Effects of Mutation or Deletion

Table of contents

Wingless and organ development

Wingless is required for cell proliferation in the Malpighian tubule anlage. The pattern of Wingless mRNA and protein in Wingless developing tubules is consistent with a requirement for wingless for cell division. Analysis of the temporal requirement for wingless using a temperature-sensitive allele confirms that the normal expression of wingless is necessary during cell proliferation in Malpighian tubules. Overexpression of wingless results in supernumerary cells in the tubules (Skaer, 1992).

The proventriculus is a multiply folded muscular organ of the foregut formed from a simple epithelial tube. It functions to grind and masticate food. Coordinated cell movements are critical for tissue and organ morphogenesis in animal development. Drosophila genes hedgehog and wg, which encode signaling molecules, and the gene myospheroid, which encodes a beta subunit of the integrins, are required for epithelial morphogenesis during proventriculus development. In contrast, this morphogenetic process is suppressed by the decapentaplegic gene (Pankratz, 1995).

A unique cell, the tip mother cell, arises in the primordium of each Drosophila Malpighian tubule by lateral inhibition within a cluster of achaete-expressing cells. This cell maintains achaete expression and divides to produce daughters of equivalent potential, of which only one, the tip cell, adopts the primary fate and continues to express achaete, while in the other, the sibling cell, achaete expression is lost. In this paper the mechanisms are charted by which achaete expression is differentially maintained in the tip cell lineage to stabilize cell fate. Initially, wingless is required to maintain the expression of achaete in the tubule primordium so that wingless mutants lack tip cells. Conversely, increasing wingless expression results in the persistence of achaete expression in the cell cluster. Then, Notch signaling is restricted by the asymmetric segregation of Numb, as the tip mother cell divides, so that achaete expression is maintained only in the tip cell. In embryos mutant for Notch, tip cells segregate at the expense of sibling cells, whereas in numb neither daughter cell adopts the tip cell fate resulting in tubules with two sibling cells. Conversely, when numb is overexpressed two tip cells segregate and tubules have no sibling cells. Analysis of cell proliferation in the developing tubules of embryos lacking Wingless, after the critical period for tip cell allocation, reveals an additional requirement for wingless for the promotion of cell division. In contrast, alteration in the expression of numb has no effect on the final tubule cell number (Wan, 2000).

In order to establish when the wg product is required for tip cell allocation, wg activity was manipulated using the temperature-sensitive allele, wgIL114. At the permissive temperature tip cells develop normally, while at the restrictive temperature Wg function is lost and tip cells fail to appear. Temperature shift experiments establish that embryos must develop at the permissive temperature between 4 and 5 h for tip cells to appear normally. In accordance with these findings, embryos shifted to the permissive temperature for this period develop with tip cells, while a shift to the restrictive temperature results in tubules with no tip cells. Given that the restoration of wild-type wg protein has been shown to take 20-30 min and that the removal of functional protein takes a similar time after shifting temperature, these results establish the window of requirement as 4.5-5.5 h into embryogenesis for functional Wg to be expressed in the tubules for tip cell allocation. Wg is normally expressed in the tubule primordia as they evert from the hindgut. From 4.5 h, expression is higher in the posterior region of the developing tubules than the anterior. The tip mother cell segregates from this posterior region, where wg continues to be expressed. Wg is lost from tubules during stage 12. Manipulation of wg expression using the temperature sensitive (ts) allele reveals a requirement for wg in cell division, separate from its role in tip cell specification. If Wg is removed after 5 or 6 h after egg lay (AEL) but before cell division ceases in the tubules, the final tubule cell number is reduced compared to wild type. This indicates that in the absence of Wg, the appearance of tip cells is insufficient to promote the normal pattern of division in the tubules (Wan, 2000).

The tip cell progenitor is selected from a group of competent cells by lateral inhibition and is demarcated by the continued expression of ac. Further extrinsic and intrinsic cues (Wg signaling and the asymmetric distribution of Nb) operate to ensure the continued expression of ac and so confirm tip cell potential. The selection of cell fate from an equivalence group by lateral inhibition alone relies on chance fluctuations in the equilibrium of signaling between cells and therefore may not be completely reliable. The activity of other genes, by biasing lateral inhibition, serves to make the selection of cells to specific fates more robust. Such mechanisms have been shown to confirm cell fate in the PNS and of the anchor cell in the nematode gonad. The results presented here indicate that wg and nb are required for the specification of the tip cell and sibling cell fate in the Malpighian tubules. The activity of these two genes biases the outcome of intercellular signaling at separate stages in this process, resulting in the reliable allocation of tip and sibling cell fates, suggesting that this distinction is important to the development of the tubules. However, it is clear that continued cell division in the tubules relies only on the allocation of the tip cell progenitor and not on the differentiation of fate between the tip cell's daughter cells, in which nb plays an important role (Wan, 2000).

This result is surprising, since Nb is active where sister cells of specific lineages are allocated to separate cell fates, for example, in the PNS, in the CNS, and in myogenesis. Separation between sister cell fates involves the maintenance of gene expression in one sibling and its repression in the other, for example, of Kr, eve, and S59 in sibling muscle founder cells. This pattern is also seen in the tubules: ac, Kr, and Dl continue to be expressed in the tip cell but are repressed in the tip cell's sibling. In the neural and myogenic lineages the correct allocation of sibling cell fates underpins normal tissue differentiation. In the tubules, the separate roles of the tip cells and their siblings are not yet known; they both appear to be active in regulating cell proliferation but later only the tip cell expresses genes characteristic of neuronal cells. The later function of both cell types has yet to be elucidated. By manipulating nb, Malpighian tubules that lack sibling cells can be generated, but these have two tip cells or have two sibling cells but lack tip cells, thus providing an important tool for this analysis (Wan, 2000).

Wingless and mesoderm development

In the mesoderm of Drosophila embryos, a defined number of cells segregate as progenitors of individual body wall muscles. Progenitors and their progeny founder cells display lineage-specific expression of transcription factors but the mechanisms that regulate their unique identities are poorly understood. The homeobox genes ladybird early and ladybird late are shown to be expressed in only one muscle progenitor and its progeny: the segmental border muscle (SBM) founder cell and two precursors of adult muscles. lb activity is associated with all stages of SBM formation, namely the promuscular cluster, progenitor cell, founder cell, fusing myoblasts and syncytial fiber. The segregation of the ladybird-positive progenitor requires coordinate action of neurogenic genes and an interplay of inductive Hedgehog and Wingless signals from the overlying ectoderm. The SBM progenitor corresponds to the most superficial cell from the promuscular cluster, thus suggesting a role for the overlying ectoderm during its segregation. Since epidermal Wg and Hedgehog (Hh) signaling has been shown to influence muscle formation, the SBM-associated lb expression was examined in embryos carrying hh and wg thermosensitive mutations. Wg and Hh signalings, mutually dependent at this time, are shown to be required for the promuscular lb activity and/or the segregation of SBM progenitors. The initial influence of these signals is no longer observed later in development. In addition to signals from the epidermis, the activity of the mesodermal gene tinman, initially expressed in the whole trunk mesoderm, is involved in the early events of myogenesis. In tin - embryos, the formation of SBM promuscular clusters and segregation of lb-positive progenitor cells are strongly affected, leading to the absence of the majority of SBM fibers. During promuscular cluster formation, since tin expression becomes restricted to the dorsal mesoderm, its influence on ventrolaterally located SBMs is likely to be indirect and mediated via an unknown factor. The lack of neurogenic gene function, known to be involved in cell-cell interactions during lateral inhibition, generates the opposite phenotype. Mastermind - and Enhancer of split - embryos fail to restrict promuscular lb expression to only one cell; in consequence, they display a hyperplastic lb pattern in later stages (Jagla, 1998).

During Drosophila embryogenesis, mesodermal cells are recruited to form a stereotyped pattern of about 30 different larval muscles per hemisegment. The formation of this pattern is initiated by the specification of a special class of myoblasts, called founder cells, that are uniquely able to fuse with neighbouring myoblasts. The COE transcription factor Collier plays a role in the formation of a single muscle (muscle DA3[A] in the abdominal segments; DA4[T] in the thoracic segments T2 and T3). Col expression is first observed in two promuscular clusters (in segments A1-A7), corresponding to two progenitors and then their progeny founder cells, but its transcription is maintained in only one of these four founder cells, the founder of muscle DA3[A]. This lineage-specific restriction depends on the asymmetric segregation of Numb during the progenitor cell division and involves the repression of col transcription by Notch signaling. In col mutant embryos, the DA3[A] founder cells form but do not maintain col transcription and are unable to fuse with neighbouring myoblasts, leading to a loss-of-muscle DA3[A] phenotype. In wild-type embryos, each of the DA3[A]-recruited myoblasts turns on col transcription, indicating that this conversion, accomplished by the DA3[A] founder cell, induces the ‘naive’ myoblasts to express founder cell distinctive patterns of gene expression, activating col itself. Muscles DA3[A] and DO5[A] (DA4[T] and DO5[T] respectively) derive from a common progenitor cell, the DA3[A]/DO5[A] progenitor. However, ectopic expression of Col is not sufficient to switch the DO5[A] to a DA3[A] fate. Together these results lead to a proposal that specification of the DA3[A] muscle lineage requires both Col and at least one other transcription factor, supporting the hypothesis of a combinatorial code of muscle-specific gene regulation controlling the formation and diversification of individual somatic muscles (Crozatier, 1999a).

The col-expressing promuscular clusters and progenitor cells have a distinctive position, as defined relative to morphological landmarks and ectodermal Engrailed (En) expression. The DA3[A]/DO5[A] progenitor cell lies underneath the anterior epidermal compartment, whereas the DT1[A]/DO4[A] progenitor cell lies on the anterior edge of the posterior compartment, consistent with mapping of the primordium for the somatic mesoderm. Since Wingless (Wg) and Hedgehog (Hh) signaling have been shown to be required for mesoderm segmentation and formation of a subset of muscle founder cells, col expression was analyzed in wg and hh mutant embryos. At stage 10, both mutant embryos show changes in mesodermal col expression: rather than being restricted to specific clusters in the anterior compartment, it appears almost continuous along the anteroposterior axis. Therefore, both wg and hh signalings appear to restrict col transcription to specific clusters. Lack of Wg or Hh activity does not seem, however, to impede specification of the DA3[A]/DO5[A] progenitor, which is singled out in the mutant as well as the wild-type embryos. It was noticed, however, that, while the DA3[A]/DO5[A] progenitor appears to be specified normally, more than one cell is singled out from the DT1[A] /DO4[A] cluster in hh mutant embryos (Crozatier, 1999a).

Muscle founder cells arise from the asymmetric division of muscle progenitor cells, each of which develops from a group of cells in the somatic mesoderm that express lethal of scute. All the cells in a cluster can potentially form muscle progenitors, but owing to lateral inhibition, only one or two develop as such. Muscle progenitors, and the subsequent founder cells, then express transcription factors such as Krüppel, S59 and Even-skipped, all of which confer identity on the muscle. Definition of some muscle progenitors, including three groups that express S59, depends on Wingless signaling. Lateral inhibition requires Delta signaling through Notch and the transcription factor Suppressor of Hairless. Since the Wingless and lateral-inhibition signals are sequential, one might expect that muscle progenitors would fail to develop in the absence of Wingless signaling, regardless of the presence or absence of lateral-inhibition signaling. The development of the S59-expressing muscle progenitor cells has been examined in mutant backgrounds in which both Wingless signaling and lateral inhibition are disrupted. Progenitor cells fail to develop when both these processes are disrupted. This analysis also reveals a repressive function of Notch, required before or concurrent with Wingless signaling that is unrelated to its role in lateral inhibition (Brennan, 2000).

During wild-type development, expression of S59 is first seen during stage 10 in a single muscle progenitor cell either side of the midline in every segment. By stage 11, this pattern has evolved in abdominal segments such that S59 expression is seen both in the nervous system and in two groups of muscle progenitor cells. During stage 12, a third muscle progenitor cell starts to express S59. These muscle progenitor cells give rise to three muscle founder cells that maintain the expression of S59. Fusion of these founder cells with myoblasts results in the S59-expressing muscles seen in late stages of embryogenesis (Brennan, 2000).

Disruption of lateral-inhibition signaling, in either Notch (N) germline-clone, suppressor of Hairless germline-clone or Delta zygotic mutant embryos, increases the number of cells expressing S59 compared with wild type at stage 11. Because of general degeneration of these embryos during germ-band retraction, however, it is difficult to examine the expression of S59 after stage 11, but the mesoderm clusters that can be identified are expanded (Brennan, 2000).

Unlike the disruption of lateral-inhibition signaling, attenuation of Wingless signaling, by removing either wingless (wg) or dishevelled function, blocks the expression of S59 in the mesoderm. In contrast, increasing Wingless signaling, either by overexpressing the Wingless protein in the mesoderm using the GAL4/UAS system (twist-GAL4>UASwg embryos), or by removing shaggy function (sggm11 germline-clone embryos), leads to enlarged groups of S59-expressing muscle progenitor cells during stage 11. However, during germ-band retraction, the groups are reduced in size. In the twist-GAL4>UASwg embryos the reduction in cluster size leads to a largely normal set of three muscles, whereas in the sggm11 embryos the reduction is more extreme and leads to the loss of S59-expressing muscles (Brennan, 2000).

Since Wingless signaling is required for the initiation of S59 expression in the mesoderm and lateral-inhibition signaling is required for the subsequent restriction of S59 expression to one or two cells within each cluster, it is expected that in the absence of Wingless signaling S59 will not be expressed, even if lateral-inhibition signaling is also blocked. This appears to be the case in wgS107.5;DlFX3 zygotic and wgS107.5,Su(H)SF8 germline-clone embryos. In contrast, mesodermal S59 expression is observed in Df(1)N81k1,dshv26 and Df(1)N81k1;wgCX4 germline-clone embryos, in which Wingless signaling is blocked and Notch function is removed. Finally, as with the single-mutant embryos, the double-mutant embryos degenerate during germ-band retraction, making it difficult to examine S59 expression after stage 11 (Brennan, 2000).

These results first confirm that Wingless signaling is required for the initiation of S59 expression and that a Delta-initiated lateral-inhibition signal is required for the restriction of S59 expression to one or two cells of each initial cluster. They also confirm the prediction that, in the absence of a Wingless signal, S59 is not expressed, regardless of whether lateral-inhibition signaling is occurring. Also, even though hyperactivating Wingless signaling leads to initially enlarged groups of S59-expressing muscle progenitor cells, a reasonably normal muscle pattern is obtained (Brennan, 2000).

The observed S59 expression in Df(1)N81k1, dshv26 and Df(1)N81k1; wgCX4 embryos can be explained if it is assumed that Notch has a repressive function that precedes Wingless signaling. In this situation, removal of Notch function will lead to the derepression of S59 expression before Wingless signaling. Consequently, it does not matter whether or not Wingless signaling occurs. This repressive function cannot be related to Delta signaling, however, because the removal of Delta or Su(H) function in embryos where Wingless signaling is not occurring does not result in S59 expression. The repressive function of Notch uncovered in these experiments must therefore be distinct from its repressive role during lateral inhibition (Brennan, 2000).

The second observation suggests that in response to increased Wingless signaling there is a linked increase in lateral-inhibition signaling. This would mean that increased Wingless signaling will only lead to a significant increase in the number of muscle progenitors if lateral inhibition cannot occur. The observed difference in the final muscle pattern between twist–GAL4>UASwg and sggm11 embryos is probably due to the difference in how Wingless signaling is activated in the different embryos. In the twist–GAL4>UASwg embryos, Wingless signaling is activated only transiently and is restricted to the mesoderm. In contrast, Wingless signaling is activated globally and throughout embryogenesis in sggm11 germlineclone embryos. This difference, along with the proposed linkage between Wingless signaling and lateral inhibition would mean that lateral inhibition is much greater in the sggm11 embryos. This situation would explain the greater reduction in the size of the groups of S59-expressing muscle progenitor cells observed in the sggm11 embryos and the loss of muscles if the restriction is too great (Brennan, 2000).

The link between Wingless signaling and lateral inhibition could occur in a number of ways. For example, Wingless signaling may directly alter a component of the Delta signaling pathway that would then increase the ability of this pathway to transduce the Delta signal. Alternatively, Wingless signaling could affect Delta signaling by altering the transcription of one of the components of the pathway. Either of these mechanisms would allow the organism to generate a lateral-inhibition signal appropriate to the input signal: a strong Wingless signal would lead to a strong lateral-inhibition signal and prevent unnecessary and unwanted development, whereas a weak Wingless signal would lead to a weak lateral-inhibition signal that allows development to proceed even though the input signal is weak. This would allow normal development to occur even if there are fluctuations in the input signal (Brennan, 2000).

It is thought that the muscle progenitor cells develop from a large pool of developmentally equivalent cells that is refined through two steps to produce one muscle progenitor cell. A very large group of cells is initially defined that have the potential to become muscle progenitor cells but are prevented from doing so by the novel function of Notch identified here. Wingless signaling then alleviates this repressive function of Notch within a few cells of the cluster to establish an equivalence group. This triggers the process of lateral inhibition, which subsequently selects a single cell to become a muscle progenitor. In this situation, overexpressing Wingless or constitutively activating Wingless signaling will alleviate the initial repressive function of Notch in all the cells is observed, revealing the larger extent of the initial cluster. The linked increase in lateral-inhibition signaling, however, ensures that the normal number of muscle progenitor cells develop (Brennan, 2000).

This model contrasts with others in which Wingless signaling is instructive and defines the position at which muscle progenitor cells will develop, but can explain why overexpressing Wingless leads to the development of S59-expressing muscles in their normal position. In this model the Wingless signal is permissive and not instructive: it does not define where S59 will be expressed but merely reveals places defined by earlier mechanisms. Finally, these data suggest that the loss of S59 expression in the absence of a Wingless signal is due to the early repression mediated by Notch (Brennan, 2000).

Mutations in wingless leads to the complete loss of a subset of muscle founder cells characterised by the expression of NK1/S59. Wingless acts directly on the mesoderm to ensure the formation of NK1-expressing founder cells. Wg can signal across germ layers: in the wild-type embryo, Wg from the ectoderm constitutes an inductive signal for the initiation of the development of a subset of somatic muscles (Baylies, 1995).

The somatic muscles, the heart, the fat body, the somatic part of the gonad and most of the visceral muscles are derived from a series of segmentally repeated primordia in the Drosophila mesoderm. This work describes the early development of the fat body and its relationship to the gonadal mesoderm, as well as the genetic control of the development of these tissues. The first sign of fat body development is the expression of serpent in segmentally repeated clusters within the trunk mesoderm in parasegments 4-9. Segmentation and dorsoventral patterning genes define three regions in each parasegment in which fat body precursors can develop. The primary and secondary dorsolateral fat body primordia are formed ventral to the visceral muscle primoridium in each parasegment. The ventral secondary cluster forms more ventrally in the posterior portion of each parasegment. Fat body progenitors in these regions are specified by different genetic pathways. Two dorsolateral regions require engrailed and hedgehog (within the even-skipped domain) for their development while the ventral secondary cluster is controlled by wingless. Ubiquitous mesodermal en expression leads to an expansion of the primary clusters into the sloppy-paired domain, resulting in a continuous band of serpent-expressing cells in parasegments 4-9. The observed effect of en on fat body development is seen not only on mesodermal overexpression but also when en is overexpressed in the ectoderm. Loss of wingless leads to an expansion of the dorsolateral fat body primordium. decapentaplegic and one or more unknown genes determine the dorsoventral extent of these regions. High levels of Dpp repress serpent, resulting in the formation of visceral musculature, an alternative cell fate (Reichmann, 1998).

In each of parasegments 10-12 one of these primary dorsolateral regions generates somatic gonadal precursors instead of fat body. The balance between fat body and somatic gonadal fate in these serially homologous cell clusters is controlled by at least five genes. A model is suggested in which tinman, engrailed and wingless are necessary to permit somatic gonadal develoment, while serpent counteracts the effects of these genes and promotes fat body development. In wg mutant embryos, all dorsolateral mesodermal cells, including those in parasegments 10-12, acquire fat body fate. This phenotype can be interpreted as the combined effects of two separate functions of wg: (1) wg is necessary to repress fat body development in the dorsolateral mesoderm underlying the wg domain in all parasegments; (2) wg is required in the primary cluster to permit somatic gonadal precursor instead of fat body development in parasegments 10-12. Loss of engrailed results in the absence of demonstrable somatic gonadal precursors, similar to the situation in tinman mutants. Ubiquitous mesodermal en expression leads to the formation of additional somatic gonadal precursor cells in parasegments 10-12. The homeotic gene abdominalA limits the region of serpent activity by interfering in a mutually repressive feed back loop between gonadal and fat body development. It is unlikely that abdA represses srp directly, since srp can be expressed in cells in which abdA is active. abdA might prevent srp from inhibition of a somatic gonadal precursor competence factor (Riechmann, 1998).

Wingless and terminalia

This study reports the expression pattern of Dll in the genital disc, the requirement of Dll activity for the development of the terminalia and the activation of Dll by the combined action of the morphogenetic signals Wingless (Wg) and Decapentaplegic (Dpp). In Drosophila, the terminalia comprise the entire set of internal and external genitalia (with the exception of the gonads), and includes the hindgut and the anal structures. They arise from a single imaginal disc of ventral origin which is of complex organization and shows bilateral symmetry. The genital disc shows extreme sexual dimorphism. Early in development, the anlage of the genital disc of both sexes consists of three primordia: the female genital primordium (FGP); the male genital primordium (MGP), and the anal primordium (AP). In both sexes, only two of the three primordia develop: the corresponding genital primordium and the anal primordium. These in turn develop, according to the genetic sex, into female or male analia. The undeveloped genital primordium is the repressed primordium (either RFP or RMP, for the respective female and male genital primordia) (Gorfinkiel, 1999).

During the development of the two components of the anal primordium -- the hindgut and the analia -- only the latter is dependent on Dll and hedgehog (hh) function. The hindgut is defined by the expression of the homeobox gene even-skipped. The lack of Dll function in the anal primordia transforms the anal tissue into hindgut by the extension of the eve domain. Meanwhile targeted ectopic Dll represses eve expression and hindgut formation. The Dll requirement for the development of both anal plates in males and only for the dorsal anal plate in females, provides further evidence for the previously held idea that the analia arise from two primordia. In addition, evaluation was made of the requirement for the optomotor-blind (omb) gene which, as in the leg and antenna, is located downstream of Dpp. These results suggest that the terminalia show similar behavior as the leg disc or the antennal part of the eye-antennal disc, consistent with both the proposed ventral origin of the genital disc and the evolutive consideration of the terminalia as an ancestral appendage (Gorfinkiel, 1999).

The requirement for the Hh signal in Dll activation might be mediated by Wg and Dpp signals. This occurs in other ventral discs. Dll expression arises at the juxtaposition of Wg and Dpp expressing cells as revealed by double staining for Dll and Dpp, and Dll and Wg. In both genital and anal primordia, Dll expressing cells overlap those that express wg and dpp. It has been previously reported that the ectopic expression of both Wg and Dpp produces several phenotypic alterations in both female and male terminalia. Similar types of transformations are also induced by the lack of function of either patched (ptc) or Protein kinase A (Pka). In these mutants, the Hh pathway is constitutively active giving rise to the derepression of Wg and Dpp. The lack of Pka function in the genital disc induces ectopic Dll. This Dll induction requires both Wg and Dpp signals in the same cells since Dll is not activated in Pka2;dpp2 and in Pka2;wg2 double mutant clones, as occurs in other discs of ventral origin (Gorfinkiel, 1999).

In the male repressed primordium (RMP) of the female genital disc, wg is expressed but not dpp. Consequently, Dll is not expressed because Dll is only activated in cells that express both dpp and wg. Ectopic Dpp expression in the wg expression domain driven by the MS248-GAL4 line induces Dll 'de novo' in the RMP, which shows an increase in size. However, these changes do not allow the development of adult structures from this primordium since there is no activation of the male specifc cyto-differentiation genes because the genetic sex has not changed. Dll is not activated in the repressed female primordium (RFP) of the male genital disc despite the fact that, in this primordium, both wg and dpp are normally expressed. This activation does not occur even if the levels of Dpp are increased. These results suggest that specific genes expressed in the RFP can exert a negative control of Dll expression (Gorfinkiel, 1999).

In order to find other genes involved in the development of the terminal structures, the expression pattern and the functional requirement for optomotor-blind (omb) were examined. This gene encodes a protein with a DNA-binding domain (T domain) and behaves as a downstream gene of the Hh pathway in other imaginal discs. In the genital disc, Omb is detected in the dpp expression domains, abutting the wg expressing cells. This behaviour of omb expression is similar to that found in the leg and antennal discs. In the genital disc, omb is also regulated by the Hh signaling pathway since Pka2 clones also ectopically express omb. The phenotypes produced due to omb lack of function using the allele omb282 were examined; homozygous females for this allele could not be obtained but some male pharates were analyz

ed. In males, the dorsal bristles of the claspers and the hypandrium bristles are absent. Also, the hypandrium is devoid of hairs and the hypandrium fragma is reduced. Surprisingly, the anal plates are mostly somewhat enlarged in the ventral region and reduced in the dorsal areas. The structures affected in omb2 are duplicated when omb is overexpressed in the dpp domain using the dpp-GAL4/UAS-omb combination. In males, the dorsal bristles of the clasper and the hypandrium bristles are duplicated. These phenotypes are similar to the ones obtained as a result of ectopic Dpp (Gorfinkiel, 1999).

The hindgut of the Drosophila embryo is subdivided into three major domains, the small intestine, large intestine, and rectum, each of which is characterized by specific gene expression. The expression of wingless, hedgehog, decapentaplegic, and engrailed corresponds to the generation or growth of particular domains of the hindgut. wg, expressed in the prospective anal pads, is necessary for activation of hh in the adjacent prospective rectum. hh is expressed in the prospective rectum, which forms anterior to the anal pads, and is necessary for the expression of dpp at the posterior end of the adjacent large intestine. wg and hh are also necessary for the development of their own expression domains, anal pads, and rectum, respectively. dpp, in turn, causes the growth of the large intestine, promoting DNA replication. en defines the dorsal domain of the large intestine, repressing dpp in this domain. A one-cell-wide domain, which delineates the anterior and posterior borders of the large intestine and its internal border between the dorsal and ventral domains, is induced by the activity of en. A model is proposed for the gene regulatory pathways leading to the subdivision of the hindgut into domains (Takashima, 2001).

The term 'tissue compartments' can be used to indicate the domains of the gut. In this report, the term 'domain' is used in order to avoid confusion with the term 'developmental compartment', which has been defined by clonal analysis of the wing disc. To clarify the use of anatomical descriptions, the organization of the hindgut domains, as revealed by specific gene expression patterns is described. The most anterior domain of the hindgut, which is just posterior to the midgut, is the small intestine. The small intestine is followed by the large intestine, then the rectum. The large intestine is further subdivided into a ventral and a dorsal domain. A one-cell-wide domain, which was designated as h4, forms at the anterior and posterior borders of the large intestine, as well as at the border between the dorsal and ventral domains of the large intestine. The cells in these regions are designated collectively 'border cells'. Until the end of stage 12, the hindgut tube is situated on the midline of the body, and is left-right symmetric. During early stage 13, the hindgut rotates to the left, resulting in the original dorsal and ventral domains coming to face the left and right side of the body, respectively. The orifice of the rectum (the anal slit) is surrounded by the anal pads, the development of which is tightly linked to that of the hindgut (Takashima, 2001).

wg, hh, and dpp are expressed in the hindgut of the Drosophila embryo. The expression patterns of these genes have been re-examined in detail to define their exact spatial relationship. wg is expressed throughout the proctodeum at stage 9, then soon becomes restricted to two separate regions: (1) the primordium of the anal pads, which surrounds the posterior opening of the hindgut, and (2) a narrow ring anterior to the small intestine. The expression in these two domains persists throughout embryogenesis (Takashima, 2001).

hh is expressed throughout the hindgut primordium at stage 10. Subsequently, as in the case of wg, the expression is divided into two separate regions at stage 11: the region just posterior to the anterior wg domain, which corresponds to the small intestine, and the posterior-most region of the hindgut, which corresponds to the prospective rectum and is situated just anterior to the anal pads (Takashima, 2001).

The border cells differentiate at the anterior and posterior border of the large intestine and at the border between the dorsal and ventral domains of the large intestine. The border cells are first detected at stage 12 by lacZ expression of some enhancer-trap strains, and after stage 14, the cells are distinguished by marked expression of Crb and dead ringer. By double staining for En and beta-galactosidase protein of border cell-specific enhancer trap lines, the border cells are found to abut the En-positive domain and to express no En protein, suggesting that dpp-positive cells abutting the en-positive domain differentiate into border cells. It is noteworthy that the spatial organization of en, hh, wg, and dpp domains is quite different from that of the segmented epidermis or the imaginal discs, suggesting that a different patterning mechanism is working in the hindgut (Takashima, 2001).

Strong defects occur in the hindgut of wg mutant embryos, and it has been argued that such defects are likely due to an early effect on cell proliferation. The effects of the hypomorphic mutation wg17en40 on development of hindgut patterning was examined in detail. In this mutant, proctodeal invagination is almost normal until stages 9-10, but much of the proctodeum, except the anterior-most region including the small intestine, begins to degenerate after the onset of germband shortening, resulting in a very tiny epithelial sac. The anal pads, which express wg, also degenerate in this mutant. The expression of hh in the prospective rectum is abolished in this wg mutant, while the expression of hh in the small intestine is not affected. The hindgut of a null allele of wg (wgPY40) shows essentially the same defects though overall morphology of the embryo is affected more severely. This result suggests that hh expression in the prospective rectum is activated by wg signaling. To prove this, the effect of ectopic expression of wg was examined by using the GAL4-UAS system. The UAS-wgts strain, in which functional wg can be induced at the permissive temperature of 17°C, was mated with the byn-GAL4 strain, in which GAL4 is expressed throughout the whole hindgut and anal pads. It was found that hh expression is expanded throughout hindgut upon misexpression of wg. Except for a short anterior portion corresponding to the small intestine, the lumen of the hindgut is characteristically enlarged, with no morphological boundary between the large intestine and rectum. Similar results were obtained when an active form of Armadillo is misexpressed throughout the hindgut. These results strongly suggest that the ectopic wg activity induces hh expression at the prospective large intestine, and the latter develops as a part of the rectum. Conversely, in the hh mutant, there are no drastic changes in anal pad development or wg expression (Takashima, 2001).

In wg mutants, dpp expression in the large intestine is completely abolished. The ectopic expression of wg slightly upregulates the initial dpp expression in the large intestine at stage 11, but has repressed dpp throughout the hindgut by late stage 12. The relationship between wg activity in the anal pads and dpp expression in the large intestine seems to be indirect and complicated (Takashima, 2001).

en expression in the dorsal domain of the large intestine, in contrast to that of dpp and hh, is not affected in wg embryos. These results suggest that the defects of the hindgut in wg mutants are partly mediated by failure of hh expression in the future rectum. It should be noted that the defects of the large intestine in the wg mutant are more drastic than those in either hh or dpp mutants. There may exist some pathway of wg action that is not mediated by hh and dpp (Takashima, 2001).

It should be noted that wg and hh mutations result in a short hindgut, and these mutations are associated with the reduction of dpp expression in the large intestine. It is very likely that suppression of the growth of the large intestine correlates with the decrease in dpp expression. The effect of dpp

Wingless and dorsal closure

At the end of germband retraction, the dorsal epidermis of the Drosophila embryo exhibits a discontinuity that is covered by the amnioserosa. The process of dorsal closure (DC) involves a coordinated set of cell-shape changes within the epidermis and the amnioserosa that result in epidermal continuity. Polarization of the dorsal-most epidermal (DME) cells in the plane of the epithelium is an important aspect of DC. The DME cells of embryos mutant for wingless or dishevelled exhibit polarization defects and fail to close properly. The role of the Wingless signalling pathway in the polarization of the DME cells and DC was investigated. The ß-catenin-dependent Wingless signalling pathway is required for polarization of the DME cells. Although the DME cells are polarized in the plane of the epithelium and present polarized localization of proteins associated with the process of planar cell polarity (PCP) in the wing, e.g., Flamingo, PCP Wingless signalling is not involved in DC (Morel, 2004).

The initiation of DC in Drosophila embryos correlates with the elongation and polarization of the DME cells in the DV axis of the embryo. In parallel with this polarization, a cable of F-actin assembles on the dorsal-most surface of these cells and promotes the formation of filopodia and lamellae during the final phases of the process. A functional link between the polarization and the assembly of the cable of actin is supported by the observations that in mutants in which the DME cells do not elongate, there is no actin cable and no dynamic protrusions. As a consequence, these embryos display defects and delays in the closure process. Embryos mutant for wg and dsh are good examples of this class (Morel, 2004).

The polarization of the DME cells occurs in the plane of the epithelium and can be seen as a manifestation of the phenomenon of planar cell polarity (PCP). Since a specific branch of Wg signalling has been implicated in PCP and there is evidence for an interaction between Dishevelled and JNK signalling during dorsal closure, whether there is a role for this mode of Wg signalling in the process of DC was tested. The results clearly show that the 'canonical' Wg signalling pathway that leads to activation of Armadillo and of the transcription of target genes is necessary and sufficient to restore the polarity of the DME cells and to promote a normal process of dorsal closure in a wg mutant embryo. Surprisingly, it was found that the PCP pathway does not appear to play a major role in DC or the polarization of the DME cells, since activation of the 'canonical' pathway in the absence of dsh activity rescues the polarity and function of the DME cells.

Dsh contains three highly conserved domains, the DIX, PDZ and DEP domains. The DEP domain mediates interaction of Dsh with the cell cortex and is required for PCP but not 'canonical' Wg signalling, while the DIX domain is required for the 'canonical' Wg signalling but seems dispensable for PCP. To investigate an involvement of the PCP pathway in the activities of the DME cells during DC, rescue experiments of wg embryos were carried out using truncated forms of Dsh deleted for either the DEP (DshDeltaDEP) or the DIX (DshDeltaDIX) domain. Although overexpression of DshDeltaDEP leads to the partial rescue of naked cuticle and of En expression, neither naked cuticle nor rescue of En expression are observed in wg>da>DshDeltaDIX (using da-GAL4 to drive DshDeltaDIX in wg mutants) embryos. This thus confirms that DshDeltaDEP is able to signal within the 'canonical' Wg pathway but not DshDeltaDIX (Morel, 2004).

Then the ability of either protein to rescue DC in wg embryos was tested. wg>da>DshDeltaDEP embryos are longer than wg mutants and their dorsal cuticle is improved; no hole is observed and only occasional warts can be seen. The DME cells are oriented in the DV direction and most of them show a slight elongation in the DV direction when the zippering process has started. Simultaneously, Fmi is observed at the membrane and accumulates at the level of the ANCs. Although no clear elongation of DME or ventral epidermal cells is observed, DC process is improved; two zippers, at the anterior and posterior ends of the embryo, are initiated, whereas only the posterior one is observed in wg embryos. By contrast, wg>da>DshDeltaDIX embryos have a shorter cuticle than wg mutants and show a more severe puckering and hole on the dorsal side. Furthermore, neither the shape nor the polarization of DME cells is improved in these embryos (Morel, 2004).

Thus, although DshDeltaDEP can rescue partially the DC defects of wg mutants, ubiquitous overexpression of DshDeltaDIX does not rescue any of the observed features confirming the requirement for the Wg 'canonical' pathway during DC. Thus, the conclusion that the PCP pathway does not appear to play a role in DC or in the polarization of the DME is supported by the observation that although a moiety of Dishevelled that promotes Armadillo signalling is capable of rescuing the defects of wg mutants, a moiety that promotes JNK signalling and PCP does not. Altogether, these results indicate that the polarization and activity of the DME cells during dorsal closure requires Armadillo/ß-catenin-dependent Wg signalling. Furthermore, this requirement is restricted to the epidermis because activation of Wg signalling in the amnioserosa has no effect on the epidermis (Morel, 2004).

The polarization of the DME cells and subsequent dynamics of actin at the LE can be construed since the development of the leading edge of a motile cell and to a certain extent is akin to an epidermal/mesenchymal transition (EMT), as one of the features of this process is the reorganization of the actin cytoskeleton and the acquisition of motility by the cells. In this regard, it is interesting to note that ß-catenin-dependent Wnt signalling has been implicated in EMT both in normal and cancerous cells and that therefore there are precedents for the involvement of the ß-catenin-mediated transcriptional regulation in the development of actin dynamics. However, the targets of the Wnt pathway mediating this process are not known (Morel, 2004).

It has been suggested that Dpp is a central effector of dorsal closure. Embryos mutant for dpp signalling exhibit defects in dorsal closure. dpp is expressed in the DME cells and has been proposed to act as a long range signal for the elongation of the more ventral cells. Wingless is shown to be required for the correct maintenance of dpp expression in the DME cells, although in these experiments the input is less significant than has been reported before. Altogether, these observations suggest that some of the activity of Wingless during DC is mediated by Dpp. Indeed, when the Dpp pathway was ubiquitously activated by the means of an activated form of its receptor Tkv, some rescue of the polarity of the DME cells was observed. However, although in this case the DME cells orient themselves in the DV direction and Fmi localises as it does in wild type, neither the DME nor the ventral epidermal cells elongate, and the DC process is not substantially improved. This contrasts with the full rescue of both the polarization of DME cells and the DC process following ubiquitous activation of the ß-catenin-dependent Wg pathway. Thus, if Dpp contributes to DC, it is not as the only target of Wg signalling (Morel, 2004).

Expression of Wingless from the amnioserosa in wg mutants induces high and continuous levels of dpp in the DME cells together with some rescue of the polarity of the DME cells but without any effect on the elongation of these or the more ventral cells. This rescue is very similar to the one observed with ubiquitous expression of the activated Tkv. These results indicate that Dpp does not act as a long-range signal for the elongation of the more ventral epidermal cells; rescue of Dpp expression in the DME cells or activation of Dpp signalling throughout the epidermis in wg mutants does not lead to the elongation of the more ventral cells. A similar conclusion had been suggested from the observation that epidermal cells initially elongate in the absence of Dpp signalling but resume their polygonal shape soon after. However, an alternative explanation for these observations is that the elongation of the ventral epidermal cells requires inputs from both Dpp and Wingless signalling (Morel, 2004).

Altogether, these observations indicate that Dpp is not the only effector of Wingless during DC and indicates that Wingless signalling via Armadillo controls genes that act either in parallel or together with those regulated by JNK and Dpp (Morel, 2004).

Wingless is required in the epidermal cells but does not act as a polarizing signal, since ubiquitous activation of the pathway rescues the defects of wg mutants. An important observation of these experiments is that the DME cells of wg mutant embryos display a polarity and an elongation at the very final stages of DC, suggesting that the polarization signal is received correctly by the DME cells but that in the absence of Wingless signalling there is a delay either in its interpretation or in its materialization. This, together with the lack of importance of a fixed source of Wingless for the polarization of the DME cells, suggests that Wingless makes the DME cells competent to interpret a pre-existing polarization signal (Morel, 2004).

In the case of DC, the permissive function of Wingless signalling translates itself in the correct coordination of the different events, i.e., the cells have to elongate at the right time and the activity of their cytoskeleton has to be properly linked to other events some of which are transcriptional. Failure to do this will result in defects in dorsal closure. These observations raise the question of the temporal requirements for Wg signalling during DC (Morel, 2004).

ASGal4 (the amnioserosa specific driver, 332.3-Gal4) drives expression of Wingless from the elongation of the germband to the end of DC. However, when driven by ASGal4, Wg can be detected only over the epidermal cells during the first phase of DC. This is probably due to the inability of Wingless to cross the deep fold existing between the AS and the epidermis during germband retraction and the zippering process. The provision of Wg from the amnioserosa rescues the defects of the DME cells of wingless mutants but not those of the more ventral cells. Although the DME cells, in contact with the AS, might have received Wg signal at the very onset of the overexpression (around stage 9-10), the more ventral epidermal cells seem to see the signal too late to elongate, suggesting that Wg signalling is required before the beginning of DC for the cell shape and polarity changes. A hint at the timing of Wnt requirement for DC is provided by experiments using a temperature sensitive allele of wingless. Removal of wingless function between 4 and 4.5 hours after egg laying, i.e., at stages 9-10, affects the shape of the dorsal cuticle in a way similar to DC defects. This suggests that the polarizing signal must occur very early, during germband elongation (Morel, 2004).

The notion of PCP has emerged from studies of the mechanism that determines the orientation of the hairs in the cells of the wing of Drosophila. A number of studies have revealed the existence of protein complexes that mediate this orientation by becoming asymmetrically distributed between the proximal and distal membranes of the epidermal cells. Thus, while Flamingo becomes localised equally between the proximal and the distal sides of the cell, the distal side of the cell accumulates a complex composed of Frizzled and Dishevelled and the proximal side accumulates a complex formed by Strabismus and Prickled. Genetic analysis of these complexes has led to the formulation of a model which describes the propagation of the polarity from one cell to its neighbours, but which says nothing about the origin of the polarity that is being propagated. In this model, Dsh, like Strabismus, Prickled or Frizzled, is an essential component of the mechanism that propagates the polarity (Morel, 2004).

The observation of polarized distributions of Fmi, Dsh and Fz in the DME cells during dorsal closure has led to the suggestion of a link between the polarization of these cells and the process of PCP. However, no requirement has been found for elements of this pathway in dorsal closure. In particular, the PCP function of Dsh is not required for the polarization of the DME cells and the polarized localization of Fmi, which was quite unexpected considering the interdependence of Dsh and Fmi for their asymmetric localization in the wing. This asymmetric distribution of Fmi is likely to play a role in the polarized actin dynamics in response to the polarity signal. Although this may appear surprising, it also invites a consideration of the notion of PCP (Morel, 2004).

The PCP pathway has been defined in a context of propagation of a polarity but not of its initial definition. In fact none of the experiments performed in the wing of Drosophila address the origin of the polarity that is being propagated. In DC, however, the process that was observed in the asymmetric distribution of proteins in the DME cells reflects the establishment of a polarity and not its propagation. From this perspective, the lack of a requirement for the PCP branch of Wnt signalling might not be that surprising as PCP Wnt signalling might be related to propagation or coordination of a polarity signal that has been generated in a different manner. However, the requirement for the ß-catenin-dependent Wg pathway might be significant and indicate the requirement for a transcriptional event in the establishment of PCP. This observation might also apply to the wing (Morel, 2004).

Wingless regulates adult midgut regeneration

Inducible progenitor-derived Wingless regulates adult midgut regeneration in Drosophila

The ability to regenerate following stress is a hallmark of self-renewing tissues. However, little is known about how regeneration differs from homeostatic tissue maintenance. This study examined the role and regulation of Wingless (Wg)/Wnt signalling during intestinal regeneration using the Drosophila adult midgut. Wg was shown to be produced by the intestinal epithelial compartment upon damage or stress and it is exclusively required for intestinal stem cell (ISC) proliferation during tissue regeneration. Reducing Wg or downstream signalling components from the intestinal epithelium blocked tissue regeneration. Importantly, it was demonstrate that Wg from the undifferentiated progenitor cell, the enteroblast, is required for Myc-dependent ISC proliferation during regeneration. Similar to young regenerating tissues, aging intestines required Wg and Myc for ISC hyperproliferation. Unexpectedly, the results demonstrate that epithelial but not mesenchymal Wg is essential for ISC proliferation in response to damage, while neither source of the ligand is solely responsible for ISC maintenance and tissue self-renewal in unchallenged tissues. Therefore, fine-tuning Wnt results in optimal balance between the ability to respond to stress without negatively affecting organismal viability (Cordero, 2012).

This study used the posterior adult Drosophila midgut to address the role of Wg and its downstream signalling pathway during the proliferative response of ISCs to acute damage of the intestinal epithelium. The results suggest that, in response to stress or damage, Wg production is induced in enteroblasts (EBs), which stimulates ISC proliferation and subsequent midgut regeneration in a Myc-dependent manner. The results place Wg induction downstream of the damage/stress activated kinase JNK (Cordero, 2012).

The visceral mesoderm (VM) that surrounds the Drosophila midgut was proposed to constitute the Wg niche and be the sole source of the ligand required for ISC maintenance and homeostatic tissue self-renewal. The current results suggest that Wg produced within the intestinal epithelium and not the VM is essential for intestinal regeneration in response to acute damage. Furthermore, neither source of the ligand seems solely responsible for homeostatic self-renewal. Apparent discrepancies with the previous study could have different explanations. One possibility is that the ISC phenotype of whole wg mutants may be the result of combined Wg loss in VM and midgut epithelium. Alternatively, minimal levels of wg may be sufficient to maintain ISCs during homeostasis while a higher threshold of ligand production and subsequent signalling activation may be required for regeneration. In such a scenario, wg knockdown by RNA interference may still leave enough Wg to maintain the tissue under homeostatic conditions. The latter possibility is favored over the former since combined wg knockdown in esg;how>wg-IR midguts did not show significant loss of ISCs. Dose-dependent roles of Wnt signalling have been previously reported to regulate haematopoietic stem cells and tumourigenesis. Another possible explanation to the mild or absent role of Wg in homeostatic self-renewal may be compensation by other Wnt ligands. Therefore, while Wg is the main ligand required for regeneration, different ligands may compensate for each other during homeostatic maintenance of the tissue. Recent work in the murine small intestine has shown that one of the differentiated progeny of ISCs, the Paneth cell, expresses multiple growth factors such as EGFs and Wnt3 and are sufficient to drive the formation of 'crypt-like' structures from single Lgr5-expressing ISCs in vitro. Paneth cells have therefore been proposed as an important component of the Wnt ISC niche. In vivo work involving ablation of Paneth cells suggests the presence of potential compensatory mechanisms during homeostasis, while their role in intestinal regeneration has been suggested. The current work demonstrates that Wg from the transient daughter of the ISC, the EBs, is essential for efficient ISC proliferation during regeneration of damaged midgut epithelium. Therefore, Drosophila EBs could be seen as a functional homologue of the vertebrate Paneth cell and represent an essential component of the ISC niche. In the particular case of Wg, it is proposed that EBs represent a 'regeneration-specific ISC niche'. Growth factors such as IL-6/Upds and EGF-like ligands are important components of the Drosophila ISC niche. Intriguingly, the EGF-like ligand Spitz has been shown to be expressed in the small progenitor cells (ISCs/EBs) in the midgut, which is characterized by the expression of escargot. Even though direct functional assessment of the role of Spitz and other growth factor from EBs remains to be performed the current results strongly point to these cells as a potential general source of factors essential for ISC proliferation (Cordero, 2012).

Previous work suggests that midgut regeneration involves an intricate crosstalk between multiple signalling pathways. JAK/Stat signalling seems to be a central component of this response. Current work group suggests that JAK/Stat signalling is an important mediator of the hyperplastic phenotype resulting from loss of Apc in the Drosophila midgut. The results presented in this study suggest that damage to the midgut results in parallel activation of Wg/Myc and JAK/Stat. Knockdown of either pathway does not affect upregulation of the other pathway in response to damage even though midguts are still unable to regenerate. A similar scenario has been reported in the interplay between EGFR and JAK/Stat signalling. Therefore, activation of multiple pathways is a necessary condition for proper midgut regeneration. Likewise, midgut hyperproliferation in response to ectopic Wg signalling requires Myc and involves concerted activation of EGFR and JAK/Stat (Cordero, 2012, under review). Consistently, forced overexpression of ectopic Myc only is not sufficient to drive ISC proliferation and cannot overcome the absence of other proliferating signals such as JAK/Stat or Wg during midgut regeneration (Cordero, 2012).

One important question in the stem cell arena is whether tissue homeostasis and regeneration are controlled by similar mechanisms. Work in the mammalian intestine has shown examples where genes redundant for normal homeostasis are required for intestinal regeneration and Apc-driven intestinal hyperplasia. Therefore, the regenerative process cannot be interpreted as a simple acceleration of tissue self-renewal. Consistent with this concept, the curren work shows that partial reduction of the levels of Wg and Myc prevents ISC hyperproliferation during regeneration and aging but does not lead to long-term loss of ISCs. Furthermore, components of Wnt signalling such as Pygo are required for intestinal regeneration upon damage but dispensable for ISC proliferation in homeostatic conditions. Therefore, modulating Wg levels could lead to controlled ISC proliferation in conditions of hyperplasia without crossing a threshold that affects tissue integrity (Cordero, 2012).

The Wnt signalling pathway is a central regulator of homeostasis in the mammalian intestine. Inactivating mutations in Wnt pathway components lead to a very rapid loss of intestinal tissue. The current results show that, with the exception of Tcf, knocking down Wnt/Wg signalling has a rather mild and component-dependent role in homeostatic self-renewal of the Drosophila midgut. This is indeed consistent with previous reports. Although this scenario may appear at odds with that of the mammalian intestine there are indeed many similarities. In contrast, to genetic ablation of β-Catenin or Tcf4, the impact of Wnt inhibitors such as LRP6 blocking antibodies or Frizzled traps, which partially decrease Wnt signalling is much more subtle with little impact on intestinal homeostasis. In addition, it is also possible that β-Catenin signalling that is independent of ligand may explain the strong phenotype of TcfDN midguts. For example, it is known that the phosphorylation of β-Catenin by AKT/PKB has important roles in mammalian intestinal homeostasis (Cordero, 2012).

Although the similarities between the fly gut and the intestinal systems are often highlighted, intrinsic differences in the rates of homeostatic proliferation are observed between the two systems. The mouse intestine shows a high rate of homeostatic proliferation and undergoes complete self-renewal in 3-4 days, while the homeostatic fly midgut is a much more quiescent tissue. Basal proliferation rates in young, undamaged Drosophila midguts are very low and essentially undetectable by simple pH3 staining. Lineage-tracing experiments show it takes almost a month to achieve complete self-renewal of the midgut epithelium. Therefore, the homeostatic vertebrate intestine could be more comparable to the regenerating fly midgut. On the other hand, the homeostatic Drosophila midgut resembles a lowly proliferative epithelium like that of the mammalian urinary bladder or liver, which both show remarkable regenerative potential and shift from an almost quiescent to a hyperproliferative state in response to injury. Recent work uncovering an inducible role of Wg in regeneration of the bladder epitheliu suggests potential general implications of the current work. Additionally, one can expect that the intestinal epithelium of flies living in the wild, which is subject to constant challenges, might show higher basal proliferation than that of laboratory animals. Therefore, similarities between the mammalian and Drosophila intestine are likely to outweight their differences (Cordero, 2012).

Wnt signaling is required for long-term memory formation

Wnt signaling regulates synaptic plasticity and neurogenesis in the adult nervous system, suggesting a potential role in behavioral processes. This study probed the requirement for Wnt signaling during olfactory memory formation in Drosophila using an inducible RNAi approach. Interfering with β-catenin expression in adult mushroom body neurons specifically impairs long-term memory (LTM) without altering short-term memory. The impairment is reversible, being rescued by expression of a wild-type β-catenin transgene, and correlates with disruption of a cellular LTM trace. Inhibition of wingless, a Wnt ligand, and arrow, a Wnt coreceptor, also impairs LTM. Wingless expression in wild-type flies is transiently elevated in the brain after LTM conditioning. Thus, inhibiting three key components of the Wnt signaling pathway in adult mushroom bodies impairs LTM, indicating that this pathway mechanistically underlies this specific form of memory (Tan, 2013).

This study was prompted by a previous discovery that a casein kinase Iγ homolog (CkIγ), gilgamesh (gish), is required for STM in Drosophila (Tan, 2010). CkIgγmediated phosphorylation of the cytoplasmic tail of Lrp5/6 (Arr) is crucial for Wnt/β-catenin signaling (Davidson, 2005), and it was predicted that disruption of the Wnt signaling pathway would perturb STM. Surprisingly, however, it was found that knockdown of the four Wnt signaling components leaves STM intact. The likely explanation for this discrepancy is that Gish serves other important functions in STM formation besides its role in LTM through phosphorylation of the Arr receptor (Tan, 2013).

How does Wnt signaling in the MB neurons mediate the formation of LTM? Since the normal expression of β-catenin, Wg, and Arr is required in the set of MB neurons defined by P{MB-GeneSwitch}12-1, and Wg is a short-range ligand, a model is favored in which the Wnt ligand, Wg, participates in an autocrine fashion in the MB neurons. Spaced conditioning, which produces long-term behavioral memory, but not massed or single-cycle conditioning, leads to a transient increase in wg expression in the MB neurons, perhaps as a step downstream of Creb. The subsequent secretion of Wg by the MB neurons activates the Fz/Arr receptor, leading to the accumulation of β-catenin in the MB neurons. β-catenin, in turn, orchestrates transcriptional changes in the MB neurons that are required for LTM, as well as the breaking and remaking of cell contacts through N-cadherin function, which is necessary for the reorganization of synapses for LTM storage. Recently, ribonucleoprotein particles containing synaptic protein transcripts were shown to exit the nucleus through a nuclear envelope budding process in response to Wnt signaling at the Drosophila neuromuscular junction (Speese, 2012). Wnt-dependent nuclear budding could provide the initial step for transporting RNAs to synapses for local protein synthesis and LTM formation (Tan, 2013).

Table of contents

wingless continued: Biological Overview | Evolutionary Homologs | Transcriptional regulation |Targets of Activity | Protein Interactions | mRNA Transport | Developmental Biology | References

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