region, and melanotic patches in proximal joints. The two genes therefore have similar but essential roles in leg segmentation, although their functions elsewhere appear to be distinct (Wang, 1996; de Celis Ibeas, 2003).

Neither Bowl nor Odd appear to be present within the tarsus (Rauskolb, 1999), yet mutations in either gene produce defects in this part of the leg (fusion of tarsomeres and growth defects). There are three models to explain this. (1) Bowl and Odd influence tarsomere segmentation indirectly, by regulating production of a long-range signal. (2) The two genes are expressed at intertarsomere joints but at a level too low to be detected. (3) They are involved in an earlier patterning event that influences subsequent tarsal segmentation. The first hypothesis can be ruled out, because fusions only occur within tarsal bowl clones and the effects on downstream genes are autonomous. Although it is difficult to rule out the low levels of tarsal expression implied by the second model, the data argue that this is an unlikely explanation for several reasons. (1) bowl clones that spanned only intertarsomere joints (e.g. t2/t3/t4) appeared normal. Almost all clones that resulted in observable phenotypes spanned the tibia/t1 boundary and, even within these clones, there was considerable variation in the number of tarsomere joints affected. This suggests that the effect on tarsomere joints is a secondary consequence of the mutations. (2) In the case of Odd, the pattern of expression detected in the larva persists throughout pupal leg morphogenesis (Mirth, 2002), ruling out later expression in the tarsomeres. (3) When high levels of bowl mRNA were produce using GAL4/UAS system, very little protein is detected in the tarsal domain of late-third-instar/early-pupal legs, suggesting either that the protein is unstable or that the mRNA is poorly translated in this region (de Celis Ibeas, 2003).

To investigate the third model (that bowl has an early patterning function in the leg), two questions were asked. (1) Whether E(spl)mß-CD2 expression in t2-t4 is affected by bowl mutations was tested, as predicted if Bowl acts prior to tarsomere boundary formation. As in the adult legs, there is considerable variation between clones, but clear disruptions to E(spl)mß-CD2 were observed in three out of nine clones. These defects are confined to the clone but do not strictly follow clone boundaries because some of the mutant cells retain wild-type levels of E(spl)mß-CD2 even in the most severe cases. This demonstrates that bowl function precedes Notch activation at the tarsomere boundaries and supports the hypothesis that its effects on tarsomere boundaries are secondary (de Celis Ibeas, 2003).

(2) It was asked whether mutations in bowl alter the expression of genes involved in the initial regionalization of the leg. Several genes have been identified that confer distinct regional identities and are expressed in broad domains within the leg disc. These include dachshund (dac), which is expressed in more proximal regions including t1, the two genes of the bric-a-brac (bab) complex (bab1 and bab2), which are expressed in the presumptive tarsal region, and two Bar genes that are expressed in distal tarsal segments t4 and t5 (BarH1 and BarH2). Expression of all three types of regional genes are affected by mutations in bowl. In late-third-instar/early-pupal leg discs, Bab2 expression normally extends to the proximal edge of t1. In bowl clones, ectopic Bab2 is detected in proximal parts of t1 and the levels in t2 are also altered. Conversely, when these clones also encompass the distal part of Dac domain, there is a reduction in Dac. Mutant clones that lie at the distal side of the tarsal domain again show derepression of Bab2 (in distal t5, where Bab2 expression is low or absent), and this is coupled with a reduction in the levels of BarH1. In all cases, the effects are autonomous to the clone and precisely follow clone boundaries (de Celis Ibeas, 2003).

It is therefore concluded that Bowl regulates the expression of patterning genes, promoting development of the proximal (t1/t2) and distal (t5) extremities of the tarsus. Thus, bowl mutations lead to an expansion of a 'central tarsal fate' that is characterized by uniform Bab2 and decreased BarH1 and Dac. This disruption in tarsal patterning would in turn affect the expression of Notch ligands in the tarsus and hence lead to defects in tarsomere joints, as seen in the disruption of E(spl)mß-CD2. However, there is still a discrepancy between the apparent function of Bowl and its site of expression: Bowl is necessary to inhibit/lower Bab2 expression in t1/t2 and t5, but is not present in these regions in late-third-instar/early-pupal discs. Nevertheless, the effects of bowl mutations on Bab2 and Dac are strictly cell autonomous. These observations can be reconciled if Bowl (and likewise Odd) is expressed within the cells that give rise to t1/t2 and t5 at early stages when the domains of the regional genes (bab, dac and Bar) are first established. This expression must subsequently disappear from these regions and become restricted to the boundaries of the tarsus (de Celis Ibeas, 2003).

If Bowl and Odd regulate tarsomere segmentation via effects on regional genes like bab2, they should be expressed at the boundaries of the Bab2 domain during early stages. At ~76-80 hours, both Bowl and Odd are first detected in a 2-3-cell-wide ring that surrounds the Bab2 expressing cells and corresponds to the proximal edge of the Dll domain and the distal edge of the Dac domain. Most of these Bab2-expressing cells also express BarH1; on the proximal side only a 1-2-cell-wide ring contains Bab2 and not BarH1. At this stage, therefore, the tarsus consists primarily of one identity, which has Bab2 and BarH1 expression and appears to approximate to t4. This early domain is surrounded by cells expressing Bowl and Odd (de Celis Ibeas, 2003).

Subsequently, a further ring of Bowl and Odd-expressing cells appears in the centre of the Bab2 domain, at the boundary with the pretarsus, and Bab2 is rapidly lost from within this ring. Bab2 is now flanked both proximally and distally by Bowl/Odd. At later stages, gaps appear between Bab2 and the flanking rings of Bowl/Odd. These gaps expand and, at the same time, Bab2 expression becomes more graded, with decreasing levels at the edges of its expression domain. This is most marked in the proximal (t2) direction, and is even more evident with Bab1 than with Bab2. Dac expression also extends distally beyond the proximal ring of Bowl/Odd so that it occupies t1 and a small part of t2 by the time the leg disc everts. As a consequence, a series of distinct territories is established within the tarsus by late third instar. Bowl/Odd mark the extreme (tibia/t1 and t5/pretarsal) boundaries of the tarsus and Bab2 expression spans t2-t5 with the peak of its expression in t3/t4 (de Celis Ibeas, 2003).

Both the expression patterns and the phenotypes suggest that cells within the t1/t2/t3 and t5 regions of the tarsal domain contain Bowl/Odd at 76-86 hours. It is proposed that Bowl/Odd expression is gradually lost from the tarsal cells as they proliferate, giving rise to a temporal gradient of Bowl/Odd (prolonged expression in t1, shorter period of expression in t3/t2). If this is the case, expression from the odd-lacZ line might be visible within t1/t2/t3 because of the endurance of ß-galactosidase. Indeed, in 96-hour-old odd-lacZ discs, ß-galactosidase is detected at lower levels within many cells of the tarsus. A temporal gradient cannot definitively be shown by this method, but the expression of odd-lacZ is most persistent close to the final domain of Bowl and Odd, consistent with this model (de Celis Ibeas, 2003).

Because bowl mutations result in expansion of 'central tarsal' (t3/t4) fates, it was anticipated that persistent expression of Bowl within the tarsus would have the converse effect, expanding proximal (t1/t2) and distal (t5) fates. To test this, GAL4 driver lines were used to direct expression of UAS-bowl within the tarsus, scoring phenotypes in the adult male pro-thoracic legs, using the sex comb as a marker of t1. Expression of UAS-bowl throughout the tarsal region (Dll-Gal4) gave rise to legs with expanded t1 fates manifest in the ectopic sex-combs on distal tarsal segments. In the more strongly expressing lines, the tarsus became severely distorted and carried sex-comb bristles throughout its length. Even with more restricted production of Bowl (e.g. klumpfuss-GAL4), similar transformations occurred, with ectopic sex-comb bristles present in t2 and t3 (de Celis Ibeas, 2003).

To determine whether expansion of t1 fates occur at the expense of 'central tarsal' fates, the effects of ectopic Bowl on Bab, Dac and BarH1, were assayed. In leg discs from Dll-GAL4/UAS-bowl, levels of Bab2 were strongly reduced and more patchy than in the wild type, consistent with central tarsal identity being compromised. Conversely, the domains of Dac and BarH1 were extended so that they were almost contiguous in the middle of the tarsus, demonstrating the expansion of t1 and t5 fates. Ectopic expression of Bowl in a more restricted domain (e.g. with Ptc-Gal4) also reduced Bab specifically within the domain of ectopic expression. The inhibition of bab2 by Bowl fits with the phenotypes of bab/bab2 loss-of-function alleles, which are similar to that of ectopic Bowl (ectopic sex combs on distal segments). In analysing the levels of Bowl produced by the directed misexpression, it was noted that high levels of protein only accumulate close to the normal sites of expression. Elsewhere, such as within the tarsus, protein levels remain low and patchy, even though mRNA levels are fairly uniform throughout the domain of misexpression. Despite the low levels of protein, inhibition of Bab is still seen within the tarsus (de Celis Ibeas, 2003).

Further support for the role of bowl in patterning the proximal tarsus comes from analysing spineless mutant legs. This gene is essential for antenna development but is also expressed transiently in the tarsus of the early third-instar leg. The phenotype observed in weak spineless mutants (ssa/ss114) resembles that of ectopic Bowl (with ectopic sex combs in t2 and an ectopic joint within t1), and the domain of Bowl expression remains broader in spineless larval discs and ectopic patches of Bowl persist in t1 and t2 of early pupal discs. Sometimes, the ectopic Bowl forms a discrete ring within t1 that corresponds to the site of the ectopic joint. Persistent Bowl therefore alters P/D patterning, promoting t1-like fates and, in some cases, resulting in an ectopic tibia/t1-like joint. These data suggest that spineless is involved in keeping Bowl absent from in the tarsus. In agreement with this, ectopic Spineless results in loss of Bowl, although these conditions also result in transformation to antenna fates, complicating the interpretations. It is concluded that the translation or the stability of Bowl protein are regulated. Candidates to participate in Bowl regulation include Spineless and Lines, a protein that acts antagonistically to Bowl and Drm in hindgut morphogenesis (de Celis Ibeas, 2003).

The odd-skipped family of zinc finger genes promotes Drosophila leg segmentation

To assess the normal role of odd and its cognate genes sob and drm, during leg development, mutant clones were analyzed for those genes for which mutants are available. It was found that neither odd nor drm mutant clones had a detectable effect on leg segmentation or leg growth. Thus, odd and drm may not have a role during leg development, they may act redundantly, or their function may be masked by the presence of sob. Since neither a single mutation in sob nor a deficiency removing only odd, sob, and drm is available, the contribution of sob to leg development or potential redundancy amongst these genes could not easily be addressed. The Df(2L)drmP2 (Green, 2002), a large deficiency removing drm, sob, and odd, as well as about 30 other genes, appears to be cell lethal, as mutant clones could not be recovered in the adult leg (Hao, 2003).

Although determination of the requirement for odd, sob, and drm in leg segmentation is complicated by their potential redundancy, ectopic expression has the possibility to reveal whether any of these three genes is sufficient to promote aspects of segmentation in the developing leg. Moreover, if odd, sob, and drm are important downstream effectors of Notch activity, then ectopic expression of these genes should give phenotypes similar to those observed with ectopic Notch activation, namely induction of segmentation and/or tissue growth. To test the functional potential of odd, sob, or drm, UAS expression constructs were made, allowing for either patterned misexpression or generation of FLP-out clones during leg development, and the resulting phenotypes were examined in adult legs (Hao, 2003).

Importantly, ectopic expression of odd, sob, or drm can induce deep creases in the leg cuticle that resemble ectopic joint-like structures. The phenotype obtained upon ectopic expression of these genes along the A-P axis of the leg is similar to that observed with ectopic expression of a constitutively activated form of Notch. The ectopic expression of these genes was not associated with altered Notch ligand expression, and thus these genes appear to function solely downstream of Notch activation. In addition, broad misexpression of odd, sob, and drm resulted in tarsal segment fusions, as has also been observed with broad misexpression of activated-Notch. This result implies that, for normal leg development to occur, odd, sob, and drm must be expressed in a segmentally repeated pattern (Hao, 2003).

To address whether odd, sob, and drm behave cell autonomously, small patches of cells expressing the gene of interest were made using the FLP-out GAL4 technique. Significantly, FLP-out clones of odd, sob, or drm were each capable of inducing an indentation in the cuticle, in a largely cell-autonomous manner, and such alterations can occur anywhere along the proximodistal axis of the leg. In some instances, a crease in the cuticle was induced around the FLP-out clone, while in other instances, all the cells of the clone appeared to invaginate and form an indentation in the cuticle. In these latter instances, a lack of bristles was often found within the clone. One interpretation of these observations is that these cells are now fated, by virtue of their expression of odd, sob, or drm to become "joint-like" cells, which indent and do not make bristles. FLP-out clones of odd, sob, or drm never induce leg tissue outgrowths. By contrast, FLP-out activated-Notch clones or FLP-out four-jointed clones (another Notch target gene) do induce leg tissue outgrowths (Hao, 2003).

Together, these results indicate that the role of odd, sob, and drm during leg development may be to initiate cellular changes associated with joint formation. Since ectopic expression of these genes does not induce growth of the leg tissue, they appear to regulate only a subset of the functions downstream of Notch activation during leg development (Hao, 2003).

The formation of a joint is a complex process involving cell movements and changes in cell shape. To determine whether odd, sob, or drm might contribute to joint formation by altering cellular morphology, these genes were ectopically expressed along the A-P axis of the leg and cellular morphology was examined by staining with anti-E-cadherin or with phalloidin. Both of these reagents preferentially label the apical surface of cells, at the level of the adherens junctions. ptcGAL4 drives the expression of the gene of interest in a gradient within the anterior compartment, with the highest levels of expression at the A-P compartment boundary. The expression of each gene was monitored by coexpression of GFP (Hao, 2003).

Normally, in late third instar discs, cells at the tip of the leg, within the pretarsus, are arranged as a flat epithelial sheet, with their apical surfaces densely packed and in register. This is revealed by a straight line of either E-cadherin or phalloidin staining seen when the discs are examined in cross-section. By contrast, when constitutively activated-Notch is expressed along the A-P axis (ptcGAL4 UASGFP UASN*), cells are induced to fold into the pretarsus, as an indentation in the epithelial sheet is observed. When observed in cross-section, the cells at the A-P border expressing activated-Notch appear to have invaginated, as they are now located below the plane of the pretarsal epithelium. However, they do not lose contact with their neighbors and are still a continuous part of the epithelial sheet. This invagination occurs at the border between cells expressing activated-Notch and nonexpressing cells; in fact, cells in which Notch is activated appear to nonautonomously induce neighboring cells to invaginate. Although similar results were obtained with E-cadherin and phalloidin staining when examining the behavior of cells expressing activated Notch, it is noted that, while E-cadherin expression is not altered, the invaginating cells have higher levels of phalloidin staining than their neighbors. Interestingly, increased phalloidin staining is observed at the endogenous joints during leg segmentation (Hao, 2003).

Since odd, sob, and drm are targets of Notch activation, it was asked whether their ectopic expression might induce changes in epithelial morphology like those observed with constitutive Notch activation. Strikingly, ectopic expression of odd along the A-P axis of the leg gave identical cellular changes to those observed upon activated-Notch expression. An invagination with increased phalloidin-staining levels at its apical surface was induced and nonautonomous effects on neighboring nonexpressing cells were observed. Similar results were obtained upon ectopic expression of sob, while ptcGAL4-driven drm expression was lethal. Taken together, these data showing similar morphological changes associated with FLP-out clones of odd, sob, and drm, and similar cellular changes associated with ptcGAL4-driven odd and sob, strongly suggest that each gene can act as an effector of Notch activation to promote the epithelial cellular changes driving joint formation (Hao, 2003).

The developing leg joint is composed of different cell populations that ultimately contribute to a particular structure of the adult leg joint. These different cell populations can be identified in the developing leg disc by the genes that they express. Although these studies show that the odd-skipped family genes can promote one aspect of joint morphogenesis, epithelial invagination, it remained possible that this phenotype was the result of a more general role for these genes in regulating joint fate. However, this latter possibility does not seem to be the case, since ectopic odd expression does not activate the expression of other genes that serve as markers of the developing joint. One of these joint markers is nubbin, which is expressed in proximal joint tissue, just proximal to those cells expressing odd, sob, and drm. Ectopic odd expression does not alter the expression of nubbin in any region of larval and pupal leg discs. In particular, closer examination of the pretarsus, in which the aforementioned epithelial invaginations were characterized, shows no change in nubbin expression. Examination of other markers of the presumptive joint, including E(spl)mß, big brain, odd, and drm, indicates that the expression of these genes is not induced upon ectopic odd expression, but rather expression also remains unaffected within the pretarsus. It was noted that, during late third instar stages and pupal development, when ptcGAL4-driven odd expression results in extensive tarsal segment fusions, the expression of E(spl)mß and big brain is repressed within the developing tarsus. It is believed that this disruption of their expression is a secondary consequence of the fusion of these segments, since earlier in development their expression is unaffected by ectopic odd expression. Together, the data are consistent with the idea that the odd-skipped family genes promote the epithelial cellular changes characteristic of invaginating joint cells, without inducing the expression of other joint markers, and hence these genes appear to have a specific role in joint morphogenesis (Hao, 2003).

These studies show that the odd-skipped family is a key group of genes induced upon Notch activation that promotes morphological changes associated with joint formation during leg development. Expression of odd, sob, and drm is induced in cells responding to Notch activation; these cells lie distal to Notch ligand-expressing cells. bowl expression is also regulated by Notch. Ectopic expression of odd, as well as loss of bowl function, does not alter the expression of Notch ligands. Hence, the morphological changes induced by expression of these genes appear to be mediated downstream of Notch activity. Ectopic expression of odd, sob, and drm, like ectopic Notch activation, can cause alterations in the leg cuticle that resemble those that occur at joints, including deep creases within the cuticle and an absence of bristles. Importantly, their ectopic expression, like ectopic Notch activation, induces cells to form an invaginating furrow, while still remaining part of the disc epithelium. Interestingly, during normal leg development, mid-distal joint cells express odd; these are the same cells that will invaginate and ultimately fold under proximal joint cells. Moreover, the cells that invaginate because of their expression of odd, both in ectopic expression studies and in wild-type legs, accumulate high levels of apical filamentous actin. Further support for the idea that odd, sob, and drm control a specific aspect of cell morphogenesis, an invagination, as opposed to being more generally required for specifying joint fate, comes from the observation that ectopic odd expression does not induce the expression of other markers of joint fate, including nubbin, E(spl)mß, big brain, odd, and drm. This contrasts with the effect of Notch, which induces nubbin, E(spl)mß, big brain, odd, and drm expression. Moreover, FLP-out clones of Notch induce outgrowths of leg tissue, whereas FLP-out clones of odd, drm, and sob are not associated with leg outgrowths. Thus, while both ectopic Notch activation and ectopic odd or sob expression are capable of inducing an invagination into the disc epithelium, Notch activation must further organize additional aspects of joint formation and also leg growth. Taken together, the ectopic expression studies indicate that odd, sob, and drm are Notch target genes that mediate a subset of the activities of Notch during leg development, namely, they promote a cell morphological change, an epithelial invagination, which normally occurs during joint formation (Hao, 2003).

Interestingly, the involvement of the odd-skipped family of segmentation genes in promoting epithelial cellular changes may not be unique to the leg joint. odd-LacZ is expressed in the apodemes of the developing leg, which are tubes of invaginating cells that serve as muscle attachment sites. Thus, these genes may have a role in promoting the apodeme invagination as well. odd is also required for embryonic segmentation during which segmental borders are defined by intersegmental furrows; cells at the prospective segment boundary elongate and fold into the epidermis. While the relationship between odd expression and cell morphology during embryonic segmentation has not been elucidated, it is possible that one function of odd in segmentation is to orchestrate epithelial invaginations. Thus, the odd-skipped gene family may be required in multiple developmental contexts to induce epithelial cellular changes, such as promoting an invagination, as has been have described here. Since the odd-skipped family genes encode transcriptional regulators, it is hypothesized that they regulate the expression of genes involved in cytoskeletal architecture or cell adhesion (Hao, 2003).

These studies on the function of odd, sob, and drm suggest that these genes may have a similar function during leg development. They share a common expression pattern at all stages of leg development, consistent also with their overlapping expression in the embryo. Importantly, ectopic expression of each gene is capable of inducing the same morphological changes in the adult cuticle and, for odd and sob, the same cellular changes in the leg disc epithelium. It is thus suggested that odd, sob, and drm act redundantly during leg segmentation. Hence, it is not surprising that, when only one of the genes is removed, no effect on leg development is observed. It will ultimately be of interest to determine the phenotype of leg tissue triply mutant for odd, sob, and drm (Hao, 2003).

bowl, in contrast, may have adopted functions that are independent of and/or not obscured by the other three members of the family. bowl expression appears largely distinct from the other three genes; its expression encompasses a broader domain that overlaps that of the other genes in proximal segments and tarsal segment 5, while bowl is the only odd-skipped family gene expressed in tarsal segments 1–4. The identical expression profile of odd, sob, and drm, yet distinct pattern of bowl, is also observed in other tissues. The observation that the odd-skipped family genes are expressed in overlapping domains in a number of different developmental contexts, yet are not always genetically redundant, suggests that their contribution to a particular morphogenetic process may depend on their relative expression levels or their interaction with other proteins in that particular tissue (Hao, 2003).

In fact, recently, it has been shown that a physical interaction between one of the odd-skipped family members, Drm, and another transcriptional regulator, Lines, is important during hindgut morphogenesis [Green, 2002]. By interacting with Lines, Drm inhibits Lines activity in the embryonic hindgut, thereby allowing specification of the small intestine. As the functionally significant Drm-Lines interaction was mapped to the first zinc-finger of Drm, it is conceivable that Odd, Sob, and Bowl may also interact with Lines in other developing tissues [Green, 2002]. Indeed, this does seem to be the case, since regulatory interactions among Drm, Bowl, and Lines operate during the patterning of the embryonic dorsal epidermis and the foregut [Hatini, 2000 and Johansen, 2003b]. In these contexts, Lines inhibits Bowl, resulting in a particular cell type. The remaining cell types are controlled by Drm, which activates Bowl by causing inhibition of Lines (Hao, 2003).

These results are consistent with this molecular genetic circuit also functioning during Drosophila leg development. Clones of cells mutant for bowl are unable to participate in joint formation, resulting in melanotic protrusions from the leg cuticle in proximal segments and in a fusion of tarsal segments. The difference in the phenotype of bowl clones in proximal versus tarsal segments may be because proximal joints do not form in the same way as tarsal joints, although some of the changes in cell behavior are presumably conserved. Notably, the difference does not appear to be due to redundancy, because loss-of-function bowl mutations result in the fusion of the tibia and tarsal segment ta1, despite the fact that all four genes are expressed in the developing tibia. Thus, odd, sob, and drm are insufficient to induce tibia-tarsal 1 joint formation in the absence of bowl; the ability of these genes to induce morphogenesis might be dependent in some way on the expression of bowl. It has also recently been reported that lines has a role during leg development [Green, 2002]. These results are all consistent with a molecular model in which Bowl and Lines interact to regulate joint formation during leg development, although it remains to be determined whether Lines inhibits Bowl function or whether a Bowl–Lines complex regulates the expression of genes effecting joint formation. It is further proposed that formation of proximal leg joints requires the additional contribution of Odd, Sob, and Drm, which act redundantly to relieve the repression of Bowl by Lines. In such a model, ptcGAL4-driven ectopic bowl expression would be insufficient to induce ectopic segmentation in the leg, as was observed, since Lines would repress Bowl and hence render Bowl inactive. Also, drm bowl mutant clones would behave similar to bowl mutant clones, as was observed, since odd and/or sob would compensate for the loss of drm. While odd, sob, drm, and bowl may act together to regulate proximal leg segmentation, it appears that only bowl is essential to tarsal segmentation, as both these data and that of [Mirth, 2002] indicates that odd, sob, and drm are not expressed in tarsal segments 1–4. This would suggest that within tarsal segments 1–4 an alternative mechanism regulates Bowl activity (Hao, 2003).

drumstick, bowl, and lines are required for patterning and cell rearrangement in the Drosophila embryonic hindgut

The Drosophila embryonic hindgut is a robust system for the study of patterning and morphogenesis of epithelial organs. In a period of about 10 h, and in the absence of significant cell division or apoptosis, the hindgut epithelium undergoes morphogenesis by changes in cell shape and size and by cell rearrangement. The epithelium concomitantly becomes surrounded by visceral mesoderm and is characterized by distinct gene expression patterns that forecast the development of three morphological subdomains: small intestine, large intestine, and rectum. At least three genes encoding putative transcriptional regulators, drumstick (drm), bowl, and lines (lin), are required to establish normal hindgut morphology. The defect in hindgut elongation in drm, bowl, and lin mutants is due, in large part, to the requirement of these genes in the process of cell rearrangement. Further, drm, bowl, and lin are required for patterning of the hindgut, i.e., for correct expression in the prospective small intestine, large intestine, and rectum of genes encoding cell signals (wingless, hedgehog, unpaired, Serrate, dpp) and transcription factors (engrailed, dead ringer). The close association of both cell rearrangement and patterning defects in all three mutants suggest that proper patterning of the hindgut into small intestine and large intestine is likely required for its correct morphogenesis (Iwaki, 2001).

Focusing on hindgut elongation that occurs after stage 10, neither apoptosis nor cell proliferation contribute significantly to the process. Thus, hindgut morphogenesis occurs normally in the apoptosis-deficient DfH99 mutant, and the only cell proliferation occurring in the hindgut after stage 10 is in a small domain at the anterior of the small intestine. The morphogenesis of the hindgut after stage 10, in particular its elongation and narrowing, must therefore be driven by changes in cell size, shape, and rearrangement (Iwaki, 2001).

After the cessation of the postblastoderm mitoses, an endoreplication cycle increases the size of the cells of the large intestine (but not small intestine or rectum). Inhibition of this endoreplication by different genetic manipulations results in a shorter large intestine with a smaller cell size, but roughly normal diameter. Endoreplication thus appears to be required to bring the large intestine to its full length, but not to play a critical role in reducing hindgut diameter. There is a change in cell shape, from columnar to cuboidal, as the hindgut elongates; such a change increases epithelial surface area and thus could contribute to hindgut elongation, but not to a reduction in its diameter (Iwaki, 2001).

The threefold elongation of the hindgut is accompanied by a three- to four-fold reduction of circumferential cell number, but not by appreciable cell proliferation or apoptosis. The major process driving this stereotypic elongation and narrowing must therefore be cell rearrangement. Elongation by cell rearrangement is a morphogenetic process of broad significance: it has been shown to drive gastrulation and embryonic axis elongation, and elongation of various tissues, throughout the bilateria. To date, few molecules required for this process have been identified. Elongation by cell rearrangement of the Drosophila germband, ovarian terminal filaments, and stigmatophore requires the Evenskipped homeodomain, Bric a brac BTB, and Grain GATA proteins, respectively, while that of the C. elegans dorsal epidermis requires the DIE-1 zinc finger protein. The genetic pathways in which these presumed transcriptional regulators function have not yet been determined. Only the Xenopus Brachyury transcription factor has been shown to affect cell rearrangement by controlling expression of a specific target, Wnt11, which acts via the planar cell polarity pathway to orient cell intercalation. A fuller understanding of the molecular basis of oriented cell rearrangement clearly depends on the identification of additional required genes and genetic pathways (Iwaki, 2001).

Since the hindguts of their mutant embryos are shorter and wider than normal, drm, bowl, and lin have been identified as possible regulators of the cell rearrangement that drives hindgut elongation. Analysis of hindgut morphology and gene expression patterns in mutants indicates that drm, bowl, and lin function in hindgut development after the primordium has already been established and internalized by gastrulation. No massive apoptosis in the hindgut (as seen in fkh, cad, and byn) is observed in drm, bowl, or lin hindguts. The number of cells in the hindgut epithelium of drm, bowl, or lin mutants is within 20% of wild type, demonstrating that cell proliferation is roughly normal in these mutants. The byn and fkh genes are expressed normally throughout drm, bowl, and lin hindguts, and otp is expressed throughout drm and bowl hindguts. The hindgut visceral mesoderm, on the basis of its expression of Connectin, appears to be established normally in drm, bowl, and lin mutants. Taken together, these results indicate that early events in hindgut development, namely the establishment and maintenance of the primordium (including initiation of gene activity, and cell proliferation throughout the primordium), its internalization during gastrulation, and its investment with visceral mesoderm, all occur more or less normally in drm, bowl, and lin mutants. The shorter overall length, and the two- to three-fold greater circumferential cell number seen in drm, bowl, and lin hindguts, must therefore be a result of a failure to complete the cell rearrangement that elongates and narrows the wild-type hindgut (Iwaki, 2001).

Patterning of the Drosophila hindgut serves as a microcosm of the complex anteroposterior and dorsoventral patterning that takes place during vertebrate gut development. In the Drosophila hindgut, patterning along the anteroposterior axis gives rise to the small intestine, large intestine, and rectum; patterning along the dorsoventral axis gives rise to the large intestine ventral and large intestine dorsal domains, and the boundary cells. Previous studies described gene expression patterns in the different domains of the developing Drosophila hindgut (as well as the requirement of fkh for these expression patterns), but did not identify any genetic activity that distinguished among or specified the different domains (Iwaki, 2001).

This study shows that drm, bowl, and lin are required for the gene expression patterns that distinguish these three domains: lin is required for expression characteristic of large intestine (dpp, dri, and en) and rectum (Ser, hh, and wg); drm and bowl are required for expression characteristic of small intestine (hh and upd). By both morphological criteria (cell shape, presence or absence of boundary cells) and gene expression patterns (expanded expression of genes expressed in the small intestine), lin hindguts appear to consist of a greatly expanded small intestine and to lack the large intestine and rectum. In contrast, both morphological and gene expression characteristics of drm and bowl hindguts indicate that they lack most or all of the small intestine, and consist only of large intestine (which remains unelongated) and rectum. A model consistent with these data is that lin functions in the hindgut to repress small intestine fate and to promote large intestine and rectum fate, while establishment of the small intestine requires the activity of drm and bowl. The requirement for drm (but not bowl) for wg expression at the most anterior of the hindgut could be explained if the domain of bowl function in the small intestine does not extend to the most anterior of the hindgut (consistent with the expression of bowl). Since they have opposite effects on Ser expression, bowl and drm may function in different ways, possibly in different pathways, to promote small intestine fate (Iwaki, 2001).

The function of lin as both an activator and repressor of gene activity in the developing hindgut is consistent with molecular and genetic characterization of its function in other embryonic tissues. In the developing dorsal epidermis, lin is required for transcriptional regulation (both activation and repression) of targets downstream of wg signaling. In the developing posterior spiracles, lin is required for the activation by Abd-B of its transcriptional targets. lin encodes a novel protein that is expressed globally throughout the embryo, including the developing hindgut. When ectopically expressed, Lin protein is detected in nuclei of cells signaled by Wg. The early expression of wg throughout the hindgut primordium, starting at the blastoderm stage and continuing to stage 10, might, analogous to its effect in the dorsal epidermis, activate Lin. This might be required for Lin to carry out its function, demonstrated here, of promoting expression of genes characteristic of large intestine identity (otp, dpp, en, and dri), and repressing expression of genes characteristic of small intestine identity (hh, upd, and Ser) (Iwaki, 2001).

It has been shown by genetic analysis that bowl and drm function to establish the small intestine. bowl encodes a zinc finger protein related to Odd-skipped and is expressed strongly in the hindgut primordium starting at the blastoderm stage and continuing through stage 11. Although the Bowl protein has not been shown to be nuclear or to bind DNA, the fact that it encodes five tandem zinc fingers suggests that it is a transcription factor. Thus, Bowl might function in the hindgut as an activator or coactivator of transcription of genes characteristic of small intestine fate. Finally, drm encodes a zinc finger protein related to Bowl and Odd-skipped and is expressed during stage 10 in the anterior of the developing hindgut, consistent with its required role in establishing the small intestine. drm, like bowl, is required for gene expression characteristic of small intestine fate. The drm protein may, like Bowl, function as a transcriptional regulator in the small intestine primordium (Iwaki, 2001).

Thus drm, bowl, and lin are required for both patterning and cell rearrangement of the hindgut. At least one other putative transcriptional regulator expressed in the hindgut has similar properties: Dichaete encodes a Sox protein required for en, hh, and dpp expression in and elongation of the hindgut. The question therefore arises whether any of the genes expressed in different hindgut domains are mediators of the required role of drm, bowl, lin, or Dichaete in hindgut morphogenesis (Iwaki, 2001).

The phenotypes described for wg, hh, dpp, dri, Ser, and en do not suggest a role for these genes in hindgut elongation by cell rearrangement. Mutations in Ser, dri, and en do not appear to affect overall hindgut morphology. The hindgut in wg mutants is extremely small, suggesting that the critical function of wg in hindgut development is in establishing and maintaining the primordium, but not in elongation. dpp mutant hindguts are shorter, consistent with the role of dpp in endoreplication in the large intestine; nevertheless, dpp hindguts have a roughly normal diameter. In hh mutant embryos, the rectum degenerates and hindgut length is reduced, but the overall morphology, particularly the narrowing of the large intestine, appears normal (Iwaki, 2001).

Only in upd embryos is a defect in both elongation and narrowing of the hindgut observed; significantly, upd is expressed only in the small intestine, the same domain that is largely missing from drm and bowl mutants. Shorter and wider hindguts are seen in younger upd embryos, but the majority of hindguts in mature upd embryos appear normal. Thus, while upd may at least partially mediate drm and bowl function in the hindgut, there must be other targets of these genes that are required for cell rearrangement in the hindgut (Iwaki, 2001).

It is concluded that, if correct patterning of the hindgut is a prerequisite for its elongation by cell rearrangement, either all the targets of drm, bowl, and lin that are the essential components of the necessary patterning have not been identified, or the genes presently identified have overlapping or redundant function. Consistent with the idea that cell rearrangement in the Drosophila hindgut requires its correct patterning, convergent extension during vertebrate gastrulation has been shown to depend on patterning of cell fates along the dorsoventral axis of the embryo. It is, of course, possible that hindgut patterning and cell rearrangement, although closely associated both temporally and in the drm, bowl, and lin mutant phenotypes, do not have a necessary relationship to each other. A number of genes are known that, without affecting patterning, control cell rearrangement by directly affecting morphogenetic movements. This is a property of the Drosophila GATA transcription factor-encoding grain in stigmatophore elongation and of the zebrafish trilobite locus in body axis elongation. Thus, drm, bowl, lin, and Dichaete, in addition to patterning the hindgut, might be regulating other genes that independently control cell rearrangement. Nonetheless, the relationship between patterning of both small intestine and large intestine, on the one hand, and cell rearrangement, on the other hand, is striking. drm and bowl hindguts have a substantial cohort of large intestine cells, yet fail to complete cell rearrangement, presumably due to absence of the small intestine. lin hindguts have an excess of small intestine cells and also fail to complete cell rearrangement, presumably due to absence of the large intestine. The connection between hindgut patterning and cell rearrangement observed in drm, bowl, and lin mutants supports the idea that interaction between two correctly patterned anteroposterior subdomains, the small and large intestine, is a requirement for cell rearrangement in the hindgut tubule (Iwaki, 2001).

The Drm-Bowl-Lin relief-of-repression hierarchy controls fore- and hindgut patterning and morphogenesis

In addition to their roles in patterning and morphogenesis of the hindgut, the Drosophila genes drumstick (drm) and bowl are required in the foregut for spatially localized gene expression and the morphogenetic processes that form the proventriculus. drm and bowl belong to a family of genes encoding C2H2 zinc finger proteins; the other two members of this family are odd-skipped (odd) and sob. In both the fore- and hindgut, drm acts upstream of lines (lin), which encodes a putative transcriptional regulator, and relieves the lin repressive function. In spite of its phenotypic similarities with drm, bowl was found in both foregut and hindgut to act downstream, rather than upstream, of lin. These results support a hierarchy in which Drm relieves the repressive effect of Lin on Bowl, and Bowl then acts to promote spatially localized expression of genes (particularly the JAK/STAT pathway ligand encoded by upd) that control fore- and hindgut morphogenesis. Since the odd-family and lin are conserved in mosquito, mouse, and humans, it is proposed that the odd-family genes and lin may also interact to control patterning and morphogenesis in other insects and in vertebrates (Johansen, 2003b).

By following the spatially localized expression domains of Wg, Dead ringer (Dri), and Connectin (Con), it is possible to identify at least four processes that contribute to morphogenesis of the three-layered valve of the proventriculus. During stages 13-15, a bulge referred to as the 'keyhole' forms in the foregut ectoderm, at its junction with the endoderm of the anterior midgut; this appears to be a process of evagination, similar to that described for formation of the vertebrate optic cup. The keyhole can be distinguished from the anterior ectoderm of the esophagus and from the anterior endoderm of the anterior midgut, since it is not surrounded by visceral mesoderm and is flanked by two domains of Wg expression. During stage 16, the anterior hemisphere of the keyhole reverses its curvature, and is approached by the posterior hemisphere; this has been described as a folding process. During stage 17, the posterior portion of the keyhole moves interiorly over the anterior lip of the developing proventriculus, a movement that appears similar to involution over the dorsal lip of the blastopore in Xenopus. Also during stage 17, the most posterior portion of the esophagus, i.e. that portion just anterior to the keyhole, inserts posteriorly into the pocket consisting of the most anterior endoderm of the anterior midgut. During this step of insertion, there is significant elongation of the most interior cells, which are derived from the anterior of the keyhole. At the conclusion of proventricular morphogenesis, the domain of Wg expression initially at the anterior of the keyhole is now inserted most distally into the proventriculus, while the domain of Wg initially posterior to the keyhole is now just interior, at the most anterior of the proventriculus (Johansen, 2003b and references therein).

drm and bowl mutants, which exhibit similar defects in hindgut elongation (Iwaki, 2001), are also similar in that they have related effects on proventriculus folding; in both mutants, rather than undergoing the first step of evagination to form the keyhole, the entire foregut remains as a narrow tube (Johansen, 2003b).

In both foregut and hindgut, localized expression of signaling molecules is required for gut morphogenesis; in the hindgut, this patterned expression has been shown to depend on drm and bowl (Iwaki, 2001). In the foregut, drm and bowl are also required for patterned gene expression, specifically for the two stripes of Wg that bracket the keyhole primordium, as well as for expression of upd and dri in the posterior hemisphere of the keyhole primordium. Another indicator of foregut patterning is its investment with visceral musculature, which surrounds the foregut epithelium with the exception of the keyhole region. In both drm and bowl mutants, the entire foregut (up to the anterior midgut) is surrounded by visceral mesoderm, indicating that the keyhole region is not established in these mutants. The odd mutant has no detectable foregut or hindgut defect. In a screen of over 10,000 chromosomes, no lethal mutation was isolated in sob; it therefore seems unlikely that sob plays a significant role in gut morphogenesis (Johansen, 2003b).

The similar sequence, phenotype, and partially overlapping expression of drm and bowl suggest that these genes might play redundant roles in foregut and hindgut morphogenesis. To address this possibility, double, triple, and quadruple mutants were constructed. Proventriculus and hindgut morphologies, patterned gene expression, and investment with foregut visceral musculature indicate that the drm bowl double mutant has hindgut and foregut phenotypes similar to those of drm and bowl single mutants. Further, drm sob odd (drmP2) and drm sob odd bowl (drmP2 bowl) mutants also have foregut and hindgut phenotypes that resemble those of drm and bowl single mutants. Since no additional phenotypes were revealed when drm, sob, odd, and bowl mutants were combined, it is concluded that, in the gut, members of the odd family, in particular drm and bowl, do not have redundant or overlapping function (Johansen, 2003b).

One odd-family member, drm, has previously been shown to interact with lin; like drm, lin is required for both hindgut patterning and the cell rearrangement that elongates the hindgut (Green, 2002 and Iwaki, 2001). In the foregut, the processes of folding and involution that form the proventriculus fail to occur in both drm and lin mutant embryos; the resulting phenotypes, however, are distinct: the drm foregut is long and narrow, while that of lin is short and bloated. Consistent with what has been observed for the hindgut, the foregut phenotype of the drm lin double mutant is very similar to that of the lin single mutant. In addition, the region that will become the keyhole, i.e., that portion of the foregut bracketed by Wg expression and lacking Con-expressing visceral musculature, is expanded in both lin and drm lin embryos. Since the foregut in the drm lin double mutant (both in terms of gene expression and morphology) is similar to that seen in the lin single mutant, it is concluded that in the foregut, as has been shown in the hindgut (Green, 2002), lin is epistatic to (acts downstream of) drm (Johansen, 2003b).

Since the bowl phenotype is similar to that of drm in both foregut and hindgut (Iwaki, 2001; Wang, 1996), the epistatic relationship between bowl and lin in both fore- and hindgut was investigated. Strikingly, while the drm lin phenotype is similar to that of lin, the bowl lin mutant phenotype, in both fore- and hindgut, appears the same as that of bowl. Further, the hindgut and foregut of drm bowl lin and drm sob odd bowl lin (drmP2 bowl lin) embryos are indistinguishable from those of bowl embryos. These results are consistent with observations that odd and sob are not required for gut morphogenesis (Wang, 1996). Most importantly, they show that in both hindgut and foregut, bowl is epistatic to (acts downstream of) lin (Johansen, 2003b).

More detailed analysis of gene expression confirms the epistasis of bowl to lin in both the hindgut and foregut. upd expression, which is observed throughout the small intestine (anterior portion of the hindgut) in wild-type (Iwaki, 2001), is barely detectable in bowl and bowl lin, but greatly expanded in lin hindguts. Similarly, hh expression, seen in both small intestine and rectum, is greatly reduced in the anterior of bowl and bowl lin, but expanded in lin hindguts. En expression, seen on the dorsal side of the large intestine (Iwaki, 2002), is expanded to both dorsal and ventral sides in bowl and bowl lin, but missing from lin hindguts. dri, expressed strongly in the two boundary cell rows and at a lower level in all cells of the small intestine (Iwaki, 2001), is expressed in duplicated boundary cell rows in bowl and bowl lin hindguts, but at a low level throughout the lin hindgut. All of these results confirm the epistasis of bowl to lin in the hindgut (Johansen, 2003b).

Characterization of gene expression also supports the epistasis of bowl to lin in the foregut. The expression of upd in the foregut epithelium, in a region that will become the posterior keyhole, is not seen in bowl or bowl lin mutant foreguts, but is greatly expanded in lin mutants. hh expression, which extends throughout the keyhole and part of the foregut anterior to it, is significantly reduced in bowl and bowl lin, while it appears to be expanded in lin embryos. dri, expressed in a narrow ring in the posterior keyhole region, is missing in bowl and bowl lin, but expanded in lin embryos. It is concluded that in both foregut and hindgut, lin is epistatic to drm, and bowl is epistatic to lin; in other words, lin acts downstream of drm, and bowl acts downstream of lin (Johansen, 2003b).

The byn-GAL4 construct drives posterior gut-specific expression, specifically, a uniformly high level of expression in the hindgut starting at stage 8 and continuing through embryogenesis (Iwaki, 2002). When this construct is used to drive ectopic expression of drm throughout the hindgut, a lin-like phenotype, i.e., expansion of small intestine, is observed, while ectopic expression of lin throughout the hindgut results in a drm-like defect, i.e., loss of small intestine (Green, 2002). This is demonstrated by morphology, by expanded expression of upd and hh, and by absence of expression of En (Johansen, 2003b).

In contrast to the dramatic effect of ectopic drm, ectopic expression of bowl throughout the hindgut has little effect on morphology and patterning. The morphology of the hindgut is altered only modestly: the small intestine is somewhat wider, the large intestine shorter, and the rectum a little longer. Similarly, patterning of the hindgut does not appear different: expression of upd, hh, and En is normal. Therefore, although bowl is required to specify the small intestine and for normal hindgut elongation, it functions differently from drm (Johansen, 2003b).

A genetic hierarchy of drm, lin, and bowl activity controls the localized expression of upd in both foregut and hindgut. Previous work has shown that, most likely by establishing a gradient of JAK/STAT activity, localized expression of upd is required for hindgut morphogenesis, specifically the cell rearrangement that drives hindgut elongation (Green, 2002 and Johansen, 2003a). upd is expressed in the region of the foregut that will become the posterior hemisphere of the keyhole, and remains restricted to this region through the remainder of embryogenesis. Stat92E, a transcriptional target of JAK/STAT signaling in the embryo, is expressed in the foregut in a domain that overlaps with, but extends beyond (both anteriorly and posteriorly), the ring of upd expression in the keyhole. Analysis of anti-Crb stained embryos shows that, in upd mutants, the evagination and folding steps of proventriculus morphogenesis occur, but the involution and insertion steps do not take place. It is concluded that upd is required for late steps in proventricular morphogenesis, and that Upd is likely to affect cells beyond the domain in which it is expressed (Johansen, 2003b).

For each of the Drosophila odd-family genes drm, sob, odd, and bowl, a specific ortholog could be identified in the Anopheles gambiae genome; this was possible because of the high similarity in amino acid sequence between specific zinc fingers. Three of the four Anopheles odd-family genes (the fourth was not mapped at the time of this submission) are, as in Drosophila, clustered on one chromosome arm (Johansen, 2003b).

There is a high degree of identity not only between the sequence of each of the five zinc fingers in the Drosophila and Anopheles Sob and Bowl proteins, but also between these and the five zinc fingers encoded by a splice variant of mouse Odd-skipped related 2. Remarkably, these proteins share 88% identity over the 135 amino acids that comprise five zinc fingers, implying that this group of fingers may have conserved molecular function (Johansen, 2003b).

Like the odd-family genes, lin is highly conserved between Drosophila and Anopheles, showing 43% identity over 858 amino acids, and a striking 76% identity in the 165 amino acid 'Lines homology domain' at the carboxy terminus (Johansen, 2003b and references therein).

From the data presented here and previously, it is concluded that drm, lin, and bowl have the same relationship to each other in both the foregut and the hindgut. drm and bowl in one case, and lin in the other, affect specification of the small intestine in different ways: bowl, expressed throughout the hindgut, and drm, expressed at the anterior of the hindgut, are both required to establish the small intestine, while lin, expressed throughout the hindgut, represses the small intestine (Green, 2002; Iwaki, 2001). Similarly, in the foregut, drm and bowl are both required to establish the keyhole, while lin, expressed throughout the foregut, represses formation of the keyhole region. For mutants in all three genes, failure to establish the keyhole results in an early failure in morphogenesis of the proventriculus (Johansen, 2003b).

These observations can be integrated with the results of epistasis and ectopic expression experiments presented in this study to yield a hierarchical model for the mechanism by which drm, lin, and bowl gene activities interact to specify discrete domains in the gut. The epistasis of bowl to lin as demonstrated in this study means that the repressive activity of lin acts through bowl. Bowl promotes specification of the small intestine and keyhole, while Lin represses specification of these regions. Drm, expressed in the small intestine and in the keyhole, relieves repression of the small intestine and keyhole by repressing Lin, thereby allowing Bowl to function (Johansen, 2003b).

Although bowl is necessary to specify small intestine fate, overexpression studies suggest that it is not sufficien to specify this fate in the context of gene activity in the hindgut. At least two possible reasons can be imagined for this: either the level of bowl activity generated by the byn-GAL4 driver is not sufficient to overcome the repressive effect of lin, or another activity in addition to bowl (and present only in the anterior hindgut) is required to promote small intestine fate (Johansen, 2003b).

The drm-lin-bowl hierarchy that patterns the epithelial foregut and hindgut tubes functions by a distinctly different mechanism from the segmentation hierarchy that patterns the blastoderm embryo. During embryonic segmentation, a cascade of transcription factor-encoding genes (gap, pair-rule, and segment polarity) is sequentially expressed in more and more restricted domains. During gut development, in contrast, the activator Bowl and the repressor Lin are expressed throughout both foregut and hindgut; it is the spatially localized expression of Drm that is required for patterning of the distal foregut and hindgut (at the junction with the midgut). Drm, presumably by its direct binding to Lin (Green, 2002), relieves the repression of Bowl by Lin. The Drm-Lin-Bowl genetic hierarchy defined in this study is thus based, not on transcriptional regulatory interactions, but on protein-protein interactions (Johansen, 2003b).

Foregut and hindgut comprise the most distal portions of the gut tube, connecting the endodermal midgut to the exterior; both are ectodermal in origin, arising from the invagination of the stomodeum and proctodeum, respectively. Although the proventriculus undergoes a different type of morphogenesis than the hindgut (evagination, folding, involution, and insertion, versus elongation by cell rearrangement), it is significant that the drm-lin-bowl hierarchy in the hindgut obtains in the foregut as well. The drm-lin-bowl hierarchy acts in both ectodermal gut tissues to specify a small domain at the most interior position of the tube (i.e., the most posterior of the foregut and the most anterior of the hindgut) (Johansen, 2003b).

Specification of these domains results in the localized expression of upd, encoding the Drosophila JAK/STAT pathway ligand. In the hindgut, upd is required for morphogenetic behavior (rearrangement) of cells distant from the site of its expression, suggesting that a gradient of Upd may orient cell rearrangement (Johansen, 2003a). Since upd is required for proventriculus formation, and a domain of Stat92E expression is observed in the foregut extending beyond the domain of localized upd expression, there may also be a gradient of Upd in the foregut that plays a role in the morphogenetic processes that form the proventriculus (Johansen, 2003b).

These results support the proposal that foregut and hindgut morphogenesis are controlled in parallel by the some of the same genes. In addition to the drm-lin-bowl-upd pathway delineated here, the transcription factor encoded by forkhead (fkh) is required for expression of wg, hh, and decapentaplegic (dpp), which each contribute to aspects of both foregut and hindgut morphogenesis. Thus, in Drosophila, a number of molecular pathways are similarly deployed to promote both fore- and hindgut morphogenesis; some of this pathway conservation may extend to other organisms (Johansen, 2003b).

The one-to-one correspondence among each of the four members of the Drosophila and Anopheles (both members of the order Diptera) Odd family proteins, indicates that the four members were present prior to the divergence of the suborder Cyclorrhapha (which include Drosophila) from the suborder Nematocera (which include Anopheles), approximately 250 MYA. Drosophila chromosome 2L and Anopheles chromosome 3R, on which the odd family of each species is located, respectively, are the most conserved pair of chromosome arms between the two species. Based on their high sequence similarity to Drosophila drm and bowl, the Anopheles gambiae orthologs are likely to control similar developmental processes, in particular, gut morphogenesis (Johansen, 2003b and references therein).

odd-like genes are present in mammals and in Ciona intestinalis, a hemichordate. The mouse Osr2 and human OSR1 proteins display 65% and 70% respective identity to Drosophila Odd, Sob, and Bowl over their first three zinc fingers. The Ciona Odd-family protein, with two zinc fingers, has approximately 85% identity to the first and second zinc fingers of Drm, Sob, Odd, and Bowl. Intriguingly, human OSR1 is expressed in the adult colon, the mammalian equivalent to the Drosophila hindgut, and the Ciona homolog shows expression in the esophagus of the young adult. An important question to be addressed, therefore, is whether mammalian and Ciona odd-like genes also play roles in gut development (Johansen, 2003b).

The presence of lin-like genes in other genomes suggests that the interaction between odd family members and lin demonstrated for Drosophila may also obtain in other organisms. It is striking that the Lin ortholog in Anopheles is 45% identical overall, and 76% identical over 165 amino acids, to Drosophila Lin. Since each of the four members of the Anopheles odd family has an ortholog in the Drosophila odd family, the epistatic relationships observed between Drosophila drm, lin, and bowl are also likely to obtain in Anopheles. It is proposed that drm, lin, and bowl may play similar roles in Drosophila and mosquito gut development; analysis of expression of the Anopheles orthologs will be an important step toward testing this hypothesis. The human WINS1 and mouse Wins2 proteins show a much lower, although significant, 29 and 27% respective identity to Drosophila Lin. Since these genomes do not contain 1:1 orthologs of each of the odd family members, it is unlikely that there is a drm-lin-bowl pathway in these species. Nevertheless, interactions between Osr1/2 and Lin proteins may very well play important roles in embryonic patterning and morphogenesis (Johansen, 2003b).

Odd-skipped genes specify the signaling center that triggers retinogenesis in Drosophila

Although many of the factors responsible for conferring identity to the eye field in Drosophila have been identified, much less is known about how the expression of the retinal 'trigger', the signaling molecule Hedgehog, is controlled. This study shows that the co-expression of the conserved odd-skipped family genes at the posterior margin of the eye field is required to activate hedgehog expression and thereby the onset of retinogenesis. The fly Wnt1 homologue wingless represses the odd-skipped genes drm and odd along the anterior margin and, in this manner, spatially restricts the extent of retinal differentiation within the eye field (Bras-Pereira, 2006).

The eye disc is a flat epithelial sac. By early third larval stage (L3), columnar cells in the bottom (disc proper: Dp) layer are separated by a crease from the surrounding rim of cuboidal margin cells. Margin cells continue seamlessly into the upper (peripodial; Pe) layer of squamous cells. The Dp will differentiate into the eye, while the margin and Pe will form the head capsule. In addition, the posterior margin produces retinal-inducing signals (Bras-Pereira, 2006).

By examining gene reporters it was found that the zinc-finger gene odd is expressed restricted to the posterior margin and Pe of L3 eye discs. Since the odd family members drumstick (drm), brother of odd with entrails limited (bowl) and sister of odd and bowl (sob) are similarly expressed in leg discs, they were examined in eye discs. In L2, before retinogenesis has started, odd and drm are transcribed in the posterior Pe-margin, and this continues within the posterior margin after MF initiation. bowl is transcribed in all eye disc Pe-margin cells of L2 discs, but retracts anteriorly along the margins and Pe after the MF passes. In addition, bowl is expressed weakly in the Dp anterior to the furrow. sob expression in L2 and L3 is mostly seen along the lateral disc margins. Therefore drm, odd and bowl are co-expressed at the posterior margin prior to retinal differentiation initiation (Bras-Pereira, 2006).

Odd family genes regulate diverse embryonic processes, as well as imaginal leg segmentation. In embryos, the product of the gene lines binds to Bowl and represses its activity, while Drm relieves this repression in drm-expressing cells. Since drm/odd/bowl expression coincides along the posterior margin around the time retinal induction is triggered, it was asked whether they controlled this triggering. First, bowl function was removed in marked cell clones induced in L1. bowl- clones spanning the margin, but not those in the DP, cause either a delay in, or the inhibition of, retinal initiation and the autonomous loss of hh-Z expression. Correspondingly, there is a reduction in expression of the hh-target patched (ptc). These effects on hh and ptc are not due to the loss of margin cells, since drm is still expressed in the bowl- cells. The requirement of Bowl for hh expression is margin specific, since other hh-expressing domains within the disc are not affected by the loss of bowl (not shown). As expected from the bowl-repressing function of lines, the overexpression of lines along the margin phenocopies the loss of bowl. Nevertheless, the overexpression of bowl in other eye disc regions is not sufficient to induce hh. This suggests that, in regions other than the margin, either the levels of lines are too high to be overcome by bowl or bowl requires other factors to induce hh, or both (Bras-Pereira, 2006).

drm and odd are expressed together along the posterior disc margin-Pe, and drm (at least) is required for Bowl stabilization in leg discs. Nevertheless, the removal of neither drm nor odd function alone results in retinal defects. odd and drm may act redundantly during leg segmentation and this may also be the case in the eye margin. To test this, clones were induced of DfdrmP2, a deficiency that deletes drm, sob and odd, plus other genes. When DfdrmP2 clones affect the margin, the adjacent retina fails to differentiate, suggesting that drm and odd (and perhaps sob, for which no single mutation is available) act redundantly to promote bowl activity at the margin (although the possibility that other genes uncovered by this deficiency also contribute to the phenotype cannot be excluded). To test the function of each of these genes, drm, odd and sob were expressed in cell clones elsewhere in the eye disc. Only the overexpression of drm or odd induced ectopic retinogenesis, and this was restricted to the region immediately anterior to the MF, which is already eye committed. Interestingly, bowl is also expressed in this region of L3 discs. The retina-inducing ability of drm requires bowl, because retinogenesis is no longer induced in drm-expressing clones that simultaneously lack bowl function. Therefore, it seems that in the eye, drm (and very likely also odd) also promotes bowl function (Bras-Pereira, 2006).

The expression of hh or activation of its pathway anterior to the furrow is sufficient to generate ectopic retinal differentiation. Since (1) bowl is required for hh expression at the margin, (2) this hh expression is largely coincident with that of odd and drm, and (3) drm (and possibly odd) functionally interacts with bowl, whether drm- and odd-expressing clones induced the expression of hh was examined. In both types of clones hh expression is turned on autonomously, as detected with hh-Z, which would thus be responsible for the ectopic retinogenesis observed. That the normal drm/odd/bowl-expressing margin does not differentiate as eye could be explained if margin cells lack certain eye primordium-specific factors (Bras-Pereira, 2006).

These results indicate that the expression of odd and drm defines during L2 the region of the bowl-expressing margin that is competent to induce retinogenesis. How is their expression controlled? wingless (wg) is expressed in the anterior margin, where it prevents the start of retinal differentiation. drm/odd are complementary to wg (monitored by wgZ) during early L3, when retinal differentiation is about to start, and also during later stages. In addition, when wg expression is reduced during larval life in wgCX3 mutants, drm transcription is extended all the way anteriorly. This extension precedes and prefigures the ectopic retinal differentiation that, in these mutants, occurs along the dorsal margin. Therefore, wg could repress anterior retinal differentiation by blocking the expression of odd genes in the anterior disc margin, in addition to its known role in repressing dpp expression and signaling (Bras-Pereira, 2006).

Interestingly, the onset of retinogenesis in L3 is delayed relative to the initiation of the expression of drm/odd and hh in L1-2. This delay can be explained in three, not mutually exclusive, ways. (1) The relevant margin factors (i.e. drm/odd, hh) might be in place early, but the eye primordium might become competent to respond to them later. In fact, wg expression domain has to retract anteriorly as the eye disc grows, under Notch signaling influence, to allow the expression of eye-competence factors. (2) Building up a concentration of margin factors sufficient to trigger retinogenesis might require some time. In fact, the activity of the Notch pathway along the prospective dorsoventral border is required to reinforce hh transcription at the firing point. (3) Other limiting factors might exist whose activity becomes available only during L3. Such a factor might be the EGF receptor pathway, which is involved in the triggering and reincarnation of the furrow along the margins during L3 (Bras-Pereira, 2006).

Reciprocal roles for bowl and lines in specifying the peripodial epithelium and the disc proper of the Drosophila wing primordium

Central to embryonic development is the generation of molecular asymmetries across fields of undifferentiated cells. The Drosophila wing imaginal disc provides a powerful system with which to understand how such asymmetries are generated and how they contribute to formation of a complex structure. Early in development, the wing primordium is subdivided into a thin layer of peripodial epithelium (PE) and an apposing thickened layer of pseudostratified columnar epithelium (CE), known as the disc proper (DP). The DP gives rise to the wing blade, hinge and dorsal mesothorax, whereas the PE makes only a minor contribution to the ventral hinge and pleura. The mechanisms that generate this major asymmetry and its contribution to wing development are poorly understood. The Lines protein destabilizes the nuclear protein Bowl in ectodermal structures. This study shows that Bowl accumulates in the PE from early stages of wing development and is absent from the DP. Broad inhibition of Bowl in the PE resulted in the replacement of the PE with a mirror image duplication of the DP. The failure to generate the PE severely compromised wing growth and the formation of the notum. Conversely, the activation of bowl in the DP (by removal or inhibition of lines function) resulted in the transformation of the DP into PE. Thus, this study provides evidence that bowl and lines act as a binary switch to subdivide the wing primordium into PE and DP, and assigns crucial roles for this asymmetry in wing growth and patterning (Nusinow, 2008).

The wing PE can be identified molecularly and morphologically as a thin epithelial sheet overlying the thickened DP epithelium. Mapping studies show that the distribution of Bowl and Lines correlates with the establishment of this asymmetry. The wing primordium inherits its subdivision into en-expressing cells that form the posterior compartment and adjacent anterior compartment cells from the embryonic epidermis. Bowl accumulates in the posterior en-expressing cells in the embryonic epidermis, suggesting that the wing primordium also inherits the PE/DP subdivisions from pre-existing asymmetries across this tissue (Nusinow, 2008).

lines and odd-skipped genes act as a switch to specify alternative cell fates across fields of cells. bowl and lines specify the alternative 1°-3° and 4° cell fate across the dorsal embryonic epidermis. bowl and lines also specify alternative cell fates in the developing gut, leg and eye imaginal discs. The asymmetric distributions of Bowl and odd-skipped genes, and the reciprocal distribution of Lines in the wing primordium are also used to specify the alternative PE and DP fates. Indeed, functional studies show that ectopic lines expression or the inhibition of bowl function in the PE transforms PE into DP fate. Reciprocally, the removal of lines function from the DP transforms DP into PE fate. The data further suggest that lines exerts its function by controlling the stability of the Bowl protein. Thus, lines and bowl act as a switch to specify alternative DP and PE fates across the wing primordium. The distribution of Lines and Bowl correlates with the subdivision of the wing primordium into a thin squamous and a thickened columnar epithelial sheet. The activation of EGF receptor and Wg signaling in the DP may specify the formation of a columnar epithelial morphology. The pathways that specify the squamous morphology of the PE downstream to bowl remain to be elucidated (Nusinow, 2008).

Previous studies that relied on surgical and genetic ablations of the PE, and on inhibition of certain signaling pathways within the PE, suggested important roles for the PE in disc growth and patterning. It is now possible to examine wing development in discs lacking PE. These discs were significantly smaller than wild type, and the notum was dramatically reduced in size relative to the pouch and hinge. Progenitor cells that originate in the PE may stream laterally to populate the growing notum, and the loss of this progenitor cell population may account for the severe reduction in notal growth. The reduction in wing growth could have resulted from the disruption of Wg or Dpp signaling activities, as these morphogens control cell survival and cell proliferation in the wing. Indeed, a block to Dpp or Wg signaling results in formation of tiny wing rudiments. However, the expression of Wg and Dpp and their target genes was normal in these discs, indicating that the reduction in wing growth was not a consequence of the loss of wg or dpp expression, or of signaling activities. These findings instead suggest that the PE acts in parallel to the AP, DV and PD patterning systems, in part, by promoting cell survival in the DP, and in part by promoting the growth of the notum (Nusinow, 2008).

The results presented in this study argue that lines and bowl function as field-specific selector genes to specify the identity and the behavior of the DP and the PE of the wing primordium, respectively. Selector genes control cell fate, cell affinity and the competence to send and respond to patterning signals in a cell autonomous manner. lines and bowl act cell autonomously to control the fate, affinity and the interaction between the PE and the DP. As a putative transcription factor, Bowl may regulate a developmental program to control the identity and the behavior of the PE. By inhibiting Bowl accumulation, lines may allow the execution of an alternative developmental program in the DP. These studies define a new system to identify, in a systematic way, these developmental programs (Nusinow, 2008).

Lines is required for normal operation of Wingless, Hedgehog and Notch pathways during wing development

The regulatory Lines/Drumstick/Bowl gene network is implicated in the integration of patterning information at several stages during development. This study shows that during Drosophila wing development, Lines prevents Bowl accumulation in the wing primordium, confining its expression to the peripodial epithelium. In cells that lack lines or over-expressing Drumstick, Bowl stabilization is responsible for alterations such as dramatic overgrowths and cell identity changes in the proximodistal patterning owing to aberrant responses to signaling pathways. The complex phenotypes are explained by Bowl repressing the Wingless pathway, the earliest effect seen. In addition, Bowl sequesters the general co-repressor Groucho from repressor complexes functioning in the Notch pathway and in Hedgehog expression, leading to ectopic activity of their targets. Supporting this model, elimination of the Groucho interaction domain in Bowl prevents the activation of the Notch and Hedgehog pathways, although not the repression of the Wingless pathway. Similarly, the effects of ectopic Bowl are partially rescued by co-expression of either Hairless or Master of thickveins, co-repressors that act with Groucho in the Notch and Hedgehog pathways, respectively. It is concluded that by preventing Bowl accumulation in the wing, primordial Lines permits the correct balance of nuclear co-repressors that control the activity of the Wingless, Notch and Hedgehog pathways (Benítez, 2009).

The Drosophila wing is a discrete organ that has been used to study the coordination of signaling pathways during development. The developing wing disc is a sac-like structure composed of the columnar epithelium or disc proper cells (DP), the cuboidal marginal cells (MC) and the overlying squamous cells (SC); MC and SC constitute the peripodial epithelium (PE). During larval development, imaginal cells proliferate extensively and are patterned. After metamorphosis, the DP cells differentiate into the cuticle that forms the adult wing and notum, whereas PE cells contribute little to these structures (Benítez, 2009).

The Lin/Drm/Bowl cassette is emerging as an important molecular mechanism with which to coordinate various pathways in different developmental contexts. In all cases, the steady-state accumulation of Bowl is regulated by the relative levels of Drm and Lin proteins. High levels of Drm impede binding of Lin to Bowl and, thus, this transcriptional repressor becomes stabilized in the nucleus. In this study it was found that regulatory interaction Lin/Drm/Bowl also functions during wing development. In lin- or Drm GOF cause ectopic expression of Bowl and dramatic overgrowths within the wing disc. These overgrowths frequently showed altered cell identity, resembling more proximal disc margin cells. Some of the effects can be explained by the ability of Bowl to interact with Gro co-repressor through the eh-1 motif, forming a complex that sequesters Gro from other repressors complexes such as Su(H)/H/Gro and Mtv/Gro (Benítez, 2009).

Although Bowl is ubiquitously transcribed in the wing disc, Bowl protein is present only in the SC and MC, being normally absent from the DP cells. The spatial distribution of nuclear Bowl is dependent on Drm, which causes Lin to relocalize to the cytoplasm. Drm is absent from most of the DP cells and, therefore, Lin turns down the steady-state accumulation of Bowl protein in these cells. In the absence of Lin, Bowl accumulates in the DP cell nuclei and elicits the dramatic alterations observed in lin- mutant cells. Therefore, the main function of Lin is to prevent Bowl accumulation in the DP cells, restricting Bowl protein to MC and SC of the PE (Benítez, 2009).

The main alterations in lin-, Drm GOF or Bowl GOF clones can be classified according to the signaling pathways temporally affected. The earliest defect observed is the repression of Wg pathway responses and the evidence suggests that Bowl functions as a repressor of the Wg pathway. However, activated forms of nuclear Wg pathway components, such as ArmS10 or dTcf, cannot restore the expression of the proximal-distal markers owing to repression of the Wg targets in lin-, indicating that Bowl must act in parallel to or downstream of Arm and dTcf (Benítez, 2009).

Bowl is a zinc-finger protein that can interact with the co-repressor Gro directly through the eh-1 motif. The results indicate that this mechanism is also important under conditions where Bowl accumulates in the wing disc. Most of the alterations observed in lin- or Drm GOF clones can be explained by Bowl sequestering Gro from other repression complexes (causing activation of N targets and Hh). Several results support this model. First, the strong genetic interaction between lin and gro alleles, where trans-heterozygous combinations between lin and gro alleles result in dramatic phenotypes, argue that Gro is a limiting factor. Second, removal of eh-1 motif that recruits Gro, eliminates the effects of Bowl on the Hh and N pathways. Third, ectopic expression of Gro, H or Mtv partially suppress the phenotypes of ectopic Drm or Bowl. These observations imply a 'tug of war' between Bowl, H and Mtv for Gro. Increased H or Mtv would shift the balance back in favor of N target repression and Hh repression (Benítez, 2009).

By contrast, the repression of Wg pathway observed in lin- cells appears to involve a different mechanism. Although the effect is Bowl dependent, repression of Wg targets also occurs with Bowleh1-, indicating that Gro sequestration is not required. Similarly, co-expression of Bowl with H or Mtv cannot re-establish the repression of the Wg targets. These results show that Bowl is able to repress Wg targets independently of Gro and the observation that Bowleh1- VP16 can cause some ectopic expression of Sens suggests that this may involve a direct effect of Bowl on Wg targets (Benítez, 2009).

Wnt/Wg, N and Hh signaling represent major conserved signaling channels to control cell identity and behavior during development. An antagonistic interaction between the Wg and Hh has also been described in the embryo and at the intersection of the D/V and A/P compartment borders of the wing disc. Similarly, Wnt/Wg and N activities are closely entangled in many different systems. Mutual dependent interactions between N and Wnt signaling have been observed in vertebrate skin precursors, in rhombomere patterning and in somitogenesis. It has also been reported that orthologues of the Odd-skipped family, Osr1 and Osr2, function as transcriptional repressors during kidney formation. It is possible therefore that Lin/Bowl/Gro interaction is evolutionary conserved and it will be interesting to discover whether lin is an important regulatory factor in other systems (Benítez, 2009).

By analyzing lin- clones in the wing primordium, this study has uncovered the consequences of stabilizing Bowl in the DP cells. There are, however, two regions where Bowl accumulates normally, in the MC and SC within the PE. Removal of Bowl in the PE might lead to ectopic Wg protein and thus to ectopic activity of the Wg signaling to transform PE from squamous to columnar cells. In this context, recently, it has shown that Bowl inhibition by ectopic expression of Lin results in the replacement of the PE by a mirror image duplication of the DP cells. However, not much alteration has been observed in cell morphology nor in the expression of markers such as Ubx or Hth when Bowl was depleted in PE cells (bowl- clones and UAS-BowlRNAi). It could be that the recovered bowl- clones were not induced early enough or that the levels of Bowl-RNAi were not sufficient to completely eliminate the Bowl function in these cells. Nevertheless, these manipulations revealed that bowl- phenotypes in the proximal wing and notum are consistent with a functional role in MC. Therefore, it is concluded that Lin has an important role in restricting Bowl to the MC (and PE), delimiting a Bowl-free territory that forms the DP cells and enables their responsiveness to key signaling pathways such as Wg (Benítez, 2009).


Genes in the odd-skipped family encode a discrete subset of C2H2 zinc finger proteins that are widely distributed among metazoan phyla. Although the initial member (odd) was identified as a Drosophila pair-rule gene, various homologs are expressed within each of the three germ layers in complex patterns that suggest roles in many pathways beyond segmentation. To further investigate the evolutionary history and extant functions of genes in this family, a characterization, was initiated of two homologs, odd-1 and odd-2, identified in the genome of the nematode, C. elegans. Sequence comparisons with homologs from insects (Drosophila and Anopheles) and mammals suggest that two paralogs were present within an ancestral metazoan; additional insect paralogs and both extant mammalian genes likely resulted from gene duplications that occurred after the split between the arthropods and chordates. Analyses of gene function using RNAi indicate that odd-1 and odd-2 play essential and distinct roles during gut development. Specific expression of both genes in the developing intestine and other cells in the vicinity of the gut was shown using GFP-reporters. These results indicate primary functions for both genes that are most like those of the Drosophila paralogs bowel and drumstick, and support a model in which gut specification represents the ancestral role for genes in this family (Buckley, 2004).


Search PubMed for articles about Drosophila bowel

Benítez, E., Bray, S. J., Rodriguez, I. and Guerrero, I. (2009). Lines is required for normal operation of Wingless, Hedgehog and Notch pathways during wing development. Development 136(7): 1211-21. PubMed Citation: 19270177

Bras-Pereira, C., Bessa, J. and Casares, F. (2006). Odd-skipped genes specify the signaling center that triggers retinogenesis in Drosophila. Development 133(21): 4145-9. PubMed citation: 17021046

Brás-Pereira, C, and Casares, F. (2008). An antennal-specific role for bowl in repressing supernumerary appendage development in Drosophila. Mech. Dev. 125: 809-821. PubMed Citation: 18662773

Buckley, M. S., Chau, J., Hoppe, P. E. and Coulter, D. E. (2004). odd-skipped homologs function during gut development in C. elegans. Dev. Genes Evol. 214(1): 10-18. 14648222

de Celis Ibeas, J. M. and Bray. S. J. (2003). Bowl is required downstream of Notch for elaboration of distal limb patterning. Development 130: 5943-5952. 14573519

Goldstein, R. E., et al. (2005). An eh1-like motif in Odd-skipped mediates recruitment of Groucho and repression in vivo. Mol. Cell. Biol. 25(24): 10711-20. 16314497

Green, R. B., et al. (2002). Drumstick is a zinc finger protein that antagonizes Lines to control patterning and morphogenesis of the Drosophila hindgut. Development 129: 3645-3656. 12117814

Hao, I, Green, R. B., Dunaevsky, O. Lengyel, J. A. and Rauskolb, C. (2003). The odd-skipped family of zinc finger genes promotes Drosophila leg segmentation. Dev. Biol. 263: 282-295 14597202

Hart, Wang. L. and Coulter, D. E. (1996). Comparison of the structure and expression of odd-skipped and two related genes that encode a new family of zinc finger proteins in Drosophila. Genetics 144: 171-182. 8878683

Hatini, V., et al. (2000). Tissue- and stage-specific modulation of Wingless signaling by the segment polarity gene lines. Genes Dev. 14: 1364-1376. PubMed Citation: 10837029

Hatini, V., Green, R. B., Lengyel, J. A., Bray, S. J. and Dinardo, S. (2005). The Drumstick/Lines/Bowl regulatory pathway links antagonistic Hedgehog and Wingless signaling inputs to epidermal cell differentiation. Genes Dev. 19(6): 709-18. 15769943

Iwaki, D. D., et al. (2001). drumstick, bowl, and lines are required for patterning and cell rearrangement in the Drosophila embryonic hindgut. Dev. Biol. 240(2): 611-26. 11784087

Iwaki, D. D. and Lengyel, J. A. (2002). A Delta-Notch signaling border regulated by engrailed/invected repression specifies boundary cells in the Drosophila hindgut. Mech. Dev. 114: 71-84. 12175491

Johansen, K. A., Iwaki, D. D. and Lengyel, J. A. (2003a). Localized JAK/STAT signaling is required for oriented cell rearrangement in a tubular epithelium. Development 130: 135-145. 12441298

Johansen, K. A., Green, R. B., Iwaki, D. D., Hernandez, J. B. and Lengyel, J. A. (2003b). The Drm-Bowl-Lin relief-of-repression hierarchy controls fore- and hindgut patterning and morphogenesis. Mech. Dev. 120(10): 1139-51. 14568103

Mirth, C. and Akam, M. (2002). Joint development in the Drosophila leg: cell movements and cell populations. Dev. Biol. 246: 391-406. 12051824

Nusinow, D., Greenberg, L. and Hatini, V. (2008). Reciprocal roles for bowl and lines in specifying the peripodial epithelium and the disc proper of the Drosophila wing primordium. Development 135(18): 3031-41. PubMed Citation: 18701548

Rauskolb, C. and Irvine, K. D. (1999). Notch-mediated segmentation and growth control of the Drosophila leg. Dev. Biol. 210: 339-350. 10357895

Rauskolb, C. (2001). The establishment of segmentation in the Drosophila leg. Development 128: 4511-4521. 11714676

Wang, L. and Coulter, D. E. (1996). bowel, an odd-skipped homolog, functions in the terminal pathway during Drosophila embryogenesis. EMBO J. 15: 3182-3196. 8670819

Biological Overview

date revised: 11 June 2022

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