Effects of Mutation or Deletion

Table of contents

Wingless and segmentation

In the trunk of the Drosophila embryo, the segment polarity genes are initially activated by the pair-rule genes; later, the segment polarity genes maintain one another's expression through a complex network of cross-regulatory interactions. These interactions, which are critical to cell fate specification, are similar in each of the trunk segments. To determine whether segment polarity gene expression is established differently outside the trunk, the regulation of the genes hedgehog (hh), wingless (wg), and engrailed (en) was studied in each of the segments of the developing head. The cross-regulatory relationships among these genes, as well as their initial mode of activation in the anterior head are significantly different from those in the trunk. In addition, each head segment exhibits a unique network of segment polarity gene interactions. It is proposed that these segment-specific interactions evolved to specify the high degree of structural diversity required for head morphogenesis (Gallitano-Mendel, 1997).

The proposed interactions between hh, wg and en are described below.

1. The intercalary segment. In this cephalic segment, hh expression is en-independent. In addition, ptc mutations cause the loss of wg rather than ectopic wg expression The dependence of wg, en, and hh expression on ptc indicates a unique role for segment polarity genes in the intercalary segment. Unlike wg action in the trunk and gnathal segments, wg restricts rather than maintains en and hh expression in this segment. Finally, en expression, as it occurs in the trunk, depends on hh function. However, this dependence cannot be mediated through wg, since wg does not maintain en expression in the intercalary segment.

2. The antennal segment. As in the trunk, hh antennal expression depends on en, while wg expression requires hh. The requirement for hh is presumably mediated through ptc, which represses wg in this segment. Unlike in the trunk, wg restricts the expression domains of both en and hh. As in the intercalary segment, regulation of en by hh is wg- independent.

3. The ocular segment. In this segment, hh is en-independent and wg expression does not require hh. Although the wg domain (the head blob) does not expand in ptc mutant embryos, noncontiguous ectopic wg expression appears in its vicinity. Unlike its action in the trunk and the other head segments, wg is required to initiate en expression in the ocular segment. However, hh expression still expands in wg mutant embryos (as in the intercalary and antennal segment). As in the intercalary and antennal segments, regulation of en by hh does not depend on wg.

It is concluded that cross-regulatory interactions among the segment polarity genes in the anterior head are very different from those in the posterior head and trunk segments. The mode of patterning of the anterior head (the acron and cephalic segments) is thought to be more ancient than that of the posterior head (the gnathal segments). This distinction appears to be reflected in the segmentation mechanism used by certain present day short germ insects and primitive arthropods. In these organisms, the early germ band includes only the acron, cephalic segments, and tail. Gnathal and trunk segments are generated later in embryogenesis by a progressive budding process (Gallitano-Mendel, 1997).

A model for formation of the anterior and posterior compartments maintains that cells at the inter-compartment interface drive pattern formation by becoming the source of a morphogen. Does this model apply to the ventral embryonic epidermis of Drosophila? First, it is shown that interfaces between posterior (engrailed ON) and anterior (engrailed OFF) cells are required for pattern formation. Second, evidence is provided that Wingless could play the role of the morphogen, at least within part of the segmental pattern. The cuticular structures are examined that develop after different levels of uniform Wingless activity are added back to unsegmented embryos (wingless- engrailed-). Unsegmented embryos are small and spherical, carry a low number of unpolarised denticles and have no Keilin's organs, presumably because they have no functional parasegmental boundary. Because it is rich in landmarks, the T1 segment is a good region to analyse. Here, the cuticle formed depends on the amount of added Wingless activity. For example, a high concentration of Wingless gives the cuticle elements normally found near the top of the presumed gradient. Unsegmented embryos are much shorter than wild type. Alternation of cells expressing engrailed in the "on" and "off" state is necessary for segmentation. If Wingless is added in stripes, the embryos are longer than if it is added uniformly. The response to Wingless depends on whether engrailed is on or off. When en is on, Wingless determines an anterior segmental identity, and when en is off, WG determines a posterior identity. For example when en is off, the ventral abdomen makes naked cuticle instead of denticles. It is suggested that the Wingless gradient landscape affects the size of the embryo, so that steep slopes would allow cells to survive and divide, while an even distribution of morphogen would promote cell death. Supporting the hypothesis that Wingless acts as a morphogen, it is found that at a distance these stripes affect the type of cuticle formed and the planar polarity of the cells. There are two functions of engrailed: (1) the interface between en on and en off ensures sustained expression of the Wingless morphogen; (2) the presence or absence of en determines the cells as posterior or anterior and selects the responses to the morphogen (Lawrence, 1996).

Signaling by the Epidermal growth factor receptor (EGFR) plays a critical role in the segmental patterning of the ventral larval cuticle in Drosophila: by expressing either a dominant-negative EGFR molecule or Spitz, an activating ligand of EGFR, it is shown that EGFR signaling specifies the anterior denticles in each segment of the larval abdomen. rhomboid, spitz and argos are expressed in denticle rows 2 and 3, just posterior to denticle row 1 in the engrailed expression "posterior" domain of larval ectoderm. These denticles derive from a segmental zone of embryonic cells in which EGFR signaling activity is maximal. Expression of a dominant negative form of Egfr (DN-DER) leaves just two of the six rows of denticles, corresponding to rows 5 and 6. Expression of ectopic Egfr throughout the ventral epidermis results in larval cuticles showing considerably widened ventral denticle belts, with only narrow, naked stretches between them. Within these belts, the normal rows 1-4 are still recognisable by the morphology and orientation of their denticles. However, posterior to these, row 5 and 6 denticles are not apparent; these are replaced by a wide field of small denticles, apperently of the row 1-4 type. A similar phenotype of excessive denticles is seen after ubiquitous expression of an activated form of Ras (Stutz, 1997).

There is a competition between the denticle fate specified by EGFR signaling and the naked cuticle fate specified by Wingless signaling. The final pattern of the denticle belts is the product of this antagonism between the two signaling pathways. In the absence of Wg signaling, extra denticles form, instead of naked cuticle, whereas, ectopic activation of Wg causes naked cuticle to form, instead of denticles. Expressing DN-DER (that is removing Egfr) in wingless mutants (producing 'double-mutant' conditions) produces denticle belts with almost exclusively large denticles. Mostly based on their size, it is believed that the large denticles are of the row 5 type. This cuticle phenotype demonstrates that Egfr activity is responsible for the row 1-4 denticles that are seen in wingless mutants. There may be very little Egfr signaling in wingless loss of function mutants; the segmental stripes of rhomboid expression are weaker, and those of argos completely disappear. Thus, the segmental activation of Egfr signaling seems to be an indirect consequence of an early function of wingless. Egfr signaling activity can produce denticles in the complete absence of Wg signaling. Expressing Spitz ectopically produces denticles of the row 1-4 type. Egfr signaling does not require armadillo function to specify these small denticles. (Szuts, 1997).

How are precise boundaries formed between different cell types without early compartmentalization? One such boundary occurs between the wingless-expressing cells of the wing margin and the adjacent proneural cells, which give rise to margin sensory bristles. This boundary arises in part by a mechanism termed "self-refinement," by which Wingless protein represses wg expression in adjacent cells. Reduction or removal of WG activity, in this case accomplished using a temperature sensitive allele of wg, results in a failure to resolve this boundary properly. Dishevelled protein is required for the reception of WG signals. It is thought that DSH is activated by the WG signal. Expression of wg in dsh- clones results in expanded wg expression over as many as six cells from the normal wg boundary. Thus, reception of WG signaling is required to repress wg expression in a broad region of competence along the margin. Self-refinement is one of two identified signaling functions of margin Wingless. In the other function, WG signaling is necessary and sufficient for proneural gene expression and the subsequent formation of margin bristles in cells flanking the wg-expressing stripe. One might expect that the ectopic wg expressed within the clone might be able to induce ectopic proneural gene expression in wild-type cells surrounding the clone, leading to the formation of margin bristles abnormally distant from the margin. This phenotype has indeed been reported. One possible way that WG self-refinement might act is by repressing the Notch signaling pathway. It is suggested that heightened Notch activity along the dorsoventral boundary is responsible for the localized expression of boundary-specific genes, including wg. Recent evidence indicates that DSH-mediated WG signalling can inhibit Notch activity (Rulifson, 1996).

wingless (wg) and its vertebrate homologs, the Wnt genes, play critical roles in the generation of embryonic pattern. In the developing Drosophila epidermis, wg is expressed in a single row of cells in each segment, but it influences cell identities in all rows of epidermal cells in the 10- to 12-cell-wide segment. Wg signaling promotes specification of two distinct aspects of the wild-type intrasegmental pattern: the diversity of denticle types present in the anterior denticle belt and the smooth or naked cuticle constituting the posterior surface of the segment. The expression of wild-type and mutant wg transgenes have been manipulated to explore the mechanism by which a single secreted signaling molecule can promote these distinctly different cell fates. Evidence is presented consistent with the idea that naked cuticle cell fate is specified by a cellular pathway distinct from the denticle diversity-generating pathway. The protein encoded for by mutant allele wgPE2 is unable under any circumstances to promote the naked cuticle cell fate in the ventral epidermis, while it can promote denticle diversity (Hays, 1997).

Cells receiving the same level of Wg signaling respond differently depending on their position within the segment: some cells are directed to secrete naked cuticle, while others secrete diverse denticle types. This observation hints at a differential distribution of molecules that interact with Wg to effect its signaling activity. All cells within the segment, however, are competent to secrete naked cuticle even in a wg null mutant background. When expression levels of either mutant allele wg PE4 or wg + are driven at higher levels with a ubiquitous promoter, all cells in the ventral epidermis produce naked cuticle with few or no denticles. This indicates that all cells in the segment can be directed into the naked cuticle cell fate, and therefore presumably would express a naked cuticle specifying Wg receptor. This cell fate pathway appears to be triggered when Wg activity exceeds a certain threshold level. Dose sensitivity for naked cuticle specification is also observed under a variety of other circumstances (Hays, 1997).

When expressed uniformly, wg PE4 transgenes rescue the wg mutant phenotype in a manner similar to the wg + transgenes, promoting both denticle diversity and naked cuticle specification. This contrasts with the original wg PE4 mutant cuticle pattern, which shows little denticle diversity. One potential explanation for these disparate results is that the wg PE4 mutant protein may have a limited range of action. To test this possibility, the transgenic protein was expressed in the native wg domain, and its distribution was examined. UAS-wg +and UAS-wg PE2 transgenic protein can be detected over several cell diameters surrounding the wg-expressing row of cells; UAS-wg PE4 cannot. Rather, this transgenic protein shows limited movement away from the wg-expressing row of cells. The transgenic protein also appears to accumulate to higher levels within the wg-expressing domain, suggesting that it is not exported as efficiently as the full-length Wg molecule. Reduced secretion would contribute to the limited distribution of the mutant protein, but functional analyses indicate that at least some wg PE4 mutant protein reaches neighboring cell populations (Hays, 1997).

Since cuticle and denticle pathways are differentially activated by mutant Wg ligands, it is propose that at least two discrete classes of receptors for Wg may exist, each transducing a different cellular response. Broad Wg protein distribution across many cell diameters is required for the generation of denticle diversity, suggesting that intercellular transport of the Wg protein is an essential feature of pattern formation within the epidermal epithelium. An 85 amino acid region not conserved in vertebrate Wnts is dispensable for Wg function; structural features of the Wingless protein are discussed which are required for its distinct biological activities (Hays, 1997).

The basis for denticle diversity is still a mystery. Denticle type is not specified simply by level of Wg activity received, since uniform low levels of Wg can promote correct diverse denticle identities. Previous work has indicated that other segment polarity genes, such as patched and engrailed, play a role in determining denticle identity. These gene activities may be involved in dictating the "pre-pattern" observed when uniform wg expression is provided to a wg mutant embryo. Indeed, when ectopic wild-type Wg is provided in a wg en double mutant background, naked cuticle is rescued but very little denticle diversity is observed (Hays, 1996 and Lawrence et al., 1996).

The dorsal median cells are unique mesodermal cells that reside on the surface of the ventral nerve cord in the Drosophila embryo. The Buttonless homeodomain protein is specifically expressed in these cells and is required for their differentiation. Proper buttonless gene expression and dorsal median cell differentiation require signals from underlying CNS midline cells. Thus, dorsal median cells fail to form in single-minded mutants and do not persist in slit mutants. Through analysis of rhomboid mutants and targeted rhomboid expression, it has been shown that the EGF signaling pathway regulates the number of dorsal median cells. wingless-patched double mutants exhibit defects in the restriction of dorsal median cells to segment boundaries and alterations in CNS midline cell fates (Zhou, 1997).

A key step in development is the establishment of cell type diversity across a cellular field. Segmental patterning within the Drosophila embryonic epidermis is one paradigm for this process. At each parasegment boundary, cells expressing the Wnt family member Wingless confront cells expressing the homeoprotein Engrailed. The Engrailed-expressing cells normally differentiate as one of two alternative cell types. In investigating the generation of this cell type diversity among the 2-cell-wide Engrailed stripe, it has been shown that Wingless, expressed just anterior to the Engrailed cells, is essential for the specification of anterior Engrailed cell fate. In a screen for additional mutations affecting Engrailed cell fate, anterior open (aop) (also known as yan) was identified, a gene encoding an inhibitory ETS-domain transcription factor that is negatively regulated by the Ras-MAP kinase signaling cascade. Anterior open must be inactivated for posterior Engrailed cells to adopt their correct fate. This is achieved by the EGF receptor (Egfr), which is required autonomously in the Engrailed cells to trigger the Ras1-MAP kinase pathway. Localized activation of Egfr is accomplished by restricted processing of the activating ligand, Spitz. Processing is confined to the cell row posterior to the Engrailed domain by the restricted expression of Rhomboid. These cells also express the inhibitory ligand Argos, which attenuates the activation of Egfr in cell rows more distant from the ligand source. Thus, distinct signals flank each border of the Engrailed domain, since Wingless is produced anteriorly and Spitz posteriorly. Since En cells have the capacity to respond to either Wingless or Spitz, these cells must choose their fate depending on the relative level of activation of the two pathways (O'Keefe, 1997).

The larval cuticle comprises a repeated array of precisely patterned denticle belts interspersed with smooth cuticle. In abdominal segments, each of these belts is made up of 6 rows of denticles, where each row is of a characteristic size and orientation reflecting fate decisions made by the underlying cells. Using a lacZ reporter gene expressed in the En cells, it has been demonstrated that the anterior En cells normally produce smooth cuticle, while the posterior En cells produce denticles and, thereby, form the first row of each belt. Thus, cells in the En domain adopt either a smooth or denticle fate depending on their position. To identify genes involved in specifying En cell fates, existing collections of mutants were screened for those in which anterior En cells inappropriately produce denticles. Ectopic denticles are observed immediately anterior to the denticle belts in aop mutants. The extra denticles are located at the lateral edges of denticle belts, and are more commonly observed in the posterior segments. To determine whether the En cell fates were altered in these mutants, the En cells were visualized with a lacZ reporter construct. Anterior En cells produce denticles instead of the normal smooth cuticle. Thus, aop function is required for some anterior En cells to adopt the smooth cell fate (O'Keefe, 1997).

Spitz and Wingless signaling have been shown to have competing affects on En cell fate. Anterior En cells assume a denticle fate when wg function is eliminated at 8 hours AEL. Wg is expressed just anterior to the En domain, in a region of smooth cuticle. Thus, while Wg input instructs cells to adopt the smooth fate, activation of Egfr instructs cells to adopt denticle fates. The opposite response of En cells to these two signals raises the question of what fate these cells would adopt in the absence of both signals. To determine this, Egfr signaling was blocked by expressing Aop Act in En cells while concomitantly removing wg function using a conditional allele. When wg ts embryos carrying both En-GAL4 and UAS-Aop Act are shifted to non-permissive temperature at 8 hours AEL, the En cells adopt smooth fates. This suggests that smooth cuticle is the default cell fate. Wg signaling in this context is required primarily for antagonizing the effect of DER signaling in anterior En cells (O'Keefe, 1997).

The posterior En cells, which adopt a denticle fate, either cannot respond to Wg due to the absence of key signal transducers, or they do not see effective concentrations of Wg. In fact, it appears that the posterior En cell does not receive Wg input. The presence of downstream signal transducers was tested in posterior En cells. Cells expressing either an activated form of Armadillo or higher levels of wild-type Disheveled respond as if they have received the Wg signal. In embryos carrying both En-GAL4 and UAS-Arm S10, the expression of activated Armadillo causes the posterior En cells to inappropriately adopt the smooth cell fate. Identical results were obtained expressing Disheveled. Thus, Wg signal transducers downstream of Disheveled are present in posterior En cells. During normal patterning, these cells are probably not exposed to sufficient Wg levels to antagonize the effects of Egfr in these cells (O'Keefe, 1997).

A model is presented for the cooperation between Wingless and Spitz in specifying cell fate in Engrailed expressing cells. The En-expressing cells are flanked anteriorly by a cell row producing Wg and posteriorly by a cell row expressing Rhomboid, which produces secreted Spitz. The En cell nearest the Spi source receives a higher concentration of Spi, and thus activates the Egfr pathway sufficiently to specify a denticle fate. Reciprocally, the En cell nearest the Wg source receives a higher concentration of Wg and adopts a smooth fate. Spi also activates the Egfr pathway in the Rho-expressing cell, which therefore produces and secretes Argos. Argos can inhibit Spi activation of the Egfr pathway at a distance. As a consequence, the Egfr pathway is not sufficiently activated in the anterior En cell to out compete Wg signaling in this cell, and it adopts a smooth fate. In fact, the specific targets of Egfr signaling responsible for conferring the denticle fate are unknown (O'Keefe, 1997).

In cell culture assays, Frizzled and Dfrizzled2, two members of the Frizzled family of integral membrane proteins, are able to bind Wingless and transduce the Wingless signal. To address the role of these proteins in the intact organism and to explore the question of specificity of ligand-receptor interactions in vivo, a genetic analysis of frizzled and Dfrizzled2 in the embryo has been conducted. These experiments utilize a small gamma-ray-induced deficiency that uncovers Dfrizzled2. Dfz2-deficiency homozygotes die shortly after hatching and exhibit a subtle disorganization of denticle patterning with occasional ectopic denticles in posterior compartments. These data suggest that Dfz2 and/or other genes removed by the 469-2 deficiency play a minor or largely redundant role in cuticle patterning during embryogenesis. Mutants lacking maternal frizzled and zygotic frizzled and Dfrizzled2 exhibit defects in the embryonic epidermis, CNS, heart and midgut that are indistinguishable from those observed in wingless mutants. Epidermal patterning defects in the frizzled, Dfrizzled2 double-mutant embryos can be rescued by ectopic expression of either gene. In frizzled;Dfrizzled2 double mutant embryos, ectopic production of Wingless does not detectably alter the epidermal patterning defect, but ectopic production of an activated form of Armadillo produces a naked cuticle phenotype indistinguishable from that produced by ectopic production of activated Armadillo in wild-type embryos. These experiments indicate that frizzled and Dfrizzled2 function downstream of wingless and upstream of armadillo, consistent with their proposed roles as Wingless receptors. The lack of an effect on epidermal patterning of ectopic Wingless in a frizzled;Dfrizzled2 double mutant argues against the existence of additional Wingless receptors in the embryo or a model in which Frizzled and Dfrizzled2 act simply to present the ligand to its bona fide receptor. These data lead to the conclusion that Frizzled and Dfrizzled2 function as redundant Wingless receptors in multiple embryonic tissues and that this role is accurately reflected in tissue culture experiments. The redundancy of Frizzled and Dfrizzled2 explains why Wingless receptors were not identified in earlier genetic screens for mutants defective in embryonic patterning (Bhanot, 1999).

In the wild-type epidermis, wg functions in an autocrine pathway to maintain its own expression and in a paracrine regulatory loop to maintain expression of en in adjacent cells. In the epidermis at gastrulation, when wg function is first detected, a stripe of cells in the anterior half of each parasegment expresses wg and an adjacent stripe of cells in the posterior half express en. This pattern is initiated by pair-rule and gap genes, but its maintenance requires paracrine signaling by Wg to the en expressing cells and both paracrine signaling by Hh and autocrine signaling by Wg to the wg expressing cells. Thus, in wg mutant embryos the pattern of wg and en gene expression is initiated correctly but is not maintained. In fz;Dfz2 double-mutant embryos, the En stripes begin to fade at stage 9/10 and are completely absent from the epidermis by mid stage 10, similar to wg mutants. By contrast, en expression within the CNS is maintained as it is in wg mutants. Consistent with a defect in Wg signaling, Wg expression is greatly reduced in fz;Dfz2 mutants (Bhanot, 1999).

At the end of gastrulation, wg participates in the morphogenesis of various embryonic structures. In the embryonic central nervous system, wg is expressed by row 5 neuroblasts (NBs) and its function is required to specify NBs in rows 4 and 6. Null mutants of wg show a loss or duplication of several NBs, the most extensively studied being NB-4. The NB-4 lineage gives rise to two RP2 motoneurons per segment that innervate the dorsal musculature and are missing in wg mutant embryos. RP2 neurons are marked by their expression of even-skipped (eve). Mutant embryos missing maternal fz and zygotic fz and Dfz2 or missing only zygotic Dfz2 were examined using an antibody against Eve. fz;Dfz2 double-mutant embryos show a complete loss of RP2 neurons in all hemisegments. As observed in the epidermis, the fz^R52 allele shows residual activity: in fz;Dfz2 double mutants carrying the fz^R52 allele, approximately 26% of the double-mutant embryos show Eve-positive RP2 staining in 1-3 hemisegments. Interestingly, 469-2 homozygous embryos also show a weakly penetrant RP2 phenotype. In approximately 21% of the 469-2 homozygous embryos, an RP2 neuron is either missing or misplaced in 1-3 hemisegments. It is concluded that fz and Dfz2 are largely but not entirely redundant in specifying RP2 identity (Bhanot, 1999).

Wnt genes are often expressed in overlapping patterns, where they affect a wide array of developmental processes. To address the way in which various Wnt signals elicit distinct effects, the activities of two Wnt genes in Drosophila, DWnt-4, and wingless, were compared. These Wnt signals produce distinct responses in cells of the dorsal embryonic epidermis. Whereas wingless acts independently of hedgehog signaling in these cells, DWnt-4 requires Hh to elicit its effects. Expression of Wg signal transduction components does not mimic expression of DWnt-4, suggesting that DWnt-4 signaling proceeds through a distinct pathway. The dorsal epidermis may therefore be useful in the identification of novel Wnt signaling components (Buratovich, 2000).

DWnt-4 and wg are expressed in many of the same cells during Drosophila embryogenesis, including the ventral epidermis. However, in the cells of the dorsal epidermis each gene is expressed in distinct groups of cells. Whereas wg is expressed in the most posterior row of cells in each parasegment throughout most of embryogenesis, DWnt-4 is expressed in the anterior region of the parasegment. This expression is transient, beginning at stage 10 and fading by the end of stage 12. The wg-like ventral expression of DWnt-4 is dependent on hh, which may be due to shared regulatory elements between the two genes. However, since the dorsal expression of the two genes is nonoverlapping, this aspect of DWnt-4 expression appears to be regulated differently. Since the dorsal stripes of DWnt-4 lie in between the hh and wg stripes, the effect these genes have on dorsal DWnt-4 expression was examined. In wg mutants DWnt-4 is expressed normally, indicating that its expression in the epidermis is not dependent on the activity of wg. However, in hh mutants, both dorsal and ventral expression is eliminated. The regulation of DWnt-4 by hh within the anterior half of the dorsal parasegment suggests that it acts in concert with hh to pattern these cells (Buratovich, 2000).

Unlike the ventral epidermis, where hh and wg cooperate in the specification of naked cuticle, the dorsal epidermis is patterned through complementary activities of wg and hh. The anterior half of each parasegment consists of three cells types (1^o, 2^o, and 3^o) that are all dependent on the activity of hh. The position of the DWnt-4 expressing cells relative to the wg-expressing cells indicates that DWnt-4 is expressed in the presumptive 3^o cells. The posterior half of the parasegment consists of a single cell type, 4^o, and is dependent on wg activity. The dorsal epidermis is patterned beginning at 6 h of development, the time at which the expression of DWnt-4 is initiated in these cells. Prior to this point, between 3 h and 6 h, the expression of wg and hh are mutually dependent (Buratovich, 2000).

To determine whether DWnt-4 is able to modulate the patterning of the dorsal epidermis, and whether it mimics or otherwise regulates wg signaling in these cells, it was ubiquitously expressed using the GAL4 system. The results of ubiquitous expression of wg or DWnt-4 were compared. Ubiquitous expression of wg driven by a GAL4 insertion under the control of a daughterless enhancer (daGAL4) results in a uniform lawn of 4^o cells. Thus the hh-dependent cell types are deleted or transformed to 4^o fates. Ubiquitous expression of DWnt-4 elicits a distinct response in the hh-dependent cells, while having no effect on the wg-dependent cells. The phenotype produced by ectopic DWnt-4 is variable and dependent on levels of ectopic expression. With one copy of ectopic DWnt-4 expressed at 29^oC, 21% (23/108) of the segments exhibit a 2^o-3^o-4^o pattern, in which 1^o cells are missing and 3^o cells are expanded. In contrast, 62% of the segments exhibit either a 1^o-3^o-4^o or a 3^o-4^o pattern; it was found that 1^o and 3^o cells are difficult to distinguish. Lower levels of expression produced by rearing the flies at a lower temperature produces a higher percentage of embryos with a pattern that is more clearly 1^o-3^o-4^o along the dorsal midline, since the 2^o cell fate is still apparent laterally. Nevertheless, the 2^o-3^o-4^o phenotype shows that DWnt-4 can abolish 1^o cells, and indicates that the primary effect of DWnt-4 is to expand 3^o cells at the expense of the other two cell types (Buratovich, 2000).

These data show that cells in the anterior half of each parasegment have the ability to respond to both Wnt genes, but that each gene elicits a distinct response. Whereas Wg transforms these cells to 4^o cells or deletes them, DWnt-4 appears to modulate the specification of cell fate within the hh-dependent domain but has no effect on cell fate specification by wg. The phenotypes produced by ectopic DWnt-4 and wg therefore appear to be qualitatively distinct, in that each gene induces ectopic specification of different cell types (Buratovich, 2000).

The alteration in pattern by DWnt-4 suggests three possible interactions with hh. (1) DWnt-4 might affect the anterior half of the parasegment through modification of hh expression. However, analysis of hh transcripts following ectopic DWnt-4 expression has revealed that hh expression is not affected. (2) Since DWnt-4 expression requires hh activity, it could be a downstream effector of hh in pattern specification. (3) DWnt-4 could act in concert with hh to alter pattern. To address these possibilities, DWnt-4 was ectopically expressed in a hh temperature sensitive mutant shifted to the restrictive temperature at 6 h. Under these conditions the entire anterior half of the parasegment is missing in hh mutants. When DWnt-4 is ectopically produced in this background, anterior cell fates still fail to be specified, indicating that DWnt-4 does not simply act downstream of hh but requires hh for its activity after 6 h of development. If hh ts mutants are shifted to the restrictive temperature at 7 h, one row of 3^o denticles typically forms, while 1^o and 2^o fates are still missing. If DWnt-4 is ectopically expressed under these conditions, the number of 3^o rows increases, supporting the conclusion that DWnt-4 acts in concert with hh to specify 3^o cell fates (Buratovich, 2000).

Wingless activity is continuously required in the posterior part of parasegments to antagonize segmental groove formation

The formation of segmental grooves during mid embryogenesis in the Drosophila epidermis depends on the specification of a single row of groove cells posteriorly adjacent to cells that express the Hedgehog signal. However, the mechanism of groove formation and the role of the parasegmental organizer, which consists of adjacent rows of hedgehog- and wingless-expressing cells, are not well understood. This study reports that although groove cells originate from a population of Odd skipped-expressing cells, this pair-rule transcription factor is not required for their specification. It was further found that Hedgehog is sufficient to specify groove fate in cells of different origin as late as stage 10, suggesting that Hedgehog induces groove cell fate rather than maintaining a pre-established state. Wingless activity is continuously required in the posterior part of parasegments to antagonize segmental groove formation. These data support an instructive role for the Wingless/Hedgehog organizer in cellular patterning (Mulinari, 2009).

It has been reported that segmental groove formation requires the activity of engrailed (en) and hh and that en has a function that is independent of its role in hh activation. More recently, it was been found that en is not expressed in groove cells, thus creating a non-cell-autonomous requirement for en. To address this issue, the role of hh and en in segmental groove formation was reinvestigated (Mulinari, 2009).

It was found that segmental grooves do not form in hh mutants. When hh was overexpressed, the four to five cell rows posterior to the Hh source constricted apically, elongated their apical-basal axis and took on a shape characteristic of segmental groove cells. Very similar cell behavior was observed in patched (ptc) mutants or when activated Ci, which mediates hh activity, was expressed. These observations suggest that Hh can organize segmental groove formation. No cell constrictions were observed in the ventral epidermis, indicating that a different mechanism might regulate cell shape there (Mulinari, 2009).

To address the proposed hh-independent function of en, en, invected (inv) double mutants were investigated in which hh expression was maintained using prd-Gal4. Segmental grooves were rescued in these mutants, suggesting that en is not required for segmental groove formation independent of its role in hh activation. By contrast, it was found that en represses groove cell behavior when ectopically expressed together with hh. A previous study that reported a requirement of en in groove formation was based on the analysis of en, inv, wg triple mutants, in which hh expression was maintained but did not rescue groove formation. This result was confirmed, but it is proposed that wg may be required in en mutants to allow the morphological differentiation of grooves (Mulinari, 2009).

Analysis of ptc mutants, or embryos overexpressing hh, reveals that a broad region of cells posterior to the en expression domain are specified as groove cells. However, groove-like invaginations form only at the edges of these regions. This is even more obvious in double mutants of ptc and the segment polarity gene sloppy paired 1 (slp1), which is required for maintained wg expression. In slp1, ptc mutants, wg expression fades prematurely and Hh signaling is constitutively active. This results in a substantial expansion of the number of groove cells. However, furrows differentiate only at the edges of groove cell populations. It is proposed that the morphological differentiation of segmental grooves can occur only at the interface between groove and non-groove cells (Mulinari, 2009).

To test this, wg, ptc double mutants were used in which Hh signaling is active throughout the epidermis and all cells take on a groove fate. Interestingly, these embryos did not differentiate grooves. A similar observation has been reported in en, inv, wg mutants, in which hh expression is sustained, leading to the suggestion that en might be required for groove specification (Mulinari, 2009).

Analysis of cell behavior in wg, ptc mutants showed, however, that cells throughout the tissue constrict their apices but fail to form invaginating furrows. The failure of wg, ptc mutants and en, inv, wg; UAS-hh embryos to differentiate grooves might be due to the absence of non-groove cells in the epidermis and the concomitant absence of an interface with groove cells (Mulinari, 2009).

The pair-rule gene odd is initially expressed in 4- to 5-cell wide stripes in even-numbered parasegments. At early gastrulation, odd expression expands to segmental periodicity and is subsequently refined to a single row of prospective groove cells located posterior to en. Continued expression of odd in these cells requires hh. In odd⁵ mutant embryos, grooves are unaffected in odd-numbered parasegments, but partially missing in even-numbered parasegments, and residual grooves coincide with regions in which odd expression is detectable (Vincent, 2008). These observations have been interpreted as indicating that groove fate might be specified prior to the requirement of Hh and differentiation of the groove. Thus, the later activity of Hh might not induce, but merely maintain, groove cell identity that has been pre-established in the odd-expressing cell population (Vincent, 2008). However, this hypothesis is based on the presumption that odd has a function in groove cell specification and this has not been demonstrated (Mulinari, 2009).

Residual grooves in odd⁵ mutants have been attributed to the hypomorphic nature of the odd⁵ allele; however, the molecular lesion in odd⁵ is unknown. Therefore the nucleotide sequence of odd⁵ was determined and a substitution was found that mutates codon 84 from CAG to a TAG stop codon. The resulting truncated peptide, which lacks all four putative zinc fingers encoded by wild-type odd, is no longer restricted to the nucleus but uniformly distributed in the cell. Thus, odd⁵ is likely to be a null allele (Mulinari, 2009).

To exclude the possibility that groove formation may be rescued by read-through of the stop codon in odd⁵ mutants, or that odd may be required redundantly, segmental grooves were investigated in Df(2L)drm^P2 mutants, in which odd and its sister genes drumstick (drm) and sister of odd and bowl (sob) are entirely deleted. In these embryos, normal grooves formed in odd-numbered parasegments in the complete absence of odd function (Mulinari, 2009).

Next even-numbered parasegments were investigated in which grooves are partially missing. odd encodes a transcriptional repressor that regulates the expression of other segmentation genes in the early embryo. In odd mutants, derepression of the en activator fushi-tarazu in even-numbered parasegments results in the formation of an ectopic en stripe posterior to the normal stripe. Simultaneously, wg expands anteriorly and becomes expressed adjacent to the ectopic en-expressing cells. This results in the formation of an ectopic parasegment boundary with reversed polarity. Thus, the outward-facing edges of both en stripes are genetically anterior and lined by wg-expressing cells that do not form grooves. The inward-facing edges of the normal and ectopic en stripes fuse in some areas, and these corresponded to areas in which grooves were missing, as cells that were genetically posterior to en and could respond to the Hh signal had been replaced by en-expressing cells. The fusion of normal and ectopic en stripes was more severe in Df(2L)drm^P2 mutants; however, islands of invaginating groove cells could still be observed, demonstrating that groove fate is specified in the absence of odd, drm and sob function in all parasegments. It is concluded that all cells that are genetically posterior to en are specified as groove cells in the absence of odd function and the partial absence of grooves in even-numbered parasegments in odd mutants is a secondary consequence of the pair-rule phenotype of these embryos. The slightly more severe pair-rule phenotype seen in Df(2L)drm^P2 mutants might be due to a contribution from one of the odd sister genes, most likely sob, to pair-rule function, or could be caused by low-level read-through of the stop codon in the odd⁵ allele (Mulinari, 2009).

Finally, to investigate whether odd is sufficient to trigger cell shape changes, a UAS-odd transgene was expressed either alone or together with hh in the epidermis. No induction of groove cell behavior other than that triggered by hh was observed. Together, the data show that odd plays no essential role in groove cell specification and that odd paralogs are unlikely to act redundantly in this process (Mulinari, 2009).

The identification of odd as a groove cell marker led Vincent to suggest that groove fate might be specified prior to Hh requirement and that Hh may merely maintain groove fate instead of having an inducing role (Vincent, 2008). This study demonstrate that grooves are specified in the absence of odd function; however, this could be due to an odd-independent, early-acting mechanism present in the cells from which grooves arise (Mulinari, 2009).

In order to address whether groove fate is pre-established in the odd-expressing cell population, it was asked if groove fate could be induced in cells of a different origin at a later point in time. lines (lin) mutants were used in which late wg expression is altered resulting in the formation of an ectopic segment boundary at the anterior edge of the en domain in the dorsal epidermis. Importantly, the early expression of pair-rule or segment polarity genes is not affected (Mulinari, 2009).

In lin mutants, ectopic expression of the groove marker odd was initiated at stage 12 in a single row of groove-forming cells anterior to en that are derived from a previously non-odd-expressing cell population that does not contribute to grooves in the wild type. Ectopic grooves require hh as they were not induced in hh, lin double mutants, and ectopic odd expression was not induced in this background. An increase in hh levels in lin mutants resulted in the specification of groove fate in all cells except those expressing en. These results suggest that hh is sufficient, late in development, to specify groove cell fate in cell populations of different origins and that earlier-acting factors present in the population of odd-expressing cells posterior to en are not required. Very similar results have been reported by Piepenburg (2000), who showed that segment border cells form solely in response to the Hh signal that emanates from the en domain (Mulinari, 2009).

The findings are consistent with the role of Hh in the regulation of cell shape in other systems. Thus, during Drosophila eye development, Hh has been shown to control cell shape in the morphogenetic furrow, and Hh activation in other tissues is sufficient to induce apical constriction and groove formation. It is likely that Hh plays a similar role in tissue morphogenesis in other organisms. During neural tube closure in vertebrates, cells undergo similar shape changes involving apical-basal elongation and apical constriction, which is likely to be in response to Hh sources in the notochord and floor plate. Accordingly, knockout of sonic hedgehog is associated with defects in neural tube closure in mice. These observations suggest that Hh might be a principal inducer of cell shape across species (Mulinari, 2009).

It has previously been established that wg antagonizes the activity of hh in the specification of segment border cells (Piepenburg, 2000). However, it is not clear whether wg has a similar role in segmental groove formation, and a late requirement of wg to antagonize Hh-mediated groove specification has been questioned (Vincent, 2008). To investigate a direct role of wg in groove specification, a dominant-negative form of the transcription factor pan (pan^DN), which suppresses Wg signaling, was expressed. For this, pnr-Gal4, which initiates expression in the dorsal epidermis at stage 10-11 and thus does not affect early wg function, was used. Embryos that express pan^DN formed a single row of ectopic groove cells anterior to the en domain, confirming the results in lin mutants. Strikingly, inactivating Wg signaling and increasing Hh levels at the same time by co-expression of pan^DN and hh resulted in the expansion of groove fate to all cells except those expressing en. These results show that Wg signaling is required after stage 10 to repress groove specification anterior to en, thus making the activity of Hh asymmetric. These results also confirm observations that Hh is sufficient to induce groove fate in cells from different positions along the anterior-posterior axis and suggest that groove fate is not determined before stage 10 (Mulinari, 2009).

To confirm the ability of wg to repress groove fate, wg was expressed posterior to en in cells that normally take on groove fate. This resulted in the loss of Odd from many cells, suggesting that wg indeed antagonizes hh activity. Interestingly, these cells still formed grooves. However, these grooves appeared much earlier than segmental grooves, suggesting that they are ectopic parasegmental grooves caused by ectopic wg expression, as recently suggested (Larsen, 2008). Together, these data therefore support the contention that Wg signaling is required to repress Hh-mediated induction of groove fate after stage 10, thus permitting the formation of segmental grooves posterior, but not anterior, to en in the wild type (Mulinari, 2009).

SoxNeuro acts with Tcf to control Wingless signaling activity during segmentation

Wnt signaling specifies cell fates in many tissues during vertebrate and invertebrate embryogenesis. To understand better how Wnt signaling is regulated during development, genetic screens were performed to isolate mutations that suppress or enhance mutations in the fly Wnt homolog, wingless (wg). This study finds that loss-of-function mutations in the neural determinant SoxNeuro (also known as Sox-neuro, SoxN) partially suppress wg mutant pattern defects. SoxN encodes a HMG-box-containing protein related to the vertebrate Sox1, Sox2 and Sox3 proteins, which have been implicated in patterning events in the early mouse embryo. In Drosophila, SoxN has been shown to specify neural progenitors in the embryonic central nervous system. This study shows that SoxN negatively regulates Wg pathway activity in the embryonic epidermis. Loss of SoxN function hyperactivates the Wg pathway, whereas its overexpression represses pathway activity. Epistasis analysis with other components of the Wg pathway places SoxN at the level of the transcription factor Pan (also known as Lef, Tcf) in regulating target gene expression. In human cell culture assays, SoxN represses Tcf-responsive reporter expression, indicating that the fly gene product can interact with mammalian Wnt pathway components. In both flies and in human cells, SoxN repression is potentiated by adding ectopic Tcf, suggesting that SoxN interacts with the repressor form of Tcf to influence Wg/Wnt target gene transcription (Chao, 2007).

SoxN downregulates the Wg/Wnt pathway to reduce target gene expression. Downregulation is a crucial process because it sensitizes the signal response to allow rapid on/off switching and also keeps the system off in cells that are not actively responding to signal. Many genes have been shown to negatively regulate Wg/Wnt pathway activity through the destabilization of Arm/beta-catenin. Far fewer are known to exert negative regulatory effects downstream of Arm. The vertebrate Sox proteins -- Sox9, XSox3, XSox17α and XSox17ß -- as well as Chibby, a conserved nuclear factor, antagonize Wg/Wnt signaling by binding to Arm/beta-catenin and preventing it from partnering with Tcf to activate target gene expression. SoxN, however, does not bind beta-catenin in cell-culture assays, and does not share strong homology with the C-terminal sequences through which vertebrate Sox proteins bind this protein. Furthermore, SoxN function is not influenced by Arm levels. No difference was observed in SoxN-mediated TOPflash repression when cells were induced by co-transfection with a constitutively stabilized beta-catenin versus with Wnt-induced medium. Instead, both TOPflash and genetic experiments indicate that SoxN function depends on Tcf and Gro, its co-repressor (Chao, 2007).

One way to explain these observations is that SoxN contributes to the assembly or stability of the Tcf repressor complex on DNA. The consensus-sequence recognition for HMG domains in the Sox and Tcf families is reported to be similar, although XSox3 and XSox17ß fail to bind a consensus Tcf DNA sequence. It is shown that SoxN does not compete for Tcf-binding sites as a means of repressing target gene transcription, but the data support a model in which SoxN might bind DNA elsewhere or might bind Tcf sites transiently to initiate or stabilize the assembly of a repressor complex (Chao, 2007).

A similar model may explain the results from Xenopus that showed that XSox3-mediated repression does not require interaction between XSox3 and beta-catenin. XSox3 strongly interferes with dorsal fate specification in Xenopus embryos and represses TOPflash-reporter activity in vitro. HMG-domain mutations render XSox3 inactive in embryos without affecting its interaction with beta-catenin or its repression in TOPflash assays. Thus, it is the DNA-binding domain, not the beta-catenin-interacting C-terminus, that is relevant to its in vivo function in dorsal determination in Xenopus. XSox3 represses the expression of the dorsal-specific Nodal-related gene Xnr5 through optimal core binding sequences adjacent to and partially overlapping with Tcf sites in the Xnr5 promoter (Zhang, 2003). By contrast, the fly SoxN shows no discrepancy between its behavior in TOPflash assays and its in vivo effects. This suggests that the synthetic Tcf-binding sites arranged in the TOPflash-reporter plasmid are sufficient to support SoxN repressor function (Chao, 2007).

Because adding Tcf-site competitor DNA does not diminish the repressive capacity of limiting amounts of SoxN, the role of SoxN in repression does not appear to be stoichiometric. Therefore, the idea is favored that Sox proteins may act in a catalytic fashion during repressor-complex assembly at Wnt target gene promoters, rather than forming a structural part of the repressor complex itself. It was not possible to detect direct binding of SoxN with either Tcf, Gro or Arm, raising the possibility that SoxN interacts with some as yet unidentified protein that chaperones assembly of the repressor complex. A SoxN-binding cofactor, SNCF, has been identified in Drosophila (Bonneaud, 2003), but this gene is expressed only in pre-gastrulation embryos. Because Wg signaling occurs exclusively post-gastrulation, and specification of naked cuticle begins more than 4 hours after gastrulation, it is not thought that SNCF is a likely candidate for mediating this aspect of SoxN function. Rather, it is likely to play a role in the neuronal specification events promoted by SoxN at earlier stages of embryogenesis (Chao, 2007).

It is curious that uniformly overexpressed SoxN represses Wg signal transduction in dorsal epidermal cells more severely than in ventral cells. This effect is evident in both cuticle pattern elements and in en expression, and is reminiscent of defects observed in the 'transport-defective' class of wg mutant alleles, which includes wg^NE2. These mutations restrict Wg-ligand movement ventrally to promote only local signaling response while simultaneously abolishing all dorsal signaling, suggesting a fundamental difference in ventral and dorsal cell response. Perhaps it is not a coincidence that the NC14 mutation was isolated in the wg^NE2 mutant background. Further analysis of SoxN function may help to determine the molecular basis for dorsoventral differences in Wg signal transduction (Chao, 2007).

Wingless and tracheal development

The Drosophila tracheal tree consists of a tubular network of epithelial branches that constitutes the respiratory system. Groups of tracheal cells migrate towards stereotyped directions while they acquire specific tracheal fates. This work shows that the wingless/WNT signaling pathway is needed within the tracheal cells for the formation of the dorsal trunk (DT) and for fusion of the branches. These functions are achieved through the regulation of target genes, such as spalt in the dorsal trunk and escargot in the fusion cells. The pathway also aids tracheal invagination and helps guide the ganglionic branch. Moreover the wingless/WNT pathway displays antagonistic interactions with the Dpp pathway, which regulates branching along the dorsoventral axis. Remarkably, the wingless gene itself, acting through its canonical pathway, seems not to be absolutely required for all these tracheal functions. However, the artificial overexpression of wingless in tracheal cells mimics the overexpression of a constitutively activated Armadillo protein. The results suggest that another gene product, possibly a WNT, could help to trigger the wingless cascade in the developing tracheae (Llimargas, 2000).

Null wg mutants show a tracheal phenotype that is significantly weaker than is found when other members of the wingless pathway are removed: in wg mutants, fragments of DT are formed and fused, yet, in other mutants of the pathway, the DT is eliminated. This suggests, in acting during DT development, the wingless cascade does more than just respond to wg. But whereas loss of function shows that wg is not absolutely required for DT formation, its misexpression in tracheal cells indicates that it is sufficient to confer DT identity. These unconventional results are difficult to reconcile with a simple model. A possible explanation is that an additional factor or factors might help to trigger the Wg cascade to form the DT, the other Drosophila Wg genes being the most likely candidates. Intriguingly, in porcupine mutants, the DT also fails to form, suggesting that por mutants lack this factor/s. Therefore, if the activation of the pathway depends, at least in part, on other Wnt genes, the results indicate that por would be required for their secretion too. The Wg cascade could be activated by a particular Wnt or Wnt combination or alternatively, Wnt genes could be all functionally redundant. Wnt4 maps near wg and both genes are deficient in Df(2R)RF. However, the tracheal phenotype of this deficiency is almost identical to that of wgCX4, ruling out the possibility that zygotic Wnt4 might act on its own or in combination with wg in forming the DT. Wnt2 mutants are homozygous viable, suggesting that Wnt2 is dispensable in DT formation. However, it might contribute to DT formation in combination with wg or other Wnts. So far, there are no point mutations for Wnt3, and thus any possible tracheal phenotype could not be assessed. Other Wnt genes have recently been identified by the genome sequence project, and these also remain candidates (Llimargas, 2000).

One can envisage a model where the Wg pathway has a basal activity stimulated upon binding of any Wnt or Wnt gene combination to the receptor complex. These low levels of Wg pathway activity would be sufficient to allow DT formation and DT fusion (presumably through activation of some fusion genes). Tracheal invagination, activation of other fusion genes, and guidance of GBs would require higher levels of activity of the pathway -- these would depend on the binding of wg itself, sourced from nearby tissues (Llimargas, 2000).

arm has been shown to play dual but separable roles: one in Wg signal affecting gene expression and the other in cell adhesion. Null arm mutants have a tracheal phenotype that is, in part, due to loss of its adhesive function. This phenotype can not be simply explained by a decrease in cell adhesion. The results of this work provide several indications that at least part of arm function in the trachea is indeed due to its action in Wg signaling: (1) an arm allele that specifically impairs Wg signal produces a similar phenotype to that of an arm null allele; (2) some tracheal defects are related to a direct or indirect regulation of tracheal target genes; (3) other members of the Wg pathway participate in the same tracheal events (Llimargas, 2000).

Which part of the arm tracheal phenotype is due to its adhesive role? arm has a role in cell adhesion by interacting with shotgun, which encodes DE-cadherin. shg and arm mutants are both defective in branch fusion. However, the cause of these two phenotypes does not appear to be the same, since esg (a marker of fusion fate) is normally expressed in shg mutants but not in arm mutants. This indicates that the fusion cells are normally specified in shg mutants, whereas arm is required within the fusion cells themselves to regulate fusion markers. Therefore, arm might have a dual function during branch fusion: it is first required to activate the fusion fate through the Wg signaling and later, through its interaction with shg, it is required for the cell reorganizations that lead to branch fusion (Llimargas, 2000).

The results indicate that the Wg pathway acts in DT formation by regulating sal expression. Although this regulation could be direct, it is also possible that the Wg pathway acts by regulating the number of cells that will later express sal under a Wg-independent control. Two pieces of evidence show that normally the Wg pathway is active only in the DT during primary branching. (1) The constitutive activation of Arm cause integration of the visceral branches (VBs) into DT. Such transformation would not be expected if the pathway were normally active in the VBs. (2) In the absence of tkv, the pathway must be constitutively activated to observe sal expression in every tracheal cell. If the pathway were normally active in all tracheal cells, the mere absence of tkv should be sufficient to allow sal expression. Moreover, the pathway seems to be required in the DT cells themselves. The expression of dominant negative Pangolin in all tracheal cells impairs DT formation, indicating that there is an autonomous requirement within the tracheal cells. When the expression of dominant negative Pangolin in the tracheae is maintained only in the DT cells, similar DT defects are observed (Llimargas, 2000).

The Dpp pathway is required to form the dorsal and ventral branches while the Wg pathway is required for DT formation. Accordingly, only the VBs and transverse connectives (TCs), which are independent of these two pathways, form in their absence. The Wg and the Dpp pathways are known to act antagonistically in several structures, such as the wing or the leg disc. Similarly, the Wg and the Dpp pathways have opposing activities during primary branching. Only when the Dpp pathway is not active is the Wg pathway able to confer DT identity to the tracheal cells. This antagonism is likely to be mediated through the negative regulation of sal by kni. sal is positively regulated by the Wg pathway and kni is activated by the Dpp pathway. However, when the Wg signal is constitutive in a wild type background, the VBs (which express kni) seem to adopt a DT identity. Remarkably, in the VBs, the Dpp pathway is neither active nor does it control kni expression. This indicates that kni expression is not sufficient to prevent sal regulation by the Wg pathway and that other targets of Dpp might also antagonize the WG/WNT pathway (Llimargas, 2000).

Sequencing of the Drosophila genome has revealed that there are 'silent' homologs of many important gene family members that were not detected by classic genetic approaches. Why have so many homologs been conserved during evolution? Perhaps each one has a different but important function in every system. Perhaps each one works independently in a different part of the body. Or, perhaps some are redundant. This study takes one well known gene family and analyzes how the individual members contribute to the making of one system, the tracheae. There are seven DWnt genes in the Drosophila genome, including wingless. The wg gene helps to pattern the developing trachea but is not responsible for all Wnt functions there. Each one of the seven DWnts was tested in several ways and evidence was found that wg and DWnt2 can function in the developing trachea: when both genes are removed together, the phenotype is identical or very similar to that observed when the Wnt pathway is shut down. DWnt2 is expressed near the tracheal cells in the embryo in a pattern different from wg's, but is also transduced through the canonical Wnt pathway. The seven DWnt genes vary in their effectiveness in specific tissues, such as the tracheae; moreover, the epidermis and the tracheae respond to DWnt2 and Wg differently. It is suggested that the main advantage of retaining a number of similar genes is that it allows more subtle forms of control and more flexibility during evolution (Llimargas, 2001).

DWnt proteins bind as ligands to a family of receptor proteins -- four Frizzled (Fz) homologs in Drosophila, of which Fz and Fz2 are the most important and act through a cascade of genes [e.g., disheveled, armadillo (arm), pangolin] on the nucleus. If, therefore, Wg is the only ligand acting from the outside of the cell on the receptors, the wg^- phenotype should be identical to the phenotype when fz and fz2 are removed -- in some organs, this is so. However, in the trachea, although removal of the two receptor proteins or one of the intracellular proteins in the cascade eliminates all dorsal trunk (DT), removing only Wg leaves some DT intact. Therefore, it seems that another molecule, presumably a DWnt, acts through the canonical Wnt pathway to build DT. This study asks which DWnt is responsible (Llimargas, 2001).

Overexpression of wg or other downstream elements of the Wnt pathway in the tracheal cells results in increased DT at the expense of the ventral branch. To investigate further, each one of the seven DWnts was overexpressed locally in the embryonic trachea in a normal background. Overexpression of five DWnt genes (DWnt5, -4, -6, -8, and -10) had no detectable effects; indeed, the flies were viable, fertile, and seemed normal. This experiment suggests that the tracheae are not particularly sensitive to these five proteins. To check whether these proteins are made properly and can function, they were tested in other assays. DWnt6 and DWnt8 are able to affect tracheal development in a sensitized background. DWnt5 produces a phenotype in the ventral nerve cord when expressed with the neural specific driver 1407Gal4, in agreement with the phenotype produced by a HS-DWnt5. Moreover, protein expression is detected in the tracheae when DWnt5 is expressed in tracheal cells. DWnt4 produces ectopic denticles in the ventral epidermis when overexpressed with armGal4, and the flies die as pharate adults, showing several defects in the wings when crossed to ptcGal4. No noticeable phenotype was found when overexpressing DWnt10 in several structures, and, thus, the activity of this line awaits confirmation. However, DWnt10 together with three other DWnts (DWnt4, -6, and wg) were removed in Df(2L)RF embryos and a phenotype similar to wg^- was found, because there still was some DT. This experiment argues that at least zygotic DWnt 4, -6 and -10 do not have a significant function in the trachea under normal conditions. However, overexpression of DWnt2 locally in the tracheal cells does affect its development in a way similar to that of wg, producing an excess of DT cells and DT material at the expense of the ventral branch. These tracheae are defective; they fail to fill with air and the flies die as embryos and young larvae. This result suggests that both wg and DWnt2 act or can act in the developing trachea (Llimargas, 2001).

The tracheal placodes are specified by stage 10 in a specific part of the dorsal ectoderm and express several markers such as trachealess. The expression of DWnt2 is suggestive: it is expressed close to and dorsal to the tracheal placode by stage 10 and early stage 11 but later disappears (Llimargas, 2001).

The spalt (sal) gene (coding for a transcription factor) is expressed in the dorsal ectoderm, including some tracheal cells, during stage 10 and persists later in those tracheal cells that form the DT. sal is absolutely required for DT formation and is thus a good marker for DT cell identity. The most dorsal cells that express sal also coexpress DWnt2. The pattern of wg expression differs strikingly from that of DWnt2, although both gene products are made near the tracheal cells. In arm mutants, Sal is not expressed in tracheal cells and no DT is formed, suggesting that sal expression in tracheal cells depends on activation of the Wnt pathway. Thus, sal can be induced in the tracheal cells wherever either Wg or DWnt2 proteins are received (Llimargas, 2001).

The above results suggest that wg and DWnt2, made near the tracheal cells, together sponsor DT formation. In wg^- embryos, some DT is still formed. However, the tracheal phenotype of wg^-DWnt2^- embryos is significantly different from that of wg^- embryos: in 40%-45% of hemisegments, the DT is completely missing, and in the remaining 55%-60%, only some reduced and thin DT forms. Interestingly, in practically all hemisegments of Df(2L)RF DWnt2^- embryos, the DT is completely missing, indicating that other DWnts account for these traces of DT. Nevertheless, wg^-DWnt2^- double-mutant embryos are very similar to or indistinguishable from fz^-fz2^- embryos, suggesting that wg and DWnt2 sponsor virtually all DT formation (Llimargas, 2001).

Removal of Wg and DWnt2 proteins (in wg^-DWnt2^- embryos) eliminates detectable expression of sal in the presumptive tracheal cells of the DT, whereas in wg^- embryos, very low levels of sal still can be detected in some embryos. The early expression of sal in the dorsal ectoderm still is observed in both wg^- and wg^-DWnt2^- embryos. In wg^-DWnt2^- embryos, late kni expression in tracheal cells is normal, as is the case in arm mutants. In addition, dpp expression also is normal -- Dpp has been shown to inhibit sal expression by activating kni in tracheal cells. Thus, the lack of sal must be caused by the absence of direct or indirect stimulation by the DWnt pathway and not due to repression by the Dpp pathway (Llimargas, 2001).

Does DWnt2 act through the canonical Wnt pathway? It seems so, because the ectopic effects of DWnt2 protein are blocked in embryos that lack the arm gene. Moreover, in wg^-DWnt2^- embryos, the DT can be substantially rescued by expressing a constitutively active form of Arm in the tracheal cells. Also, the tracheal phenotype of porcupine (por) mutants is very similar to that of wg^-DWnt2^- embryos, indicating that por also might be required for DWnt2 secretion (Llimargas, 2001).

If DWnt2 sponsors at least part of DT formation, one might expect that loss of DWnt2 alone would affect trachea in some noticeable way. Surprisingly, DWnt2^- embryos and larvae have normal trachea and normal expression of sal. However, the flies have reduced viability and the males are sterile (Llimargas, 2001).

In normal embryos, the wg gene is expressed in a row of cells at the rear of the A compartment, whereas DWnt2 is expressed at the front. Wg protein spreads to make a gradient that patterns the anterior compartment. DWnt2 protein is expressed where the concentration of Wg is low or absent; that is where the tracheal placodes form and where the cuticle secretes denticles. Thus, in wg^- embryos, where there is no Wg protein and the denticles are continuous, one might expect the tracheal placodes and DWnt2 expression to form one continuous stripe and, indeed, they do (Llimargas, 2001).

This adventitious expression of DWnt2 in a broad domain in wg^- embryos could compensate at least in part for the lack of wg itself. Indeed, in these embryos, it must be mainly DWnt2 that activates some sal and determines most or all of the DT found. No change could be detected in the pattern of wg RNA or protein distribution in DWnt2 mutants (Llimargas, 2001).

The potency of DWnt2 and Wg in the tracheae was assayed: DWnt2^-wg^- double mutants were taken and each of the two missing proteins were added back in the normal pattern of expression for the wg gene. DWnt2 and Wg were found to both rescue some DT in the trachea; however, only Wg can partially rescue the various embryonic defects in morphology found in wg^- embryos. When either DWnt2 or Wg is expressed locally in the tracheal cells, each gives strong rescue, and more DT is made (Llimargas, 2001).

The DWnt2 gene was also expressed in wild-type embryos either universally and strongly (arm VP16 Gal4) or in stripes (ptcGal4), and in both cases, the tracheae are altered to the same extent as when DWnt2 is expressed in the tracheal cells alone. However, DWnt2 fails to alter the cuticle pattern, whereas wg produces a naked cuticle phenotype. This lack of effect of DWnt2 on the epidermis is remarkable since both the drivers used are strong and, when wg is driven, are more than adequate to make a naked cuticle. Interestingly, when DWnt2 is missexpressed in the eye, it also does not emulate the phenotype produced by missexpression of wg. Moreover, the effects of overexpressing DWnt2 in the ovary are stronger than when overexpressing wg. All these results argue that the tracheal cells and other tissues, including the epidermis, the eye, and the ovary are differentially sensitive to the two DWnt molecules, the trachea and the ovary being particularly responsive to DWnt2 (Llimargas, 2001).

There are several ways this difference could be achieved. Perhaps DWnt2 does not act through the canonical Wnt pathway in some tissues, such as the ectoderm or the eye. Perhaps DWnt2, on its way to the tracheal cells, could be secreted or processed differently. Perhaps the tracheal cells have something that allows efficient presentation of the ligand to the Fz receptors, or they lack a component that, in other tissues, impedes DWnt2 binding or transduction. One possibility is that glucosaminoglycans help breathless (btl, an FGF receptor expressed in tracheal cells) and are needed for Wnt signaling. Maybe Btl helps to gather or modify the heparan sulfate glucosaminoglycans, thereby altering the presentation of DWnt2 to the two receptors, Fz and Dfz2. Whatever the explanation, the tracheal cells are more responsive than other tissues to the DWnt2 signal (Llimargas, 2001).

The other DWnts were examined. DWnts5, -6, -8, and -10 were drived in the epidermis of wild-type embryos with one copy of ptcGal4; none of these affected the cuticle pattern in a noticeable way. Are these DWnts able to affect tracheal development in the wg^-DWnt2^- double mutants? Each of these five DWnts were added back to either the tracheal cells themselves or in the pattern of normal wg expression. DWnts6 and -8 are each able to rescue DT partially, whereas -4, -5 and -10 did not. Note that DWnt6 and DWnt8 are not able to produce a tracheal phenotype when expressed in tracheal cells of normal embryos, but they can do so in a sensitized background (Llimargas, 2001).

The results indicate that wg and DWnt2 make the main contribution to DT formation, as the absence of both genes completely eliminates DT in many cases. However, traces of DT still are formed in about half the hemisegments of wg^-DWnt2^- embryos, indicating that contributions of other genes might help. Also, rescue experiments show that some other DWnts are able to activate the pathway. In agreement with this result, in most Df(2L)RF DWnt2^- embryos, all DT is missing, indicating that DWnt6 and/or -4 and/or -10 can compensate weakly for the absence of wg and DWnt2. However, expression of DWnt6 and DWnt10 does not suggest that they act in tracheal development in the wild type. DWnt4 is expressed in a similar pattern to that of wg but does not seem to assist wg during embryogenesis. In addition, none of DWnt4, -6, or -10 affected tracheal development when expressed in tracheal cells of wild-type embryos. Most likely, they produce traces of DT in the wg^-DWnt2^- embryos, because those embryos offer a very sensitive test of stimulation of the Wnt pathway. It remains unclear whether these DWnts make any residual contribution to DT in the wild type (Llimargas, 2001).

However, several observations suggest that DWnt2 contributes to tracheal development in the wild-type fly. Notably, DWnt2 is expressed near the tracheal cells at the appropriate stage, and when overexpressed in tracheal cells, it mimics the effects of overexpressing wg or a constitutively activated Arm. But, most importantly, the phenotype of wg^-DWnt2^- embryos indicates that wg and DWnt2 together are responsible for virtually all DT formation. Thus, DWnt2 probably cooperates with Wg or reinforces its main action (Llimargas, 2001).

Nevertheless, DWnt2^- embryos do not show a visible tracheal phenotype, indicating, at first sight, that the gene does not normally contribute to DT formation. This lack of abnormality suggests that wg alone (or with some help from different DWnts) is sufficient to sponsor normal development in these mutant flies. Nevertheless, it remains possible that DWnt2 could act in the wild-type. There are at least two alternative hypotheses that could explain the lack of tracheal phenotype in DWnt2^- embryos (Llimargas, 2001).

(1) The loss of DWnt2 could induce compensatory changes in the amount, distribution, or activity of the other DWnts. As in the case of DWnt6, -4, and -10, the expression of DWnt5 and DWnt8 (expression is detected only in the CNS at early stages) does not suggest that they act in tracheal development, although contributions under the level of detection cannot be discarded. Moreover, although some small changes have been detected in the expression of some DWnts in wg^- and wg^-DWnt2^- embryos (e.g., the loss of DWnt5 expression in the labial segment at stage 10 as well as loss of expression in lateral clusters of the thoracic segments at stage 11), no changes have been detected in the pattern of expression that might account for any strong tracheal rescue of DWnt2^- embryos. Therefore, it is not clear how other DWnts could contribute to the complete DT formation in DWnt2^- embryos (Llimargas, 2001).

(2) It is supposed that all DWnts bind the receptor with different affinities, with Wg binding most strongly. In the wild type, the DWnts could compete, but Wg would be most effective: the contribution of DWnt2 to DT formation would be minor. However, in embryos lacking Wg, mainly DWnt2 (which is expressed in a broader domain in wg^- embryos and is not now competing with Wg) could bind and partially substitute for Wg. In the absence of DWnt2, Wg (and maybe other DWnts) would have no competition from DWnt2 and would become even more efficient, compensating for the contribution to DT formation that DWnt2 has in the wild type. Finally, in the absence of both Wg and DWnt2, other DWnts, even if they did not act in the wild type, could now bind to the unoccupied receptors and have some tiny effect on DT formation (Llimargas, 2001).

Complications of this kind may bedevil attempts to analyze the precise wild type contributions of individual members of other gene families (Llimargas, 2001).

Thus DWnt2 can act in tracheal development, whereas Wg acts in both developing epidermis and trachea. The other five DWnts do little for the trachea. As with the achaete/scute homologs (which are alike in structure and function but have different patterns of expression and, therefore, act in different places, it may be that the DWnts are preserved fundamentally because seven genes, even if they do similar things, can be regulated in a more sophisticated way than one. Perhaps, like DWnt2, they perform specialized tasks, acting locally to help Wg in ways that could not be provided by any additional regulatory control of wg itself. In the case of tracheae, this tissue can have differential sensitivity to specific homologs, a property that may allow even more intricate forms of control (Llimargas, 2001).

Wingless, the BTB/POZ protein Ribbon, and tracheal branch migration

During development, directed cell migration is crucial for achieving proper shape and function of organs. One well-studied example is the embryonic development of the larval tracheal system of Drosophila, in which at least four signaling pathways coordinate cell migration to form an elaborate branched network essential for oxygen delivery throughout the larva. FGF signaling is required for guided migration of all tracheal branches, whereas the Dpp, Egf receptor, and Wingless/WNT signaling pathways each mediate the formation of specific subsets of branches. ribbon encodes a BTB/POZ-containing protein required for specific tracheal branch migration. In ribbon mutant tracheae, the dorsal trunk fails to form, and ventral branches are stunted; however, directed migrations of the dorsal and visceral branches are largely unaffected. The dorsal trunk also fails to form when FGF or Wingless/WNT signaling is lost, and ribbon is shown to function downstream of, or parallel to, these pathways to promote anterior-posterior migration. Directed cell migration of the salivary gland and dorsal epidermis is also affected in ribbon mutants, suggesting that conserved mechanisms may be employed to orient cell migrations in multiple tissues during development (Bradley, 2001).

Two observations have been made that support a model proposing that the primary role for Egfr signaling is the invagination of tracheal primordia and that defects in branch migration may be an indirect result of reduced invagination. (1) All branches contained fewer cells in rho mutants, hence no particular branch identity is lost. (2) sal expression in the dorsal tracheal cells of rho mutants during primary branch outgrowth is normal, suggesting that Egfr is not required to specify cell fate within the placode (at least as measured by expression of this dorsal trunk-specific gene). Thus, Egfr signaling may only regulate invagination, which would position cells to receive subsequent signals specifying branch fate (Bradley, 2001).

Analysis of Wg signaling in tracheal branching suggests that cells are allocated to branches (cell allocation) independently from cell fate specification. (1) In Wg signaling mutants the 'pre-dorsal trunk' cells are positioned correctly, but fail to migrate away from the transverse connective. (2) Wg signaling mutants do not express sal, a dorsal trunk-specific marker. Thus, the cells are allocated to the dorsal trunk (DT), but do not express DT markers or behave like DT cells. rib mutants, like Wg/WNT signaling mutants, also fail to form the DT, and 'pre-DT'cells are stalled at the transverse connective (TC); however, unlike embryos lacking Wg signaling, rib mutants express sal in DT cells. Thus, rib is not required for cell allocation or cell fate specification (as monitored by sal), but is only required for branch migration. In summary, these observations suggest that, at least for the tracheal DT, cell allocation is independent of cell fate specification, and cell fate can be further subdivided into branch identity (controlled by genes such as sal that specify branch features) and branch migration, which involves rib (Bradley, 2001).

The similarity of the tracheal DT phenotypes in rib mutants and Wg signaling mutants raises the possibility that rib functions with Wg signaling for migration of DT cells. sal is the only known early downstream target of Wg/WNT signaling in the DT. Because the DT phenotype is more severe in embryos lacking Wg/WNT signaling than in sal mutants, there must be additional downstream targets of Wg signaling. Indeed, it can be predicted that these other genes control migration based on two findings. (1) DT cells are capable of migrating in sal mutants, but move in the wrong direction (dorsally). (2) When both Wg and Dpp signaling are activated in wild-type embryos (activated arm and activated tkv in all tracheal cells), a complete longitudinal DT forms that does not express sal, suggesting that sal may be dispensable for anteroposterior migration in some cases. Loss of rib results in a DT phenotype identical to that observed in loss of Wg/WNT signaling and rib functions in parallel to Wg/WNT-dependent sal expression. Together these results suggest that rib is working with Wg/WNT signaling, either in parallel or potentially as a downstream target, to direct DT migration (Bradley, 2001).

It is hypothesized that rib may respond to signals from multiple pathways based on analysis of a ventral cuticle phenotype. In rib mutants, the defects in ventral cuticle patterning appeared most similar to the phenotype reported for the combined loss of late Wg signaling and Egfr signaling. In this tissue, rib could be integrating signaling from Wg and Egfr. In several other tissues requiring rib function, Wg signaling and signaling through a MAPK cascade are also required; however, in these cases, loss of either of the individual pathways results in phenotypes similar to those of rib mutants. For instance, rib is required for the cell shape changes in the leading edge cells during dorsal closure, a process that requires both Wg signaling and JNK signaling. The second midgut constriction and the morphogenesis of the Malpighian tubules are defective in rib mutants, and both events also require both Wg and Egfr signaling. Similarly, in the trachea, rib could respond to Wg signaling and either of the two pathways (FGF or Egfr) that activate the MAPK cascade in tracheal cells. Since the rib phenotype is distinct from Egfr signaling mutants, a role for rib downstream of FGF signaling is favored. Indeed, the stalled outgrowth of all tracheal branches and stunted ventral branches observed in rib mutants may be linked to FGF signaling. Consistent with the idea that rib responds to MAPK signaling, the Rib protein has seven consensus MAPK phosphorylation sites (Bradley, 2001).

rib is thought to be required for generating specialized cell shapes. For instance, during dorsal closure, leading edge cells of the lateral epidermis fail to elongate in rib mutants. rib mutants also show abnormal dilation of salivary gland lumina in late embryogenesis, suggesting that either rib is also required at late stages to maintain organ shape or loss of early rib function indirectly causes the late lumenal dilation. rib appears to control cell shapes by regulating the cytoskeleton. During dorsal closure, a band of actin and myosin forms at the dorsal margin of leading edge cells. In rib embryos, the actin band is narrower and myosin heavy chain (MHC) is absent from leading edge cells. Thus, rib may be required for the localization or organization of cytoskeletal components. zip encodes a nonmuscle MHC and is required in many of the same tissues as rib; however, strong loss-of-function mutations in zip suppress the distended lumenal phenotype of rib salivary glands, suggesting that rib does not positively regulate myosin activities. Instead, rib may repress myosin contraction or regulate the direction of contraction, perhaps by providing a balancing force to the direction of basal myosin contractions. These studies reveal a role for rib in coordinating directed cell migration, a process that clearly involves actin/myosin dynamics. Thus, rib may modulate actin/myosin behavior for cell movement and cell shape during both tissue formation and tissue homeostasis. If rib is responding to signaling pathways, rib could be a critical factor linking signaling events to changes in the cytoskeleton (Bradley, 2001).

The rib gene encodes a novel protein with two protein-protein interaction domains, an N-terminal BTB/POZ domain and a C-terminal coiled-coil region. The BTB/POZ domain mediates dimerization, and BTB/POZ proteins often contain additional domains that define protein function and/or subcellular localization. Many BTB/POZ proteins contain multiple DNA binding zinc fingers and function as transcriptional regulators. For example, the Drosophila Tramtrack protein is required to represses transcription of pair-rule genes in early embryogenesis. BTB/POZ domain proteins can also mediate cytoskeletal organization. For instance, the Drosophila Kelch protein, which oligomerizes via its BTB domain and binds actin through its kelch domains, is required to maintain cytoskeletal organization of ring canals during oogenesis. BTB/POZ proteins can also function outside the cell; the mammalian BTB/POZ protein Mac-2 binding protein (M2BP) localizes to the extracellular matrix (ECM) and forms multivalent ring structures proposed to be important for its interactions with collagens IV, V and VI, fibronectin, and other ECM proteins. One family of Arabidopsis BTB/POZ-containing proteins has a composition very similar to that of RIB: a BTB/POZ domain at the N-terminus and a coiled-coil at the C terminus. One family member, RPT2, appears to respond to signals that promote phototropism. RPT2 also contains an NLS; however, it is not yet known where RPT2 functions. Based on the four putative NLSs, it is speculated that RIB may function in the nucleus, where it would be positioned to regulate the expression of genes required for cytoskeletal changes during morphogenesis. Alternatively, RIB may reside in the cytoplasm and more directly regulate cytoskeletal organization. Since BTB/POZ domains can heterodimerize, RIB may have a partner(s) providing additional functional motifs. rib RNA is expressed in a dynamic pattern during development, including expression in cells that appear phenotypically normal in rib embryos. Thus, rib function is likely to be post-transcriptionally regulated, perhaps through the phosphorylation of its MAPK sites or through limited expression or activation of cofactors. Further investigation of rib may lead to a better understanding of the mechanisms by which cells control the direction of migration during development (Bradley, 2001).

Wingless signaling and induction of neoplastic tumors

In Drosophila, stresses such as x-irradiation or severe heat shock can cause most epidermal cells to die by apoptosis. Yet, the remaining cells recover from such assaults and form normal adult structures, indicating that they undergo extra growth to replace the lost cells. Recent studies of cells in which the cell death pathway is blocked by expression of the caspase inhibitor P35 have raised the possibility that dying cells normally regulate this compensatory growth by serving as transient sources of mitogenic signals. Caspase-inhibited cells that initiate apoptosis do not die. Instead, they persist in an 'undead' state in which they ectopically express the signaling genes decapentaplegic and wingless and induce abnormal growth and proliferation of surrounding tissue. Using mutations to abolish Dpp and/or Wg signaling by such undead cells, it has been shown that Dpp and Wg constitute opposing stimulatory and inhibitory signals that regulate this excess growth and proliferation. Strikingly, when Wg signaling is blocked, unfettered Dpp signaling by undead cells transforms their neighbors into neoplastic tumors, provided that caspase activity is also blocked in the responding cells. This phenomenon may provide a paradigm for the formation of neoplastic tumors in mammalian tissues that are defective in executing the cell death pathway. Specifically, it is suggested that stress events (exposure to chemical mutagens, viral infection, or irradiation) that initiate apoptosis in such tissues generate undead cells, and that imbalances in growth regulatory signals sent by these cells can induce the oncogenic transformation of neighboring cells (Perez-Garijo, 2005).

Cells that initiate apoptosis in response to x-irradiation normally disappear rapidly, but, as shown in this study, they can persist indefinitely with the help of the caspase inhibitor P35 and maintain characteristics of the apoptotic program, such as Hid, Dronc, and Drice activities and ectopic wg and dpp expression. The same is also true of cells that initiate apoptosis in response to severe heat shock, a stress that is unlikely to change the integrity of the genome, in contrast to x-irradiation. Because undead cells can divide, albeit at a much lower rate than live ones, this epigenetic condition seems to be inherited through cell division. Thus, the developmental aberrations induced by undead cells in surrounding tissue are likely caused by their continuing to send mitogenic signals that might normally be sent only transiently by dying cells (Perez-Garijo, 2005).

The results indicate that the ability of undead cells to induce growth and proliferation depends on their being able to send Dpp signal, a finding that fits with the proposal that Dpp normally regulates cell growth and proliferation in the wing disc. Conversely, the excessive growth induced by undead cells within wg^– P35 clones indicates that Wg acts to inhibit the mitogenic action of Dpp, a role consistent with the finding that Wg functions as a growth repressor during some phases of wing development (Perez-Garijo, 2005).

It is proposed that Dpp and Wg regulate the abnormal growth induced by undead cells by exerting opposite stimulatory and inhibitory effects: Dpp promotes cell division whereas Wg inhibits the response to Dpp, thus constraining the production of new cells and limiting the extent of overgrowth. In normal circumstances in which caspase activity is not blocked by P35 expression, apoptotic cells disappear rapidly, and the transient production of Dpp and Wg may play a role in restoring, but not exceeding, the missing cells. When the apoptotic cells are kept alive with P35, the persistent production of Dpp and Wg and their slightly imbalanced effects cause local overproliferation and outgrowth, which are most clearly observed when an entire compartment is affected. If they cannot send the Dpp signal (e.g., because they are mutant for dpp), there is no proliferative stimulus, but if they can send Dpp but not Wg, the growth promoting function of Dpp acts unimpeded and induces a dramatic over-production of tissue (Perez-Garijo, 2005).

This model accounts at least in part for the remarkable capacity of undead cells within wg^– P35 clones to induce tumors: absence of the proposed inhibitory action of Wg would remove a constraint on the growth-promoting action of Dpp. The lack of wg activity itself may also be a growth-promoting factor, because there is evidence that wg normally acts as a dMyc repressor in cells flanking the prospective wing margin; the increased dMyc levels in the absence of wg activity promote cell cycle progression and growth. However, the tumorous behavior of stressed wg^– P35 clones cannot be explained simply by the uninhibited action of Dpp emitted by undead cells or the consequent elevation of dMyc activity in the surrounding cells, because neither ectopic Dpp signaling nor overexpression of dMyc is sufficient to cause neoplastic transformation in the imaginal discs. It is suggested that undead cells send additional signals that act together with Dpp to induce the neoplastic transformation (Perez-Garijo, 2005).

Another prerequisite for the induction of tumors by undead cells seems to be that the responding cells must also be unable to execute the cell death pathway. It is speculated that unregulated growth within wg^– P35 clones may create new cellular stresses both inside and outside the clones that induce secondary apoptotic events. Apoptotic cells outside the clones would be rapidly eliminated, but those within would be protected by P35 and join a growing population of undead cells that become new sources of Dpp and other tumor-inducing factors. The populations of both undead and live cells within the clone would thus expand at the expense of the surrounding wild-type tissue, eventually eliminating all of the cells that do not express P35. Circumstantial evidence in favor of this view is the large number of undead (Hid-expressing) cells found in discs overgrown by wg^– P35 clones. Given that undead cells proliferate at a low rate, it seems likely that at least some if not most of the undead cells in wg^– P35 tumors will have arisen by secondary apoptotic events, rather than by descent from the initial founder population of stress-induced, undead cells (Perez-Garijo, 2005).

Such a mechanism can account for the dramatic expansion of wg^– P35 clones at the expense of surrounding wild-type tissue, once they are seeded by the initial induction of undead cells. However, it does not explain why only P35-expressing cells, and not neighboring wild-type cells, develop neoplastic properties such as the failure to maintain a normal epithelial morphology. This difference in behavior raises the possibility that P35 expression may have additional consequences, aside from the direct block of the cell death pathway, that predispose cells to neoplastic transformation (Perez-Garijo, 2005).

These results have potential implications for models of tumor transformation in mammals. It is normally argued that oncogenesis is a multistep process that requires a number of successive somatic mutations, but there are also indications that, in some instances, the transformation of cancer cells is associated with epigenetic phenomena, that is, heritable changes in gene function not caused by somatic mutations. This study has provided an example in which cell populations that cannot execute the cell death pathway are predisposed to oncogenic transformation by just such an epigenetic event, namely the induction of undead cells in response to cellular stress. In Drosophila, the ability of such undead cells to induce neighboring cells to become tumorous seems to depend on their sending an abnormal balance of growth regulatory signals that up-regulate activity of the proto-oncogene dMyc in neighboring cells. As a consequence, the responding cells behave as supercompetitors that overproliferate and eventually eliminate surrounding wild-type cells. These findings suggest a mechanism for generating neoplastic tumors in caspase-inhibited cells (Perez-Garijo, 2005).

Evading apoptosis is widely recognized as a hallmark of cancer cells. There is also evidence that caspase activity is inhibited in some aggressive human cancers. These findings may therefore provide a paradigm for the formation of neoplastic tumors in tissues that are unable to die (Perez-Garijo, 2005).

Heart- and muscle-derived signaling system dependent on MED13 and Wingless controls obesity in Drosophila

Obesity develops in response to an imbalance of energy homeostasis and whole-body metabolism. Muscle plays a central role in the control of energy homeostasis through consumption of energy and signaling to adipose tissue. MED13, a subunit of the Mediator complex, acts in the heart to control obesity in mice. To further explore the generality and mechanistic basis of this observation, this study investigated the potential influence of MED13 expression in heart and muscle on the susceptibility of Drosophila to obesity. This study shows that heart/muscle-specific knockdown of MED13 or MED12, another Mediator subunit, increases susceptibility to obesity in adult flies. To identify possible muscle-secreted obesity regulators, an RNAi-based genetic screen of 150 genes was performed that encode secreted proteins; Wingless inhibition was also found to cause obesity. Consistent with these findings, muscle-specific inhibition of Armadillo, the downstream transcriptional effector of the Wingless pathway, also evoked an obese phenotype in flies. Epistasis experiments further demonstrated that Wingless functions downstream of MED13 within a muscle-regulatory pathway. Together, these findings reveal an intertissue signaling system in which Wingless acts as an effector of MED13 in heart and muscle and suggest that Wingless-mediated cross-talk between striated muscle and adipose tissue controls obesity in Drosophila. This signaling system appears to represent an ancestral mechanism for the control of systemic energy homeostasis (Lee, 2014).

The results reveal a role of muscle in systemic regulation of obesity via the function of MED13 in Drosophila. A genetic screen identified muscle-secreted obesity-regulating factors, including Wg, and demonstrated that Wg signaling in muscle is necessary and sufficient to suppress obesity. Furthermore, it was shown that a skd-null mutation dominantly enhances the arm phenotype in muscle and that wg is epistatic to skd, suggesting that Wg is a downstream effector of MED13 in muscle (Lee, 2014).

The results reveal that MED13 in Drosophila muscle functions to suppress obesity based on several criteria, such as histology, measurement of whole-body triglycerides, tolerance to starvation stress, and susceptibility to high-fat diet. Similarly, muscle-specific knockdown of MED12 also increases fat accumulation, suggesting that MED12 and MED13 function similarly in the control of fat deposition in Drosophila. The finding that MED12 and MED13 modulate energy homeostasis adds to a growing number of examples in which components of the kinase module of the Mediator complex influence metabolic signaling on an organismal level. For example, the other two components of the kinase module, Cyclin-dependent kinase 8 and Cyclin C, have also been reported as negative regulators of fat accumulation in flies and mice. The finding that the activity of MED13 in cardiac muscle regulates fat accumulation in Drosophila is consistent with earlier observation with mice and suggests that the function of cardiac MED13 in systemic regulation of fat storage represents an ancestral mechanism conserved in metazoans. Although it seems most likely that the effect of MED13 on obesity is mediated by overall changes in metabolism, it is also conceivable that changes in feeding behavior contribute to the obesity phenotypes that were observed. Knockdown of MED12 and MED13 using drivers that are active specifically in the heart using Tin-Gal4 or generally in all muscles using Mef2-Gal4 or Mhc-Gal4 commonly evoked obesity and MED13 can control metabolic signaling from the heart, consistent with prior conclusions regarding the functions of MED13 in the mouse heart. However, these Gal4 drivers do not enable reaching of conclusions regarding the specific role of somatic or visceral muscle in this signaling process because Mhc-Gal4 and Mef2-Gal4 are active in diverse muscle-cell types. Given that MED12 and MED13 are ubiquitously expressed, it is possible that they also act in nonmuscle tissues to regulate metabolic homeostasis (Lee, 2014).

It is hypothesized that muscle-secreted factors mediate the function of MED13 in Drosophila muscle to suppress systemic fat accumulation. To identify such factors, a screen was carried out for muscle-secreted obesity-regulating proteins using two different muscle drivers, Mef2-Gal4 and Mhc-Gal4. Six genes were identified that increased fat accumulation of flies in both screens by >60%, including the genes encoding (1) an antimicrobial peptide, Diptericin B; (2) a Drosophila homolog of Angiotensin converting enzyme; (3) a G protein-coupled receptor ligand SIFamide; (4) one of seven Drosophila Insulin/IGF homologs, Insulin-like peptide 4; (5) a JAK/STAT signaling ligand, Unpaired 3; and (6) Wg. Interestingly, it has been shown recently that MED13 and MED12 are required for the expression of Diptericin B in response to Immune Deficiency (IMD) pathway activation, suggestive of additional regulatory functions of MED13 and the genes identified from the current screens beyond obesity control (Lee, 2014).

This study demonstrated that Wg and its autonomous signaling activity, controlled by Arm, in muscle are necessary and sufficient for systemic regulation of obesity in vivo. Previously, the correlation between obesity and the expression of genes involved in the Wnt signaling pathway in heart has been raised from transcriptome analyses using heart biopsies from obese patients. Similarly, correlations between obesity and differential expression of genes for Wnt signaling, as well as genes for insulin sensitivity and myogenic capacity, were also found in skeletal-muscle samples from obese rats. These findings suggest that Wg signaling activity in muscle serves as an intrinsic rheostat for obesity control (Lee, 2014).

Muscle-specific arm knockdown caused partial-patterning defects in the embryonic musculature, and a skd-null allele dominantly enhanced this phenotype to complete lethality. Given the central role of Arm in Wg target gene expression, the findings are consistent with the established function of wg in the development of mesoderm and the embryonic musculature. The findings reveal a close functional connection between MED13 and Arm, suggestive of the role of MED13 in Wg target gene expression. In fact, in the developing Drosophila eye and wing, MED13 and MED12 are essential for Wg target gene expression, and the MED13/MED12 complex physically interacts with Pygopus, a component of the Wg transcriptional complex. Furthermore, MED12 hypomorphic mutant mice are embryonic lethal with impaired expression of Wnt targets. Therefore, the genetic interaction data along with these previous reports suggest that MED13 is a general component of the canonical Wg/Wnt pathway (Lee, 2014).

epistasis experiments indicate that muscle-secreted Wg functions downstream of MED13 in muscle to suppress obesity. Because both wg and arm in muscle are crucial for obesity regulation, one function of muscle-secreted Wg might be to act on muscle. Accordingly, the nonautonomous function of Wg to suppress obesity may occur through autonomous Wg signal activity in muscle. However, if MED13 functions at the level of transcriptional control of Wg target genes and the sole function of muscle-secreted Wg ligand is to activate the Wg signal 'in' muscle, Wg should be upstream of MED13, which is contrary to the epistasis studies. Based on the data, it stands to reason that muscle-secreted Wg should also act directly on a tissue other than muscle for its nonautonomous effect. If so, which tissue may be the target? Ectopic expression of Wg using a fat body-specific Dcg-Gal4 decreased larval abdominal fat body mass, which demonstrates the role of Wg signaling in the fat body for fat-mass regulation. Similarly, in mammals, autonomous activation of the Wnt pathway in adipose tissue decreases fat mass. Wnt signaling blocks mammalian adipogenesis in vitro, and, in mice, activation of the canonical Wnt pathway in adipocytes by ectopic expression of Wnt10b, a Wnt ligand, inhibits obesity. Furthermore, autonomous activation of the Wnt pathway in adipose progenitors with constitutively active β-catenin expression decreases fat mass. Therefore, the reduced fat mass in Dcg > wg larvae indicates that autonomous Wg signaling activity in the fat body serves as a regulator of fat mass. Considered together with the data showing that muscle-secreted Wg contributes to nonautonomous regulation of adiposity in vivo, it is concluded that muscle serves as a source of Wg to regulate adiposity by modulating Wg signaling activity in fat body. However, the possibility cannot be ruled out that the systemic effect of Wg from muscle is mediated through an alternative tissue, such as nervous system (Lee, 2014).

Wg acts on short- and long-range targets. Wg is highly hydrophobic and has been shown to diffuse through the extracellular space and act on long-range targets by associating with solubilizing molecules such as lipoprotein particles and Secreted Wg-interacting molecule. Furthermore, Wnt-1 has been identified in serum, and decreased Wnt-1 levels in serum correlate with premature myocardial infarction and metabolic syndrome, suggesting that Wg may act on remote organs as an endocrine factor. Therefore, this study proposes a model in which muscle-secreted Wg is a downstream effector of MED13 and acts both to activate the signal in muscle and to act on the fat body ultimately to achieve systemic inhibition of obesity (Lee, 2014).

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