drumstick: Biological Overview | References
Gene name - drumstick
Cytological map position - 24A1-24A1
Function - putative transcription factor or transcriptional co-factor
Symbol - drm
FlyBase ID: FBgn0024244
Genetic map position - 2L: 3,539,251..3,548,086 [+]
Classification - zinc finger, odd skipped family
Cellular location - unknown
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 physically 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. 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).
Drm, Lines, and Bowl are the core components of a novel regulatory pathway. Drm and Bowl are structurally related proteins, and this forms the basis for the post-translational regulation of Bowl by Drm and Lines. Lines binds directly to Bowl and decreases the abundance of the Bowl protein, while, in cells that express drm, Drm binds directly to Lines and reverses this effect. Drm uses its N-terminal zinc finger in order to outcompete the binding of Lines to the N-terminal zinc finger of Bowl (Hatini, 2005).
Drm and Bowl most likely originated from a common ancestral gene by a process of gene duplication and divergence, as these genes map near one another and have a common aspect to their splicing pattern (Wang, 1996; Green, 2002, Johansen, 2003). Duplicated gene pairs typically perform redundant functions in a given tissue, or distinct but essential functions in different tissues as a consequence of diversification of cis-regulatory elements. However, in this particular case, drm and bowl do not appear to share cis-regulatory elements. Furthermore, the activities of Drm and Bowl are not redundant. Rather, Bowl is a nuclear protein (in cells in which it accumulates) and likely regulates target gene expression. In contrast, this study provides strong evidence that Drm uses a dominant interference mechanism to liberate Bowl from negative regulation. It is therefore proposed that drm and bowl provide an example of duplicated genes that evolved to carry out distinct molecular roles within the same regulatory pathway. It is interesting to note that the role for Drm, in this duplication-divergence scenario is likely to protect Bowl from degradation by Lines. While regulated protein degradation is at the heart of several developmentally important signal transduction pathways, including those of Hedgehogs and Wnts, the example revealed in this study - the likely inhibition of protein degradation by the patterned expression of a small peptide inhibitor (Drm) - appears to be novel (Hatini, 2005).
While the Drumstick/Lines/Bowl regulatory pathway operates in several tissues, it is likely that in a few instances other factors can substitute for drm. For instance, while bowl is necessary for the specification of distal leg identities and leg-joint morphogenesis (de Celis Ibeas, 2003; Hao, 2003), drm mutants do not exhibit an effect on these processes (Hao, 2003). It was therefore hypothesized that drm acts redundantly with either or both of two related genes mapping nearby, odd and sob, since these genes exhibit expression patterns similar to that of drm (Hao, 2003
Specialized groups of cells known as organizers establish the pattern of cell differentiation and morphogenesis across fields of progenitor cells. Although many organizer signals and their signal transducers have been identified, the pathways that link organizer signaling activity with subsequent cellular patterning and morphogenesis remain to be elucidated (Hatini and DiNardo 2001b). The embryonic epidermis in Drosophila has been contributing general insights into the mechanism of organizer function. The pattern of cell differentiation across this epidermis is organized by two conserved signals, Hedgehog and Wingless, produced from adjacent sources that flank the boundary between parasegments (PS). Following the establishment of the sources of Hedgehog and Wingless production, each signal inhibits cellular responses elicited by the other signal. Wingless inhibits Hedgehog activity by repressing Hedgehog target gene expression anterior to the source of Hedgehog production. Hedgehog, however, inhibits Wingless activity posterior to the source of Wingless production by several distinct mechanisms. This results in polarized activity of Hedgehog and Wingless from the organizer, with Hedgehog organizing the pattern of posterior cells and Wingless organizing the pattern of anterior cells. Ultimately, this initial polarity generates an asymmetric pattern of epidermal cell differentiation. The mechanisms by which epidermal cells respond to Hedgehog and Wingless signaling activities are not fully understood. In the ventral epidermis, Hedgehog and Wingless activity divide the PS into smaller territories, each a focus for a patterning signal. As a consequence of this subdivision, Hedgehog and Wingless along with Serrate and Spitz organize the final pattern. In dorsal epidermis, although Hedgehog and Wingless are again the primary organizing signals, Serrate and Spitz are not involved, and it is un-clear what mechanisms produce the final pattern. As an approach to identify the genes involved, mutants were selected in which the normal asymmetric pattern of epidermal cell differentiation was replaced with a symmetric pattern. Using this approach, the gene lines was selected for further analysis. It was subsequently shown that Lines exhibits asymmetric subcellular distribution across the PS, with enriched nuclear accumulation in cells signaled by Wingless and enriched cytoplasmic accumulation in cells signaled by Hedgehog (Hatini, 2000). This arises because Wingless promotes nuclear accumulation of Lines, and suggested that Hedgehog antagonizes Wingless signaling by localizing Lines to the cytoplasm. It was also shown that Lines mediates the cellular responses dependent on Wingless signaling, antagonizes those responses dependent on Hedgehog signaling, and in this manner contributes to the pattern of epidermal cell differentiation. It was therefore proposed that Hedgehog and Wingless signaling regulate the asymmetric subcellular distribution and consequent action of Lines across the embryonic epidermis (Hatini, 2000). That study, however, neither pinpointed the molecular mechanism by which Hedgehog and Wingless regulate the subcellular distribution and function of Lines, nor the mechanism by which Lines promotes Wingless signaling inputs and antagonizes Hedgehog signaling inputs. Finally, based on genetic analysis, it has been proposed that lines functions as a stage- and tissue-specific modulator of the Wingless signaling pathway by acting either in concert or in parallel to armadillo and dtcf/pangolin, the nuclear effectors of the Wingless signaling pathway. This study provides a revised model for the mechanism of Lines function (Hatini, 2005). .
More recent analysis of mutants that affect gut morphogenesis has shown that lines mutants exhibit phenotypes related to those of drm and bowl mutants, raising the possibility that the three genes act along the same pathway (Iwaki, 2001). Formal genetic analysis has suggested that the three genes act in a linear genetic pathway to regulate the morphogenesis of the hindgut and foregut epithelialines inhibits bowl to maintain large intestine or foregut fate, respectively, except in localized areas where drm is expressed. In these areas, drm antagonizes lines, thereby allowing bowl to specify small intestine fate in the hindgut primordium and proventricular fate in the foregut primordium (Green, 2002; Johansen, 2003). The current studies investigated neither the molecular mechanisms by which the Drm and Lines proteins regulate the activity of the Bowl protein, nor the possible involvement of this genetic pathway in other developmental processes. These studies however demonstrate that the three proteins interact physically and functionally along a molecular regulatory pathway in order to regulate the spatial pattern of Bowl protein accumulation. Depending on context, this molecular regulatory pathway can elicit specific responses such as epidermal differentiation, gut morphogenesis, and formation of leg joints and distal leg structures. In all these contexts, this pathway is engaged by organizer signals or other positional cues in order to specify distinct cell fates across fields of progenitor cells, either directly or indirectly through the production of new signals. In the embryonic epidermis, this pathway is engaged by and implements the antagonistic activities of Hedgehog and Wingless signaling. Thus, these findings define the mechanism of action of a novel molecular regulatory pathway, and demonstrate general roles for this pathway in patterning a variety of epithelial tissues (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 operation of the Drm/Lines/Bowl regulatory pathway was examined in the context of the epidermal organizer. Across the dorsal embryonic epidermis, Hedgehog and Wingless are the key pattern-organizing signals. Hedgehog specifies cell fate in half the PS (the 1°-3° cell fates), while Wingless specifies the remaining cell fate (the 4° cell fate) in the complementary half. To investigate whether Hedgehog and Wingless engage the Drm/Lines/Bowl regulatory pathway, drm gene expression and Bowl protein accumulation were examined under conditions of loss or excess of Hedgehog or Wingless signaling. Expression of drm was found to be decreased in hedgehog mutants, and expanded posteriorly in embryos expressing the secreted form of Hedgehog in Engrailed/Hedgehog-expressing cells. Two points are noteworthy here: (1) while Hedgehog can directly control drm expression posterior to the Hedgehog domain, control within the Hedgehog domain is likely indirect since these cells cannot themselves respond to Hedgehog signaling; (2) the fact that excess Hedgehog does not induce drm expression in anterior cells suggests that Wingless signaling represses drm expression in this region. Consistent with this prospect, it was found that drm expression is ectopically activated in wingless mutants and repressed upon ectopic activation of the Wingless pathway. It was also found that changes in drm expression due to manipulations of Hedgehog and Wingless signaling largely led to the expected changes in Bowl protein accumulation. For instance, broadened drm expression caused by excess Hedgehog leads to a broadened Bowl domain, while the ectopic stripe of drm expression in wingless mutants also leads to increased Bowl accumulation, although Bowl accumulates rather more broadly than the narrow drm stripe would suggest. These changes in Bowl accumulation correlate nicely with the patterning changes observed with inactivation or activation of Hedgehog or Wingless signaling. It is concluded that the asymmetric response of drm to Hedgehog underlies the pattern of epidermal cell differentiation since drm promotes the accumulation of Bowl in drm-expressing cells and consequent cellular responses elicited by Bowl. Note that Bowl accumulates in two rows of cells but apparently is required for patterning across a broader region. This observation implies that Bowl controls expression of a new signal that further elaborates epidermal pattern (Hatini, 2005).
Whether drum and lines regulate Bowl abundance in various epithelia was tested along with whether the restricted accumulation of Bowl in these epithelia controls distinct developmental fates, as it does across the embryonic epidermis. Initially, the regulation of Bowl accumulation was investigated in the gut. Genetically, bowl is required both in the foregut, where it distinguishes proventriculus from anterior gut, and in hindgut, where it distinguishes small from large intestine. Indeed, Bowl protein accumulates in two narrow domains in the gut: the primordia for the proventriculus and for the small intestine. In addition, these domains coincide with the sites of drm expression, and in drm mutants, Bowl protein was barely detectable across these domains. Conversely, in lines, as well as drm lines double mutants, Bowl accumulates ubiquitously across the foregut and hindgut primordia. Thus, in the gut just as in the embryonic epidermis, the restricted accumulation of Bowl appears to control distinct developmental fates (Hatini, 2005).
Next the analysis was extended to the leg imaginal disc epithelia, where bowl has been shown to regulate distal leg identities and leg-joint morphogenesis. It was found that the Bowl protein is detected at a set of five rings within the leg imaginal discs, and drm mRNA is detected at a set of five similar rings, supporting the idea that the Drm/Lines/Bowl regulatory pathway also operates in this tissue. To determine whether lines controls Bowl accumulation in the leg also, Bowl accumulation was examined in clones of cells mutant for lines. A cell-autonomous increase in Bowl protein accumulation was found in these clones. This ectopic Bowl accumulation disrupts the normal pattern of gene expression in the leg, as it leads to cell-autonomous reduction of bric-a-brac expression, a target gene repressed by Bowl. These regulatory interactions likely extend to several other imaginal disc epithelia, since a strong correlation was observed in the areas where Bowl is detected at high levels and the domains of drm expression in the wing and eye-antennal disc (Hatini, 2005).
This analysis in the epithelia of the embryonic epidermis, the foregut, the hindgut, and the imaginal discs provides compelling evidence that Drm, Lines, and Bowl are the core components of a novel regulatory pathway. Depending on context, this pathway can be engaged by a variety of positional cues. Once engaged, the pathway regulates the nuclear accumulation of Bowl and consequently patterning and morphogenesis in that tissue (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).
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).
The growth and patterning of Drosophila wing and notum primordia depend on their subdivision into progressively smaller domains by secreted signals that emanate from localized sources termed organizers. While the mechanisms that organize the wing primordium have been studied extensively, those that organize the notum are incompletely understood. The genes odd-skipped (odd), drumstick (drm), sob, and bowl comprise the odd-skipped family of C2H2 zinc finger genes, which has been implicated in notum growth and patterning. This study shows that drm, Bowl, and eyegone (eyg), a gene required for notum patterning, accumulate in nested domains in the anterior notum. Ectopic drm organized the nested expression of these anterior notum genes and downregulated the expression of posterior notum genes. The cell-autonomous induction of Bowl and Eyg required bowl, while the non-autonomous effects were independent of bowl. The homeodomain protein Bar is expressed along the anterior border of the notum adjacent to cells expressing the Notch (N) ligand Delta (Dl). bowl was required to promote Bar and repress Dl expression to pattern the anterior notum in a cell-autonomous manner, while lines acted antagonistically to bowl posterior to the Bowl domain. These data suggest that the odd-skipped genes act at the anterior notum border to organize the notum anterior–posterior (AP) axis using both autonomous and non-autonomous mechanisms (Del Signore, 2012).
In many developmental processes, signals that emanate from field borders play a crucial instructive role in patterning morphogenetic fields. The early Drosophila embryo is patterned by opposing gradients of Bicoid and Nanos that are generated from localized translation of corresponding mRNAs at the anterior and posterior poles of the embryo. In the embryonic epidermis, the pattern of cell differentiation across each segment is regulated by the secreted Wg and Hh signals that emanate from localized sources at the anterior and posterior borders of each segment. Similarly, the dorsoventral axis of the vertebrate spinal cord is organized by Shh ventrally, and BMP and Wnt signals that emanate from localized dorsal sources. By contrast, current models of notum AP patterning focus mainly on the organizing influence of Dpp, which is secreted from the posterior border of the notum. Previous work has found that odd-skipped genes are expressed along the anterior border of the notum, and that broadly inhibiting their function in early wing discs caused a severe reduction or complete loss of the notum. As this reduction occurred despite the maintenance of dpp expression (Nusinow, 2008), whether the odd-skipped genes might define a second organizing center within the developing notum was investigated. The current findings indeed suggest that signals that emanate from the anterior border of the notum act reciprocally to Dpp to promote expression of anterior notum genes and repress expression of posterior genes. Through loss- and gain-of-function clonal analyses, it was demonstrated that the odd-skipped genes pattern the notum AP axis both locally through regulation of Eyg, Bar, and Dl, and broadly through the regulation of Eyg and Tup. Finally, it was shown that lines acts antagonistically to bowl in this process (see Model of the role odd-skipped genes in notum AP patterning) (Del Signore, 2012).
drm overexpression was sufficient to promote Eyg accumulation non-autonomously within the notum. This activity suggests that drm controls expression of an unidentified signal that spreads from the drm domain to induce Eyg accumulation non-autonomously. Alternatively, drm could initiate the propagation of a cascade of local inductive interactions to induce Eyg at a distance. Recent studies have shown that recruitment of cells to the wing field is accomplished by the propagation of a feed forward signal from the DV compartment boundary. In this process signaling at the border between Vestigial (Vg) and non-Vg expressing cells is used to recruit non-Vg expressing cells to the expanding wing field, a process dependent on signaling through the Fat-Dachsous pathway. Though a functional relationship between odd-skipped genes and Ft-Ds signaling has yet to be characterized, it is interesting to note that Ds accumulates in a complex graded AP pattern across the notum, consistent with such a role (Del Signore, 2012).
In addition to the broad induction of Eyg accumulation, it was surprising to find that drm overexpression also induced Bowl in cells just adjacent to clones. Though the effect was subtle, it is noted that this pattern of activation recapitulated the endogenous nested pattern of drm and Bowl expression in the presumptive prescutum. It is unclear whether the nested expression of odd-skipped genes plays a functional role in notum AP patterning. Despite this, the concordance of endogenous and ectopic expression patterns supports the hypothesis that ectopic drm induces a physiologically relevant program of anterior gene expression in the notum. One possible clue as to the relevance of this nested pattern may come from the observation that only drm was able to promote Bowl non-autonomously. In contrast, lines−/−, odd+, and sob+ clones each induced only cell-autonomous accumulation of Bowl. Notably, these clones rounded up and segregated from the epithelium, while drm expressing clones remained integrated with the surrounding epithelium. One interpretation of these data is that abrupt discontinuities in the level of Odd-skipped proteins may alter epithelial morphology. This interpretation is further supported by the observation that bowl mutant clones within the Bowl domain adopt a compact, round morphology relative to clones outside the Bowl domain. It is hypothesized that drm promotes lower levels of Bowl in nearby cells to dampen otherwise sharp discontinuities in Bowl activity to regulate local buckling of the epithelium (Del Signore, 2012).
Alternatively, differences in the total levels or ratios of Odd family proteins along the anterior border of the notum could elicit different transcriptional outcomes. Since Odd and Bowl have been shown to interact with the transcriptional co-repressor Groucho, variation in the levels of the Odd-skipped proteins could titrate Groucho and affect Groucho-dependent transcriptional outputs. Alternatively, given their distinct structure outside the zinc finger domain, the Odd-skipped proteins could interact with distinct sets of target genes to pattern the anterior border of the notum. Though additional experiments will be required to ascertain whether such mechanisms are active in the prescutum, this study provides evidence that bowl is strictly required for the early autonomous induction of Eyg, the later expression of Bar genes, and the repression of Dl. These results provide evidence that odd-skipped genes act both independently and redundantly to organize the notum AP axis (Del Signore, 2012).
bowl is essential for patterning the prescutum, but not for broadly patterning the notum AP axis. Previous studies have revealed a variety of essential and redundant functions for odd-skipped family genes in patterning embryonic and larval tissues. In the embryo, drm and bowl antagonize lines function to pattern the dorsal embryonic epidermis, foregut, and hindgut, while odd functions as a pair rule gene to promote embryonic segmentation. In the leg imaginal disc, bowl is essential for patterning the tarsal proximodistal axis at early stages, but acts redundantly with other odd-skipped genes to control leg segmentation later in developmen. In the eye, bowl is essential for the initiation of retinogenesis from the eye margin, while odd and drm have been proposed to activate Bowl redundantly (Del Signore, 2012).
Loss-of-function analysis revealed that neither drm nor odd is necessary to stabilize Bowl. At present the possibility cannot be excluded that sob is necessary to promote Bowl accumulation because a null sob mutant is not yet available. Biochemical and genetic analysis demonstrates that not only Drm, but also Odd and Sob can each outcompete the interaction of Lines with Bowl and stabilize the Bowl proteins in S2 cells and in vivo. These results suggest that different combinations of Odd-skipped proteins could be used to activate bowl depending on context (Del Signore, 2012).
Previous work suggested reciprocal roles for lines and odd-skipped genes in subdividing the early wing disc into disc proper and peripodial epithelium. The loss-of-function analysis described in this study suggests that the odd-skipped genes act redundantly to control the early specification of the PE and the subsequent expansion of the notum, while revealing an essential role for bowl in specification of the anterior prescutum. Redundancy can increase the robustness of essential developmental processes and provide a buffer against fluctuations in activity of single genes. The redundant role of the odd-skipped genes in PE specification and notum expansion could therefore serve to ensure the optimal growth of the wing disc at early stages and that of the notum at later stages and protect these critical processes from perturbations (Del Signore, 2012).
It is concluded that the growth and patterning of the wing field are coordinated with the elaboration of the wing PD axis. The developing notum lacks an obvious PD axis, and instead is subdivided into a series of AP and mediolateral domains. The establishment of organizers that act antagonistically from opposing field borders is a robust strategy to subdivide the notum AP axis. This work demonstrates that the odd-skipped genes act autonomously at the anterior border of the notum to specify the prescutum, and non-autonomously at short and long range to control the expression of transcription factors that prefigure the differentiation of the notum AP axis. Though further experiments will be required to characterize the mechanism by which this putative organizer acts, these studies provide evidence that the anterior border of the notum exhibits the functional attributes of an organizer (Del Signore, 2012).
During development, cascades of regulatory genes act in a hierarchical fashion to subdivide the embryo into increasingly specified body regions. This has been best characterized in Drosophila, where genes encoding regulatory transcription factors form a network to direct the development of the basic segmented body plan. The pair-rule genes are pivotal in this process as they are responsible for the first subdivision of the embryo into repeated metameric units. The Drosophila pair-rule gene fushi tarazu (ftz) is a derived Hox gene expressed in and required for the development of alternate parasegments. Previous studies suggested that Ftz achieves its distinct regulatory specificity as a segmentation protein by interacting with a ubiquitously expressed cofactor, the nuclear receptor Ftz-F1. However, the downstream target genes regulated by Ftz and other pair-rule genes to direct segment formation are not known. In this study, candidate Ftz targets were selected by virtue of their early expression in Ftz-like stripes. This identified two new Ftz target genes, drumstick (drm) and no ocelli (noc), and confirmed that Ftz regulates a serotonin receptor (5-HT2). These are the earliest Ftz targets identified to date and all are coordinately regulated by Ftz-F1. Engrailed (En), the best-characterized Ftz/Ftz-F1 downstream target, is not an intermediate in regulation. The drm genomic region harbors two separate seven-stripe enhancers, identified by virtue of predicted Ftz-F1 binding sites, and these sites are necessary for stripe expression in vivo. It is proposed that pair-rule genes, exemplified by Ftz/Ftz-F1, promote segmentation by acting at different hierarchical levels, regulating first, other segmentation genes; second, other regulatory genes that in turn control specific cellular processes such as tissue differentiation; and, third, 'segmentation realizator genes' that are directly involved in morphogenesis (Hou, 2009).
This study identified Ftz targets based on a search for genes expressed in striped patterns in the early Drosophila embryo. Each of these Ftz-dependent genes is also regulated by Ftz-F1, an orphan nuclear receptor previously shown to interact with Ftz in vitro and in vivo. Unlike Ftz, which is expressed in a striped pattern in the Drosophila blastoderm, Ftz-F1 is expressed ubiquitously, in all somatic cells at the blastoderm stage. The finding in this study that all three additional Ftz-dependent genes, identified by virtue of their striped expression patterns, require Ftz-F1 for expression in stripes lends support to the model that interaction with Ftz-F1 is the key to Ftz functional specificity as a segmentation protein. The three genes characterized in this study, 5-HT2, noc and drm, are the earliest identified downstream targets of Ftz. Expression in stripes was observed at the cellular blastoderm stage when Ftz-F1 is highly expressed throughout the embryo and the seven Ftz stripes are at their peak levels. These early target gene stripes were lost in ftz and also in ftz-f1 mutants. In addition, ectopic expression was observed at early stages when Ftz was ectopically expressed throughout the embryo. En, long thought to be a major mediator of Ftz function in segmentation, is expressed later than these target genes, and it was verified that En is not required for the Ftz-dependent stripe expression of noc or drm. These findings suggest that Ftz and Ftz-F1 directly regulate expression of these three target genes. This new study brings to seven the targets of Ftz that appear to be directly co-regulated by Ftz and Ftz-F1: ftz itself, en, apt, Dsulf1, 5HT-2, noc and drm. For each gene, multiple potential Ftz-F1 binding sites were found within a 15-20 kb genomic region. In all cases, multiple potential Ftz binding sites surround the Ftz-F1 binding sites that could mediate cooperative interactions between Ftz and Ftz-F1. Many of these sites have been maintained during evolution and are present in distant Drosophila species. Other Ftz targets, such as Ubx, prd, odd and tsh are also likely to be co-regulated by Ftz-F1 (Hou, 2009).
The seven Ftz/Ftz-F1 target genes identified to date play diverse roles in segmentation and act at different levels of the embryonic hierarchy. First, Ftz acts in a cross-regulatory fashion to modulate expression of other pair-rule genes: it interacts with Ftz-F1 in autoregulation and also has been shown to regulate the pair-rule genes prd, odd and slp. Second, Ftz/Ftz-F1 directly regulate components of the segment polarity system: first, they activate en expression in alternate stripes, and, second, they regulate Dsulf1, thought to modulate Wg activity. Ftz has also been shown to repress wg expression. Ftz/Ftz-F1 thus indirectly control compartment border formation, via regulation of En and Wg. Third, Ftz/Ftz-F1 regulate transcription factors that in turn control the differentiation of specific cell types: apt, noc, drm. drm encodes an odd-skipped family zinc finger transcription factor that it is required for patterning the dorsal epidermis, thus regulating the differentiation of specific cell types. noc plays a role in trachael morphogenesis with mutants displaying defects in branch migration and expanded expression of trachael-specific genes. Similarly, apt is involved in this process as a regulator of the migration of trachael precursor cells. Finally, Ftz/Ftz-F1 regulate a target gene more directly involved in morphogenesis, 5HT-2. 5-HT2 encodes a serotonin receptor that demonstrates specific ligand binding in transfected cells and in Drosophila embryo extracts. Phenotypic analysis suggested a role for 5-HT2 and other genes involved in serotonin biosynthesis in morphogenetic movements during gastrulation: deficiency embryos lacking 5HT-2 displayed delayed and incomplete movements during germband extension accompanied by mislocalization of Armadillo protein, suggestive of abnormalities in adherens junctions. It will be of interest in the future to determine whether other pair-rule genes direct expression of additional cell surface proteins that coordinate these processes (Hou, 2009).
This study has identified enhancers of drm by combining bioinformatics with enhancer-reporter gene expression analysis in vivo. Fragments chosen for the in vivo analysis contained one or more matche(s) to a Ftz-F1 binding site. Three of the four fragments directed expression in drm-like patterns in vivo. The upstream fragment, drm1, harbors a late stage enhancer that directs segmental expression of drm. drm2 directed expression in seven strong stripes. drm34 harbors enhancers for the region-specific expression of drm in the proventriculus and hindgut, expression that is important for the development of the fore- and hindgut, as well as an early 7-stripe enhancer. Whether any of these enhancers also direct expression in the leg imaginal discs was not investigated. Two of the fragments, drm2 and drm34, directed expression in 7-stripe patterns. Surprisingly, for each of them, the set of seven stripes is in register with Ftz, suggesting that both regulate expression of the drm-primary stripes. Although unexpected, this phenomenon has been observed in other cases where it was suggested that enhancers directing the same or similar expression patterns function as shadow enhancers to enhance the precision of expression patterns and facilitate the rapid evolution of cis-regulatory sequences. Point mutations of either or both of the predicted Ftz-F1 binding sites in the drm34 Early 7-Stripe Enhancer abolished expression of lacZ fusion genes. Stripe expression was decreased but not completely abolished by mutation of the single predicted Ftz-F1 binding site in the drm2 7-Stripe Enhancer, suggesting additional inputs into regulation of the drm primary stripes by this enhancer. Together, these results suggest that Ftz-F1 activates expression in the primary drm stripes via the drm34 Early 7-Stripe Enhancer. It is speculated that following this initial activation, autoregulation by Drm may augment Ftz-F1 activation of stripes via the drm2 7-Stripe Enhancer to raise levels of transcription in drm primary stripes (Hou, 2009).
Drosophila ftz is a typical pair-rule gene: ftz mutant embryos die lacking even-numbered body segments. How this wild type function of ftz, and other pair-rule genes, is executed is not yet known. As for other segmentation mutants, the pair-rule mutant phenotype results from cell death. However, this cell death appears to be an indirect effect. Similarly, pair-rule genes regulate segment border formation indirectly, via activation of the segment polarity genes such as en and wg. In addition to this, segment-polarity-independent roles for the pair-rule genes in morphogenesis have been revealed by careful studies from the Wieschaus lab. For example, it was found that cell intercalation and germ band extension are regulated by the pair-rule genes, independent of segment polarity genes. Similarly, cellular studies defined two subtle morphogenetic processes that occur before gastrulation - one, controlled by the pair-rule gene paired. More recently, studies have shown that the planar polarity and organization of intercalating cells during germ band extension are controlled by the striped expression patterns of eve and runt and that the longitudinal division of cells during germ band extension is controlled by eve. These studies are suggestive of direct roles for the pair-rule system in cell shape changes and rearrangements during germ band extension. Together, these studies support the notion that combinatorial expression of early patterning genes assigns unique identities in the blastoderm at a single cell level. This study has shown that the pair-rule gene ftz regulates target genes prior to and independently of En. These findings support the model that the stripes of pair-rule genes play active roles in patterning the embryo rather than serving solely as intermediary patterns whose function is to produce the segmental stripes of segment polarity genes. One role for these pair-rule stripes may be to establish differential adhesiveness to groups of cells in the blastoderm embryo. Future work identifying additional pair-rule targets will be required to explain the fundamental biological roles of pair-rule patterning and to understand how the assignment of positional identities by pair-rule genes, prior to morphogenesis, translates into the development and differentiation of body segments (Hou, 2009).
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).
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).
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 foregut 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, 2003).
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, 2003 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, 2003).
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, 2003).
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, 2003).
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, 2003).
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, 2003).
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, 2003).
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, 2003).
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, 2003).
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, 2003).
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, 2003).
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, 2003).
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, 2003).
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, 2003 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, 2003).
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, 2003).
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, 2003).
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, 2003).
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, 2003).
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, 2003).
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, 2003).
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, 2003 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, 2003).
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, 2003).
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).
Search PubMed for articles about Drosophila Drumstick
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 ID: 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 ID: 17021046
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
Del Signore, S. J., Hayashi, T. and Hatini, V. (2012). odd-skipped genes and lines organize the notum anterior-posterior axis using autonomous and non-autonomous mechanisms. Mech Dev 129: 147-161. PubMed ID: 22613630
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
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 ID: 10837029
Hou, H. Y., et al. (2009). Stripy Ftz target genes are coordinately regulated by Ftz-F1. Dev. Biol. 335(2): 442-53. PubMed ID: 19679121
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
Johansen, K. A., Green, R. B., Iwaki, D. D., Hernandez, J. B. and Lengyel, J. A. (2003). The Drm-Bowl-Lin relief-of-repression hierarchy controls fore- and hindgut patterning and morphogenesis. Mech. Dev. 120(10): 1139-51. 14568103
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: 3031-3041. PubMed ID: 18701548
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
date revised: 25 March 2013
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