Interactive Fly, Drosophila



Effects of Mutation or Deletion (part 1/2)

Asymmetric divisions allow a precursor to produce the four distinct cells of Drosophila sensory organ lineages (SOLs). The sensory organ precursor (SOP) first divides into two different secondary precursor cells, IIA and IIB, which gives rise to one shaft-producing cell (trichogen) and one socket-producing cell (tormogen), and one neuron and one sheath cell (thecogen), respectively. Although this process requires cell-cell communication via the Notch (N) receptor, mitotic recombination that removes the N ligand Delta (Dl) or Serrate (Ser) in the SOL has mild or no effect. N mutant clones generated on the central region of the adult scutum are devoid of any external bristle structures, such as shafts and sockets, similar to the Nts mutant phenotype at a restrictive temperature. Whereas loss of N function during the process of lateral inhibition produces supernumerary SOPs, this balding phenotype is probably due to the requirement of N in asymmetric divisions. Without N activity the supernumerary SOPs divide symmetrically, giving rise to two IIB cells and, consequently, no external sensory structures. Dl clones typically produce a tuft of densely packed bristles in the interior of the clone. These tufts of bristles are likely due to a failure of lateral inhibition, resulting in overproduction of SOPs. The presence of the external bristle structures in these Dl mutant clones indicates that, unlike N clones, most of the supernumerary SOPs in the Dl mutant clones produce IIA cells that divide to form shaft and socket cells. Clones homozygous for three Ser null alleles produce normal external bristle structures. In contrast, clones with loss of both Dl and Ser function produce epidermal cells but not external bristle structures. This balding phenotype is clearly different from the phenotypes of the Dl or Ser mutant clones but is indistinguishable from that of N mutant clones, suggesting that Ser and Dl have overlapping functions in the N signaling pathway. Dl and Ser also have redundant functions in patterning wing veins. In contrast, Dl and Ser are known to serve distinct functions in specifying dorsal-ventral compartment boundary of the wing (wing margin). Ser in dorsal cells signals to N in ventral cells, and Dl in ventral cells signals to N in dorsal cells. For Dl and Ser to provide distinct signals from one compartment to the other without generating signals among cells within the same compartment, it may be necessary to involve other factors such as those encoded by the dorsally expressed gene fringe (fng), which inhibits a cell's ability to respond to Ser and potentiates a cell's response to Dl. It is concluded that Dl and Ser are redundant in mediating signaling between daughter cells to specify their distinct sensory organ cell fates (Zeng, 1998). Ser mutant larva often die, exhibiting a failure to differentiate the anterior spiracles, poorly developed mouth-hooks and a severe reduction in the size of wing and haltere primordia. The few mutants that successfully pupate develop into pharate adults, almost completely lacking in wings and halteres (Speicher, 1994). The dominant Ser mutation causes a gap in the posterior wing tip and margin, and a portion of the blade (Jack, 1992).

Flies hetero- or homozygous for the dominant mutation SerD exhibit scalloping of the wing margin due to cell death during pupal stages. SerD is associated with an insertion of the transposable element Tirant in the 3' untranslated region of the gene, resulting in the truncation of the Ser RNA, thereby eliminating putative RNA degradation signals located further downstream. This leads to increased stability of Ser RNA and higher levels of Serrate protein. Wing discs of SerD third instar larvae exhibit additional Serrate protein expression in the edge zone of the future wing margin, where it is normally not detectable. Expression of wing margin specific genes, such as cut and wingless, is repressed in these cells (Thomas, 1995).

Ectopic expression of Serrate during wing development induces ectopic outgrowth of ventral wing tissue and formation of an additional wing margin. In order for Serrate to elicit these responses the concomitant expression of wingless seems to be required. Ectopic expression of Delta provokes wing outgrowth and induction of a new margin, both on the dorsal and ventral side. Serrate acts downstream of apterous and induces expression of wing margin patterning genes. Serrate also has the potential to repress margin-specific genes such as wg and cut. This repression results in the failure to differentiate a proper wing margin, visible as a notch in the distal-most part of the wing margin. Thus ectopic Serrate provokes one of two responses, depending on the time and place of expression: outgrowth of wing tissue and induction of a new margin, or repression of margin specific genes that results in a nick in the wing margin. Actions of both Serrate and Delta are mediated by Notch, suggesting that the effects of the two Notch ligands depend on the cellular context, since the capability to activate Notch is spatially and temporally restricted, and expression of ligands at other times and in other places results in repression of Notch activity (Jönsson, 1996).

To ask whether the wing abnormalities caused by reducing Beadex levels might be due to an effect on Apterous activity, the effects of the Bxhdp excision mutants were examined on Ap target gene expression. In early third-instar fng-lacZ and Serrate (Ser) are expressed evenly throughout the dorsal compartment of the wing disc and are thought to be regulated by Ap. In Bxhdp mutant discs, the size of the dorsal compartment is considerably reduced, consistent with the small wing phenotype. fng-lacZ expression is not affected in Bxhdp discs. Ser expression is elevated in the dorsal compartment and does not resolve normally into stripes along the DV boundary and wing veins. Ser expression in the ventral compartment appears normal. The stripes of Ser expression along the DV boundary and wing veins are both dorsal and ventral and are under different regulation than the early dorsal-specific domain. The abnormal pattern of Ser in the dorsal compartment of the Bxhdp may be due to superimposition of the early and late expression patterns. It is suggested that this reflects a failure to down-regulate Ap activity as the disc matures. To ask whether elevated Ser levels might contribute to the defects observed in Bxhdp mutant wings, Ser was overexpressed in the dorsal compartment of an otherwise wild-type disc using ap-gal4 to direct UAS-Ser expression. The resulting wings are small and show thickened veins but do not show the abnormalities in vein pattern observed in the Bxhdp mutant wings. Overexpression of fng using ap-gal4 in a wild-type background produces no phenotype. These observations suggest that Ser overexpression contributes to the abnormalities observed in Bxhdp mutant wings but that there are likely to be additional factors. Thus, both gain-of-function and loss-of-function Bx mutant phenotypes can be attributed to abnormal regulation of Ap activity. It is concluded that Ap induces dLMO expression in the wing disc and that dLMO then functions as part of a feedback system to regulate the level of Ap activity (Milan, 1998).

Serrate is an essential gene in Drosophila melanogaster, best known for the Ser dominant (SerD) allele and its effects on wing development. Animals heterozygous or homozygous for SerD are viable and exhibit loss of wing margin tissue and associated bristles and hairs. The Beaded of Goldschmidt (BdG) allele of Ser, when heterozygous to wild type, will also produce animals exhibiting loss of wing margin material. However, animals homozygous for BdG exhibit a larval lethal phenotype comparable to animals homozygous for loss-of-function Ser alleles. BdG is a partial duplication of the Ser locus with a single 5' Ser-homologous region and two distinct 3' regions. Meiotic recombination between BdG and a wild-type Ser chromosome demonstrates that only one DNA lesion, caused by the insertion of a transposable element into the coding regions of the Ser transcript, appears capable of generating BdG phenotypes. Due to the insertion, the protein product is predicted to be prematurely truncated and lack an extracellular cysteine-rich region along with the transmembrane and intracellular domains found within the normal SERRATE (SER) protein. The loss of these protein domains apparently contributes to the antimorphic nature of this mutation (Hukriede, 1997a).

Specification of the dorsal-ventral compartment boundary in the developing Drosophila wing disc requires activation of Notch from its dorsal ligand Serrate and its ventral ligand Delta. Both Notch ligands are required in this process: one cannot be substituted for the other. In the wing disc, expression of BD(G), a dominant-negative, truncated form of Serrate, is capable of inhibiting Notch activation in the ventral but not the dorsal compartments. BD(G) can act as a general antagonist of both Serrate and Delta mediated Notch interactions. However, BD(G) retains the Serrate protein domain targeted by Fringe, hence BD(G)'s antagonistic effects are restricted in the dorsal wing disc. Implicit in these results is the suggestion that binding of a ligand to Notch is not sufficient for Notch activation. The specificity of the Notch signal generated by interactions with Serrate and Delta originates from regions residing outside of the Notch binding domains of these molecules; other properties attributable to Notch ligands are required for Notch activation. Thus, ligand binding to Notch is a necessary but insufficient step toward Notch activation (Hukriede, 1997b).

The functions of artificially constructed secreted forms of the two known Drosophila Notch ligands, Delta and Serrate, were examined by expressing them under various promoters in the Drosophila developing eye and wing. The phenotypes associated with the expression of secreted Delta (DlS) or secreted Serrate (SerS) forms mimic loss-of-function mutations in the Notch pathway. Both genetic interactions between DlS or SerS transgenics and duplications or loss-of-function mutations of Delta or Serrate indicate that DlS and SerS behave as dominant negative mutations. Expression of DlS and SerS in the eye results in a rough eye phenotype. This phenotype is enhanced by loss-of-function Delta and gain-of function Suppressor of hairless. These observations were extended to the molecular level by demonstrating that the expression of Enhancer of split mdelta, a target of Notch signaling, is down-regulated by SerS. The antagonistic nature of the two mutant secreted ligand forms in the eye is consistent with their behavior in the wing, where they are capable of down-regulating wing margin specific genes in an opposite manner to the effects of the endogenous ligands. For example, wingless expression is down-regulated where a SerS expressing stripe crosses the dorsal/ventral boundary. The secreted ligands also interfere with wing vein specification. This analysis uncovers secreted molecular antagonists of Notch signaling and provides evidence of qualitative differences in the actions of the two ligands DlS and SerS (Sun, 1997).

Dfrizzled3 is a novel member of the Frizzled family of seven-pass transmembrane receptors. Like Dfz2, Dfz3 is a target gene of Wingless (Wg) signaling, but in contrast to Dfz2, it is activated rather than repressed by Wg signaling in imaginal discs. Dfz3 is not required for viability but is necessary for optimal Wg signaling at the wing margin. Dfz3 was identified by characterizing a P-element line from a large scale Gal4 enhancer trap screen that allows direct visualization of gene expression patterns in living flies. A Gal4 insert found in the cytological position 1C exhibits an adult expression pattern resembling that of wg. The Gal4 expression pattern of this line has been visualized by a UAS-lacZ reporter gene. Depending on the tissue analysed, Dfz3-Gal4 is expressed in a broad domain centered over, or in a domain coinciding with, the wg expression domain. Dfz3 is expressed throughout the wing pouch but appears to be upregulated by Wg signaling at the presumptive wing margin and in a ring around the pouch. In the notum the expression is similar to the thoracic expression of wg. In the leg disc Dfz3 is expressed in a broad ventral wedge centered on the wg domain. Dfz3 expression in the eye disc is also coincident with wg expression and can be detected at the dorsal and ventral margins, which give rise to the head capsule Evidence supporting a functional role for Dfz3 in Wg signaling comes from a genetic interaction with Serrate (Ser), a gene that encodes a Notch ligand involved in establishing wg expression in the wing margin. SerD is a dominant mutation that results in reduced wg expression in wing margin cells. Dfz3 mutations enhance the SerD phenotype in a dose-dependent manner. With one copy of Dfz3 removed the SerD wings show stronger notching and a loss of wing margin bristles. Removing the second copy of Dfz3 enhances this phenotype further and results in additional posterior margin notching. It is suggested that Dfz3 may function in concert with Dfz2 and Fz to transduce or transport the Wg signal in imaginal discs (Sivasankaran, 2000).

Drosophila larval hemocytes originate from hematopoietic organs called lymph glands. These are composed of paired lobes located along the dorsal vessel. Two mature blood cell populations are found in the circulating hemolymph: the macrophage-like plasmatocytes, and the crystal cells that contain enzymes of the immune-related melanization process. A third class of cells, called lamellocytes, are normally absent in larvae but differentiate after infection by parasites too large to be phagocytosed. Evidence is presented that the Notch signaling pathway plays an instructive role in the differentiation of crystal cells. Loss-of-function mutations in Notch result in severely decreased crystal cell numbers, whereas overexpression of Notch provokes the differentiation of high numbers of these cells. In this process, Serrate, not Delta, is the Notch ligand. In addition, Notch function is necessary for lamellocyte proliferation upon parasitization, although Notch overexpression does not result in lamellocyte production. Finally, Notch does not appear to play a role in the differentiation of the plasmatocyte lineage. This study underlines the existence of parallels in the genetic control of hematopoiesis in Drosophila and in mammals (Duvic, 2002).

Crystal cells differentiate within the larval hematopoietic organ, the lymph glands, and the effect of the various mutations on differentiation in situ were examined. For this, an antibody raised against dipteran prophenoloxidase (proPO), a zymogen required for melanization reactions, was examined. In Drosophila, proPO is produced and stored in crystal cells and is released during host defense reactions. It was first noted that, in wild-type larvae, proPO is synthesized during the differentiation of crystal cells within the hematopoietic organ. Interestingly, a gradient of differentiation is apparent within the successive lymph gland lobes along the anteroposterior axis: the anteriormost lobes contained numerous proPO-positive cells, whereas the adjacent, more posterior lobes contain no or few positive cells and the most posterior lobes are totally devoid of differentiating crystal cells. In Nts1 mutants placed at a restrictive temperature, the number of proPO-positive cells is significantly reduced in the lymph glands, and they were occasionally totally absent. A similar phenotype was observed in a Su(H) and in a Ser mutant context. Conversely, activation of the Notch pathway in NMcd8 larvae and in larvae carrying UAS-Nic, UAS-Ser, or UAS-Dl transgenes driven by hsp-Gal4 dramatically increased the number of proPO-positive cells in lymph glands. Not only are the anteriormost lobes packed with such cells, but more posterior lobes are often found to contain large numbers of differentiating crystal cells. As expected, similar phenotypes are obtained when the e33C-Gal4 driver is used; this driver is strongly expressed in the lymph glands (Duvic, 2002).

An unexpected link between Notch signaling and ROS in restricting the differentiation of hematopoietic progenitors in Drosophila

A fundamental question in hematopoietic development is how multipotent progenitors achieve precise identities, while the progenitors themselves maintain quiescence. In Drosophila larvae, multipotent hematopoietic progenitors support the production of three lineages, exhibit quiescence in response to cues from a niche, and from their differentiated progeny. Infection by parasitic wasps alters the course of hematopoiesis. This study addresses the role of Notch (N) signaling in lamellocyte differentiation in response to wasp infection. Notch activity is moderately high and ubiquitous in all cells of the lymph gland lobes, with crystal cells exhibiting the highest levels. Wasp infection reduces Notch activity, which results in fewer crystal cells and more lamellocytes. Robust lamellocyte differentiation is induced even in N mutants. Using RNA interference-knockdown of N, Serrate, and Neuralized, and twin clone analysis of a N null allele, this study shows that all three genes inhibit lamellocyte differentiation. However, unlike its cell-autonomous function in crystal cell development, Notch's inhibitory influence on lamellocyte differentiation is not cell-autonomous. High levels of reactive oxygen species in the lymph gland lobes, but not in the niche, accompany NRNAi-induced lamellocyte differentiation and lobe dispersal. These results define a novel dual role for Notch signaling in maintaining competence for basal hematopoiesis: while crystal cell development is encouraged, lamellocytic fate remains repressed. Repression of Notch signaling in fly hematopoiesis is important for host defense against natural parasitic wasp infections. These findings can serve as a model to understand how reactive oxygen species and Notch signals are integrated and interpreted in vivo (Small, 2013).

Serrate function in the eye disc

Dorsoventral (DV) patterning is crucial for eye development in invertebrates and higher animals. DV lineage restriction is the primary event in undifferentiated early eye primordia of Drosophila. In Drosophila eye disc, a dorsal-specific GATA family transcription factor pannier (pnr) controls Iroquois-Complex (Iro-C) genes to establish the dorsal eye fate whereas Lobe (L), which is involved in controlling a Notch ligand Serrate (Ser), is specifically required for ventral growth. However, fate of eye disc cells before the onset of dorsal expression of pnr and Iro-C is not known. L/Ser have been shown to be expressed in entire early eye disc before the expression of pnr and Iro-C is initiated in late first instar dorsal eye margin cells. The evidence suggests that during embryogenesis pnr activity is not essential for eye development. Evidence that loss of L or Ser function prior to initiation of pnr expression results in elimination of the entire eye, whereas after the onset of pnr expression it results only in preferential loss of the ventral half of the eye. Dorsal eye disc cells also become L or Ser dependent when they are ventralized by removal of pnr or Iro-C gene function. Therefore, it is proposed that early state of the eye prior to DV lineage restriction is equivalent to the ventral half and requires L and Ser gene function (Singh, 2003).

Previously, L/Ser were thought to be required for ventral eye growth after the DV lineage restriction boundary was established, which corresponds to the onset of expression of dorsal eye selectors. The results, however, clearly suggest that L/Ser are required much earlier for the growth of the entire early eye disc, even before the DV patterning is established. In contrast to the function of dorsal selector genes in eye patterning, L and Ser have been shown to play a distinct role in controlling ventral-specific growth of eye disc (Singh, 2003).

Loss-of-function phenotypes of L or Ser are restricted to the ventral eye. The spatial as well as temporal requirement of these genes in the ventral eye pattern formation were examined. Extent of loss of ventral eye pattern in loss-of-function clones of L/Ser varies along the temporal scale. During early eye disc development, prior to onset of pnr expression in dorsal eye, removal of L or Ser function results in complete elimination of the eye field, whereas later when dorsal eye selector genes starts expressing the eye suppression phenotype becomes restricted only to the ventral eye. Therefore, DV lineage border in the eye can also be interpreted as the border between the cells sensitive and insensitive to the L/Ser gene function (Singh, 2003).

The eye antennal disc has the most complex origin in the embryo. The eye disc is initiated from a small group of ~70 precursor cells on each side contributed by six different head segments of the embryo. These embryonic precursors do not physically separate from the surrounding larval primordia and are therefore difficult to discern morphologically (Singh, 2003 and references therein).

Once the cells for the eye-antennal disc are committed, these discs proliferate and undergo differentiation into an adult eye, which requires generation of DV lineage restriction in eye. There are possibly three different ways by which genesis of DV lineage in the eye can be explained. Early first instar larval eye disc may initiate either from only dorsal, only ventral or from both DV lineages. Based on results from studies of expression patterns and analysis of mutant phenotypes, it is proposed that larval eye primordium initially comprises only the ventral-equivalent state rather than well-defined DV or dorsal states alone. The initial state of eye is referred to as ventral equivalent state because, at this stage, dorsal and ventral identity is not yet generated. DV lineage restriction is established later after the onset of pnr expression. The cells of the initial ventral-equivalent state are similar to the ventral eye cells that are generated after DV specification. The similarity is in terms of their requirement of L/Ser for growth and maintenance, and the absence of the dorsal selector expression. How dorsal lineage is initiated in the early eye disc is not yet clear. Once the DV lineage restriction is established, N signaling is initiated at the equator, a border between dorsal and ventral compartments. Activation of N signaling promotes proliferation, which is followed by differentiation of eye disc into adult compound eye (Singh, 2003).

The ventral-equivalent state model is supported by two observations: 1) presence of Ser and L expression in the dorsal and ventral eye disc of the early first instar larva and 2) change of dorsal eye fate to ventral upon removal of dorsal selectors. The mutants, which affect ventral eye development, show two major phenotypes in eye: either there is no or very small eye, or there is a preferential loss of ventral eye based on the time they affect their function but none of the mutants for dorsal eye selectors show phenotypes of loss of only dorsal eye. Conversely, loss-of-function clones of pnr or Iro-C causes dorsal eye enlargement or ectopic eye formation rather than loss of only dorsal eye clonal tissue. This phenotype is probably due to generation of ectopic boundary of pnr-expressing and non-expressing cells (rather than absence of pnr), which could be important for promoting eye growth. Overexpression of Ush or Fog proteins in eye discs results in loss of pnr activity, causing complete elimination of eye development. By removing pnr activity at different time points it was found that pnr activity in embryo and early first instar is not essential for eye disc development. Later, pnr becomes essential for DV patterning consistent with its strong expression in dorsal margin of eye disc after the early first instar stage (Singh, 2003).

In contrast to enlargements or ectopic eyes induced by loss-of-function clones of dorsal selectors, the loss-of-function clones of L or Ser always resulted in the elimination of the eye fate. L/Ser are primarily required for the maintenance and development of ventral or ventral-equivalent state of the eye, whereas dorsal genes establish the DV border. This suggests that dorsal genes and L/Ser, although involved in a common goal of generation of DV lineage in eye, probably affect eye development at two different tiers (Singh, 2003).

Fng, another essential component of DV patterning in eye, is expressed preferentially in the ventral domain of early eye disc and is required for restriction of N signaling to the DV border. Although fng is known to act upstream of Ser in the wing and eye discs, there is also an apparent difference between the two genes. Unlike L/Ser, the main function of fng seems to affect DV ommatidial polarity but not the growth. This suggests that fng may be selectively required for DV patterning after dorsal selectors initiate domain specification. This may be the reason why phenotypes of loss-of-function clones of fng are different from those of L and Ser in the eye. It has been observed that the pattern of fng expression is not altered in L mutants, and vice versa, supporting the independent functions of these two genes in controlling DV border formation and growth of ventral domain (Singh, 2003).

The function of Pnr in organizing the DV pattern from an initial ventral-equivalent state raises an interesting question of whether similar patterning processes occur in other developing tissues and organs. Interestingly, Pnr is expressed in a broad dorsal domain in early embryos, but later refined in a longitudinal dorsal domain extending along the thoracic and abdominal segments. During this stage, Pnr has an instructive and selector-like function, determining the identity of the medial dorsal structures. It has been shown that loss of pnr eliminates the dorsomedial pattern in the larval cuticle whereas the dorsolateral pattern extends dorsally without cell loss. This suggests that DV pattern in the larval cuticle is established with the onset of Pnr expression in the dorsomedial domain, and ventral may be the initial fate of epidermal cells (Singh, 2003).

Lobe and Serrate are required for cell survival during early eye development in Drosophila

Organogenesis involves an initial surge of cell proliferation, leading to differentiation. This is followed by cell death in order to remove extra cells. During early development, there is little or no cell death. However, there is a lack of information concerning the genes required for survival during the early cell-proliferation phase. This study shows that Lobe (L) and the Notch (N) ligand Serrate (Ser), which are both involved in ventral eye growth, are required for cell survival in the early eye disc. The loss-of-ventral-eye phenotype in L or Ser mutants is due to the induction of cell death and the upregulation of secreted Wingless (Wg). This loss-of-ventral-eye phenotype can be rescued by (1) increasing the levels of cell death inhibitors, (2) reducing the levels of Hid-Reaper-Grim complex, or (3) reducing canonical Wg signaling components. Blocking Jun-N-terminal kinase (JNK) signaling, which can induce caspase-independent cell death, significantly rescued ventral eye loss in L or Ser mutants. However, blocking both caspase-dependent cell death and JNK signaling together showed stronger rescues of the L- or Ser-mutant eye at a 1.5-fold higher frequency. This suggests that L or Ser loss-of-function triggers both caspase-dependent and -independent cell death. These studies thus identify a mechanism responsible for cell survival in the early eye (Singh, 2006).

During development, cell survival, growth, proliferation and differentiation define the final shape and size of the organ. This study used the Drosophila eye to identify the genes required for cell survival and to sustain early growth. Previously, it was shown that ventral is the ground state of the entire early larval eye primordium (Singh, 2003), and that L/Ser are required for development of the ventral eye. During early eye development, little or no cell death is observed. At the mid-pupal stage, programmed cell death plays an important role in the selective removal of a large number of excess undifferentiated cells in the interommatidial space that fail to be recruited to the ommatidia. During this time window of pupal development, EGFR and Ras act as survival cues. Surprisingly, there is little information concerning genes responsible for cell survival during early larval eye imaginal disc development. These studies show that, during early eye development, L and Ser are required for the survival of ventral eye cells. It was found that one of the major reasons for the elimination of the ventral eye cells in L/Ser mutants is due to the induction of caspase-dependent cell death. L- and Ser-mutant phenotypes can be rescued by blocking inappropriate induction of (1) Wg, (2) JNK-signaling-mediated cell death and (3) caspase-dependent cell death (Singh, 2006).

In animal tissues, Wg is required to drive developmental patterning. Wg is produced in a restricted area and is distributed either by diffusion or by transport to generate a concentration gradient throughout the tissue to induce proper differentiation. In the developing wing imaginal disc, Wg has also been shown to promote growth. By contrast, it has been shown that abnormal expression of Wg or Dpp triggers aberrant differentiation signals that result in the induction of apoptotic cell death in the wing disc. However, it is difficult to directly extrapolate results from the wing disc to the eye disc because of organ-specific functions of Wg (Singh, 2006).

In the eye, Wg has complex functions at different stages of development: (1) prior to eye differentiation, Wg is involved in growth and in the establishment of the dorsal eye fate; (2) during eye differentiation, initiation of the morphogenetic furrow by hedgehog (hh) is restricted to the posterior margin by the presence of Wg, which represses hh and dpp at the lateral eye margins; and (3) in the pupal stage, Wg is responsible for inducing apoptosis by activating the expression of hid, rpr and grim in ommatidia at the periphery of the eye (Singh, 2006).

This study found that, during early eye development, L and Ser are required to repress Wg signaling in the ventral eye disc. Genetic-interaction studies demonstrate that Wg expression is ectopically induced in the L-mutant background. This paper proposes a model in which L and Ser downregulate the level of Wg activity and expression in the eye. Loss-of-function of L/Ser induce higher levels of Wg, is coincident with the elimination of the ventral eye pattern by ectopic induction of caspase-dependent cell death. Because blocking caspase-dependent cell death in L/Ser-mutant backgrounds results in striking but incomplete rescues of the loss-of-ventral-eye phenotypes, it suggests that L/Ser-mutant eye phenotypes are not solely due to the induction of caspase-dependent cell death. It is possible that ectopic upregulation of Wg signaling in the LOF of L/Ser causes abnormal induction of JNK signaling or that L/Ser LOF can induce JNK signaling independently. Upregulation of JNK signaling can also induce caspase-independent cell death. It is possible that the loss of L/Ser can result in cell death caused by both caspase-dependent and caspase-independent mechanism. This may be one of the underlying developmental mechanisms for the early cell-survival function of L and Ser. However, there can be several other interesting possibilities. Other studies have shown that JNK can be activated downstream of rpr, and that it affects the extent of rpr-induced cell death. Also, wg can be induced downstream of hid and diap1. Therefore, one can suggest an alternative model where a low level of apoptosis induced by hid, rpr and grim is augmented by a secondary activation of JNK and Wg, which ultimately results in eye ablation (Singh, 2006).

Many animal tissues counter cell death, induced in response to injury, by triggering compensatory cell proliferation in the neighboring cells. It has been reported in flies that apoptotic or dying cells actively signal to induce compensatory proliferation in neighboring cells to maintain tissue homeostasis. In cellular injury, Diap1 is inhibited, whereas the JNK pathway and Wg/Dpp are induced in the apoptotic cells. Secretion of these factors stimulates the growth or proliferation in competent neighboring cells and leads to compensatory proliferation. In other scenarios in which morphogenetic cell death occurs, altered levels of morphogenetic signals give rise to abnormal cell types, which are frequently removed by activating apoptotic signals. Morphogenetic cell death is one of the strategies employed by the developing field to correct its morphogen gradient by eliminating cells with abnormal levels of morphogen by inducing the JNK-signaling pathway (Singh, 2006).

Cell death caused by loss of L/Ser function results in the induction of both JNK- and Wg-signaling pathways. However, the outcome is different from that seen in compensatory proliferation or morphogen-gradient correction. Instead of compensatory growth in neighboring cells, LOF of L/Ser triggers ectopic signaling, which can be neither corrected nor compensated for. As a consequence, the affected tissue, in this case the ventral half of the eye discs, cannot be rescued. It results in the loss of the ventral or entire eye field, depending upon the domain of function of these survival factors. The results demonstrate that one of the essential roles of L and Ser is their requirement for the survival of early proliferating cells in the eye (Singh, 2006).

During development, N signaling is involved in many processes, including cell-fate commitment, cell-fate specification and cell adhesion. In the Drosophila eye, N signaling plays important roles in compound eye morphogenesis, such as DV patterning, cell proliferation and differentiation in the eye. However, N signaling has not been shown to promote cell survival during early eye development. These studies raise the possibility of the role of the Ser-N pathway in cell survival during early eye development. Earlier, the extremely reduced or complete loss of eye field in N mutant eye disc was interpreted as being caused by a loss of proliferation. The current data raises another possibility: that N signaling may be playing an important role in cell survival (Singh, 2006).

The compound eye of Drosophila shares similarities with the vertebrate eye. There is conservation at the level of genetic machinery, as well as in the processes of differentiation. Thus, the information generated in Drosophila can be extrapolated to higher organisms. Since Wnt signaling induces programmed cell death in patterning the vasculature of the vertebrate eye, it will be important to study what molecules prevent Wg signaling from inducing cell death during early eye development. Mutations in Jagged1, the human homolog of Ser, is known to cause autosomal-dominant developmental disorder, called Alagille's syndrome, which also affects eye development. Hence, it would be interesting to study what roles N pathway genes play in cell survival during early eye development and in the early onset of retinal diseases (Singh, 2006).

Serrate is dispensable in the lgd mutant wing disc

During the development of the Drosophila wing, the activity of the Notch signalling pathway is required to establish and maintain the organizing activity at the dorsoventral boundary (D/V boundary). At early stages, the activity of the pathway is restricted to a small stripe straddling the D/V boundary, and the establishment of this activity domain requires the secreted molecule Fringe (Fng). The activity domain will be established symmetrically at each side of the boundary between Fng-expressing and non-expressing cells. Evidence is presented that the Drosophila tumor-suppressor gene lethal giant discs (lgd), a gene whose coding region has yet to be identified, is required to restrict the activity of Notch to the D/V boundary. In the absence of lgd function, the activity of Notch expands from its initial domain at the D/V boundary. This expansion requires the presence of at least one of the Notch ligands, which can activate Notch more efficiently in the mutants. The results further suggest that Lgd appears to act as a general repressor of Notch activity, because it also affects vein, eye, and bristle development (Klein, 2003).

It has also been observed that wingless (wg) is expressed ectopically in the pouch of lgd mutants during wing development. Similar phenotypes are observed, if the Notch pathway is ectopically activated during wing development, raising the possibility that the lgd mutant phenotype could stem from the ectopic activation of the Notch pathway. The Notch pathway is indeed ectopically active in lgd mutants, and hyperactivation as well as ectopic activation of the pathway accounts for the lgd phenotype during wing development. In lgd mutants, the expression of Notch target genes along the D/V boundary is expanded, indicating that Lgd is required for the restriction of Notch activity to the D/V boundary. Furthermore, the mutant phenotype of lgd is suppressed by concomitant loss of Presenilin or Suppressor of Hairless function, indicating that the mutant phenotype is caused by the activation of the Notch pathway. Evidence is provided that the activity of fng and Serrate seem to be dispensable in lgd mutant wing disc and that Delta can activate Notch efficiently enough to maintain its activity during wing development. The presented results indicate that the negative regulation of Notch by Lgd is not restricted to wing development and occurs during several other developmental processes, such as vein, eye, and bristle development, suggesting that Lgd suppresses the activity of the Notch pathway in a variety of developmental processes (Klein, 2003).

Loss of lgd function leads to an overgrowth of the imaginal discs, clearly noticeable in the wing region of the wing disc, which becomes enlarged and flat (Bryant, 1971). wg expression is normally restricted to the D/V boundary of the wing pouch. In lgd mutants, wg is activated ectopically in a much broader domain that extends into the wing pouch. In addition, lgd mutant wing discs often develop a second wing pouch in the region of the anlage of the scutellum. Similar phenotypes are caused by gain-of-function alleles of N (for example, Abruptex) and are also observed upon expression of the activated intracellular form of Notch, Nintra, or expression of Notch ligands, such as Dl. The ectopic activation of wg can already be observed in early third instar wing discs and precedes the visible morphological changes that occur at later stages. The deficiency Df(2L) FCK-20 deletes the lgd locus, allowing the classification of the relative strength of the available alleles. The phenotype is always variable, but the overall phenotype of lgdd7 and lgdd10 in homozygotes and in trans over Df(2L)FCK-20 is very similar, indicating that these two alleles are strong, probably amorphic alleles. lgdd4 and lgdd1 are weaker alleles. All alleles display a qualitatively similar phenotype over the deficiency as in homozygotes, indicating that the observed phenotype is probably caused by the loss-of-function of the lgd gene (Klein, 2003).

The similarity between the loss of lgd function and ectopic N activation suggests that the phenotype of lgd could be caused by ectopic activation of the Notch pathway. To examine this possibility, the expression of E(spl)m8, cut, Dl, and Ser was monitored as well as the activity of the vg-boundary enhancer (vgBE) in mutant wing discs. The expression of all these markers is initiated in cells at the D/V boundary in a Notch-dependent manner. The vgBE is initially expressed along the D/V boundary of the wing, but late in the third instar, it is activated in an additional stripe along the anteroposterior compartment boundary (A/P boundary), which is also dependent on Notch activity. Both domains depend on the presence of a single Su(H) binding site in the enhancer. Similarly, the expression of cut and E(spl)m8 is initiated in cells at the boundary by the Notch-pathway, and E(spl)m8 is also dependent on the presence of Su(H) binding sites in its promoter. As described above, the expression of Dl and Ser is more complex but always dependent on the activity of Notch in cells at the D/V boundary. In lgd mutant wing discs, the vgBE as well as cut, Dl, Ser, and E(spl)m8 are activated ectopically within the wing pouch. The activation of the vgBE is dependent on the presence of the Su(H) binding site in the enhancer, since a version lacking it shows no ectopic activity in the mutants. As in the case of wg, the expression of the vgBE is already expanded in early third larval wing discs. Altogether, these results show that the loss of lgd function leads to the ectopic expression of Notch target genes. This suggests that the Notch pathway is ectopically activated in lgd mutants (Klein, 2003).

All tested Notch-target genes are ectopically activated in lgd mutant wing discs or lgd mutant cell clones. The ectopic activation of Notch target genes as well as the observed overproliferation of lgd mutants is abolished in lgd;Psn double mutants. In addition, Notch target gene expression is also abolished in Psn or Su(H) mutant clones generated in lgd mutant wing imaginal discs. These data suggest that the Notch pathway becomes ectopically active in the absence of lgd function. Furthermore, the fact that Delta alone seems to provide sufficient Notch activity to sustain wing development in lgd mutants indicates that the pathway can be activated more efficiently in the mutant background. The activation of Notch is a consequence of loss of lgd function also in other developmental processes, such as bristle, leg, and wing vein development. Thus, the presented data make lgd a good candidate gene that regulates activity of the Notch pathway during adult development of Drosophila (Klein, 2003).

The phenotype of Ser;lgd double mutant wing discs was further analyzed to examine the effect of loss of one Notch ligand in lgd mutants. Loss of Ser function leads to the loss of most of the wing blade and the margin. The presence of a remnant of the wing pouch is due to the fact that the Notch pathway is active during early stages of wing development. This activation is achieved through a residual expression of Dl. Animals of the Ser;lgd double mutant phenotype develop very slowly, and only few larva survive until the third instar. The wing imaginal discs of the larva have expanded wing pouches and, in contrast to Ser-mutant discs, they express vg and Dl and wg in the wing blade. This shows, that in the absence of lgd function, the activity of Ser is not required to maintain Notch-dependent gene activity. In summary, the observed genetic interactions reveal a functional relationship between the Notch and lgd locus and support the conclusion that lgd is a negative regulator of the Notch pathway (Klein, 2003).

The observation that loss of lgd function can compensate for the loss of Ser function raises the possibility that Notch could be activated in a ligand-independent manner in the absence of lgd function. To test this possibility, Ser/Dl double mutant clones were generated in lgd-mutant wing discs. The clones were induced through combining the Flp/FRT and the targeted Gal4-System. In the experiments described here, the expression of UASFlp was activated with sdGal4. sdGal4 is active throughout wing development and therefore activates UAS Flp expression at all stages of development (Klein, 2003).

In the clones, the expression of the Notch-regulated genes wg and cut was interrupted in the center of the clone area, suggesting that the expression of these genes in lgd mutants depends on Notch ligands. However, several interesting additional effects were observed. (1) Surprisingly, wg and cut expression was induced on both sides of the clone boundary, which can be clearly seen in clones located outside the expanded expression domain normally observed in lgd mutants . The effect is observed in the dorsal as well as the ventral half of the pouch. This suggests that the removal of the ligands leads to the activation of Notch at the boundary of Dl/Ser-expressing and nonexpressing cells. (2) In several cases, the expression of cut and wg expands outside the clone, even far away from the clone boundary. This effect is biased, and the expansion toward the D/V boundary is stronger (Klein, 2003).

(3) The expression of the Notch targets is activated up to three-cell diameter into the clone in a graded manner. Since the ligands are membrane anchored and thought to signal to adjacent cells, an activation of Notch target gene expression beyond one-cell diameter into the clone is not expected. One possibility is that the induction of Cut by Notch is indirect and mediated by a diffusible factor that is induced at the clone boundary (Klein, 2003).

However, it was found that clones of Su(H) mutant cells in lgd mutant discs lose expression of Notch target genes, such as Cut, indicating that the cells require a functional Notch pathway to activate expression of its target genes. Similar results were obtained with Psn mutant clones, using Wg expression as a read out of Notch activity. These results rule out the possibility that the target genes of Notch are induced indirectly through a diffusible factor induced by the Notch pathway (Klein, 2003).

In summary, these results suggest that, in lgd mutant wing blades, all cells that express Notch-regulated genes require the activity of the signal cascade and receive a signal through Dl and/or Ser. In addition, they indicate that, in the Ser;lgd double mutant wing discs described above, Dl alone is sufficient not only to initiate, but also to maintain N-activity during wing development. Hence, it seems that Notch can be activated more efficiently by Dl in the absence of lgd (Klein, 2003).

Genetic interaction between deltex and Serrate

Notch is a single-pass transmembrane receptor. The N signaling pathway is an evolutionarily conserved mechanism that controls various cell-specification processes. Drosophila Deltex (Dx), a RING-domain E3 ubiquitin ligase, binds to the N intracellular domain, promotes N’s endocytic trafficking to late endosomes, and has been proposed to activate Suppressor of Hairless [Su(H)]-independent N signaling. However, it has been difficult to evaluate the importance of dx, because no null mutant of a dx family gene has been available in any organism. This study reports the first null mutant allele of Drosophila dx. dx is involved only in the subsets of N signaling, but is not essential for it in any developmental context. A strong genetic interaction exists between dx and Su(H) suggested that dx might function in Su(H)-dependent N signaling. These epistatic analyses suggested that dx functions downstream of the ligands and upstream of activated Su(H). A novel dx activity has been uncovered that suppresses N signaling downstream of N (Fuwa, 2006).

This study shows that the ability of Dl/Ser ligands to activate N signaling is partially reduced in the wing discs of the dx152 mutant. This result suggests that dx acts downstream of the Dl/Ser ligands to activate N signaling. When Dx is overexpressed, N signaling is induced independent of the presence of the Dl/Ser ligands. It is therefore possible that artificially elevated levels of Dx somehow overcome the Ser/Dl requirement for N activation. However, it is notable that both Dl and Ser showed a substantial ability to stimulate N signaling in the dx152 mutant disc, but they failed to activate N signaling in Su(H) mutant clones. Therefore, both dx and Su(H) play roles in ectopic vgBE activation, but while Su(H) is indispensable, Dx is required only for strong signal induction (Fuwa, 2006).

Genetically, dx has been considered a positive regulator of N signaling in Drosophila. Furthermore, mammalian homologues of dx activate the reporter genes of N signaling target genes. Also, it was shown that Dx and a dominant-negative form of Nedd4 activate the E(spl)mγ promoter in Drosophila cultured cells. However, in contrast, a human Dx homolog antagonizes N signaling in cortical neurons. This discrepancy could be explained by the finding that dx both activates and suppresses N signaling (Fuwa, 2006).

In this study, a novel genetic interaction involving dx was uncovered. It has been reported that N shows a dominant lethal interaction with hypomorphic alleles of dx. Here, it was found that Ser94c shows a dominant lethal interaction with dx152. This result was unexpected, because Dl, which functions more broadly than does Ser, did not show a lethal interaction. Thus, it is possible that dx and Ser have common tissue specificity. However, the developmental and molecular basis of this interaction between dx and Ser remains to be addressed (Fuwa, 2006).

Establishment of global patterns of planar polarity during growth of the Drosophila wing epithelium

Epithelial tissues develop planar polarity that is reflected in the global alignment of hairs and cilia with respect to the tissue axes. The planar cell polarity (PCP) proteins form asymmetric and polarized domains across epithelial junctions that are aligned locally between cells and orient these external structures. Although feedback mechanisms can polarize PCP proteins intracellularly and locally align polarity between cells, how global PCP patterns are specified is not understood. It has been proposed that the graded distribution of a biasing factor could guide long-range PCP. However, epithelial morphogenesis has been identified as a mechanism that can reorganize global PCP patterns; in the Drosophila pupal wing, oriented cell divisions and rearrangements reorient PCP from a margin-oriented pattern to one that points distally. This study used quantitative image analysis to study how PCP patterns first emerge in the wing. PCP appears during larval growth and is spatially oriented through the activities of three organizer regions that control disc growth and patterning. Flattening morphogen gradients emanating from these regions does not reduce intracellular polarity but distorts growth and alters specific features of the PCP pattern. Thus, PCP may be guided by morphogenesis rather than morphogen gradients (Sagner, 2012).

To study the emergence of polarity in the wing disc, the subcellular distribution of the PCP proteins Flamingo (Fmi) and Prickle (Pk) were quantified. Planar cell polarity (PCP) nematics were calculated based on Fmi staining and PCP vectors based on the perimeter intensity of EGFP::Pk clones. At 72 hr after egg laying (hAEL), the wing pouch has just been specified and is small. EGFP::Pk localizes to punctate structures at the cell cortex that are asymmetrically distributed in some cells, but PCP vectors exhibit no long-range alignment. By 96 hAEL, PCP vector magnitude increases and a global pattern emerges. Later, PCP vector magnitude increases further and the same global polarity pattern is clearly apparent. It is oriented with respect to three signaling centers: the dorsal-ventral (DV) boundary (where Wingless [Wg] and Notch signaling occur), the anterior-posterior (AP) compartment boundary (where Hedgehog [Hh] and Decapentaplegic [Dpp] signaling occur), and with respect to the hinge fold (where levels of the atypical Cadherin Dachsous [Ds] change sharply) (Sagner, 2012).

PCP vectors in the wing pouch near the hinge fold point away from it toward the center of the pouch. Within the Wg expression domain at the DV boundary, PCP vectors parallel the DV boundary and point toward the AP boundary. Just outside this domain, PCP nematics and vectors turn sharply to point toward the DV boundary in central regions of the wing pouch. However, where the DV boundary intersects the hinge-pouch interface, they remain parallel to the DV boundary over larger distances such that PCP vectors orient away from the hinge around the entire perimeter of the wing pouch (Sagner, 2012).

The AP boundary is associated with sharp reorientations of PCP. First, PCP vectors that parallel the DV boundary point toward the AP boundary in both anterior and posterior compartments. Second, although PCP vectors in the central wing pouch are generally orthogonal to the DV boundary, they deflect toward the AP boundary where Hh signaling is most active (as defined by upregulation of the Hh receptor Patched [Ptc]). On either side of this region, PCP vectors turn sharply to realign parallel to the AP boundary. Third, PCP vectors in the hinge point away from the AP boundary and align parallel to the hinge fold (Sagner, 2012).

The atypical Cadherins Fat (Ft) and Ds limit disc growth and orient growth perpendicular to the hinge. Their loss perturbs the PCP pattern in pupal wings and alters hair polarity. To investigate whether they influence the larval pattern, PCP was was quantified in ft and ds mutant discs. The PCP pattern is similar to wild-type (WT) in the central wing pouch but altered in proximal regions close to the hinge fold. Polarity vectors deviate from their normal orientation (away from the hinge fold) in many regions of the proximal wing pouch. This is especially clear near the intersection of the DV boundary with the hinge - here, PCP vectors orient toward the DV boundary rather than away from the hinge. Furthermore, near the AP boundary, vectors form a reproducible point defect, with vectors pointing away from the defect center (Sagner, 2012).

After pupariation, morphogenesis reshapes the wing disc, apposing its dorsal and ventral surfaces such that the DV boundary defines the margin of the wing blade. During reshaping the PCP pattern evolves, but specific local features are retained through pupal development. Consistent with this, hair polarity in ds adult wings proximal wing near the anterior wing margin orient toward the margin rather than away from the hinge. Near the AP boundary, hairs form swirling patterns. Thus, Ft and Ds are required during larval growth to ensure that PCP vectors in the proximal wing orient away from the hinge (Sagner, 2012).

Notch and Wg signaling at the DV boundary organize growth and patterning in the developing wing. These pathways maintain each other via a positive feedback loop; Notch induces transcription of Wg at the DV interface, and Wg signaling upregulates expression of the Notch ligands Delta (Dl) and Serrate (Ser) adjacent to the Wg expression domain, further activating Notch signaling at the DV boundary. To study how the DV boundary organizer affects PCP, Ser was ectopically expressed along the AP boundary with ptc-Gal4 (ptc > Ser). In the ventral compartment, Ser induces two adjacent stripes of Wg expression, which then upregulate Dl expression in flanking regions (dorsally, Fringe prevents Notch activation by Ser. The posterior Wg and Dl stripes are distinct, but the anterior stripes are broader due to the graded activity of ptc-Gal4. In these discs, the ventral compartment overgrows along the AP boundary, parallel to the ectopic 'organizers'. PCP nematics and vectors near the posterior Wg/Dl stripes are organized similarly to those flanking the normal DV boundary, running parallel to the stripe and turning sharply outside this region to orient toward the ectopic organizer). PCP nematics anterior to the ectopic Ser stripe run parallel to it over larger distances before turning sharply, consistent with the broader Wg/Dl expression in this region. In resulting adult wings, hairs orient toward the ectopic wing margin that forms along the AP boundary. Ectopically expressing Wg along the AP boundary also generates an ectopic organizer that reorients growth and PCP (Sagner, 2012).

To ask how loss of the DV boundary organizer affected PCP, a temperature-sensitive allele of wg was used that blocks Wg secretion (wgTS), or wings were populated with wg null mutant clones. Loss of Wg signaling severs the feedback loop with Notch such that both decay. PCP nematics were quantified in wgTS discs shifted to the restrictive temperature shortly after the second to third-instar transition (earlier, Wg is required to specify the wing pouch). wgTS discs have smaller wing pouches than WT and are missing a large fraction of the central region of the pouch where polarity orients perpendicular to the DV boundary. Polarity still orients away from the hinge, thus the PCP pattern in wgTS discs appears more radial (i.e., oriented toward the center of the wing pouch). Analogously, adult wings populated by wg null clones are missing those regions of the distal wing blade where hairs normally point perpendicular to the wing margin. The remaining proximal tissue is normally polarized except at its distal edges. Here, polarity deflects from the proximal-distal axis to parallel the edge of the wing. Normally, hair polarity in the wing blade parallels the margin only in proximal regions, where Ft/Ds influences polarity. Thus, the DV organizer is needed to orient PCP in distal regions perpendicular to the margin. Ft/Ds is required for a complementary subset of the PCP pattern in the proximal wing. Their influences largely reinforce each other (i.e., away from the hinge and toward the DV boundary or wing margin) except where the hinge and wing margin intersect. Here, loss of one signaling system expands the influence of the other. Wg is distributed in a graded fashion and is a ligand for Frizzled (Fz). Thus, it could bias the PCP pattern directly, e.g., by asymmetrically inhibiting interactions between Fz, Strabismus (Stbm), and Fmi or causing Fz internalization. If so, uniform Wg overexpression should prevent intracellular polarization or reduce cortical localization of PCP proteins. To investigate this, Wg was overexpressed uniformly (C765 > wg::HA). Uniform Wg expression elongates the wing pouch parallel to the AP boundary. It broadens the pattern of Dl expression, such that sharp Dl stripes at the DV boundary are lost, but Dl expression remains excluded from the Hh signaling domain anterior to the AP boundary. Fmi and EGFP::Pk polarize robustly in these discs; thus, the Wg gradient does not act directly on PCP proteins to induce or orient polarity. However, the pattern of PCP vectors and nematics is altered. PCP points away from the hinge (rather than perpendicular to the DV boundary) over larger distances compared to WT and then turns sharply to face theDV boundary in the middle of the wing pouch. Because specific alterations in the PCP pattern are induced by uniform Wg overexpression, Wg protein distribution does not directly specify the new PCP pattern (Sagner, 2012).

To identify signals that influence the PCP pattern near the AP boundary, the effects of uniform high-level expression of Dpp and Hh, two morphogens that form graded distributions near the AP boundary, were examined. Uniform Dpp expression does not influence the magnitude of PCP or the range over which PCP deflects toward the AP boundary. Interestingly, uniform Hh expression dramatically increases the range over which PCP deflects toward the AP boundary, suggesting that Hh is important for this aspect of the pattern. However it clearly indicates that PCP vectors are not oriented directly by the graded distribution of Hh or by the graded activity of Hh signaling, because both are uniformly high in the anterior compartment of Hh overexpressing discs. Whether the apposition of cells with very different levels of Hh signaling might produce sharp bends in the PCP pattern was therefore considered. In WT discs, Hh signaling levels change at two interfaces: one along the AP boundary and one along a parallel line outside the region of highest Hh signaling where Ptc is upregulated. PCP vectors orient parallel to the AP boundary in the cells posterior to it, deflect toward the boundary anteriorly, and then reorient sharply outside of this region to align parallel to the AP boundary. Discs uniformly overexpressing Hh have only one signaling discontinuity (at the AP boundary), because Hh signaling is high throughout the anterior compartment. This could explain why PCP in these discs remains deflected toward the AP boundary over longer distances (Sagner, 2012).

To test this, clones mutant for the Hh receptor Ptc, which constitutively activate signaling in the absence of ligand, were generated. Quantifying PCP nematics in these discs reveals reproducible patterns of polarity reorientation at interfaces between WT and ptc- tissue. In WT tissue adjacent to ptc- clones, PCP aligns parallel to the clone interface. Due to the typical clone shape, this orientation is often consistent with the normal PCP pattern. However, PCP also aligns parallel to ptc- clones in regions where this is not so. Thus, ptc- clones exert a dominant effect on adjacent WT tissue. In contrast, on the mutant side of the clone interface, polarity tends to orient perpendicular to the interface. Thus, apposition of high and low levels of Hh signaling causes a sharp bend in the PCP pattern. Corresponding polarity reorientation by ptc- clones is also seen in adult wing. Thus, Hh signaling has two effects in WT discs: within the Hh signaling domain, it deflects PCP toward the AP boundary, and just outside the Hh signaling domain, it orients PCP parallel to the AP boundary. In this region, the tendency for polarity to align parallel to Hh signaling interfaces is consistent with the orientation of polarity toward the DV boundary and away from the hinge. Thus, these three polarity cues reinforce each other throughout much of the wing pouch, making the global PCP pattern robust (Sagner, 2012).

Simulations have highlighted the difficulty of establishing long-range polarity alignment in a large field of cells from an initially disordered arrangement. The pattern typically becomes trapped in local energy minima, forming swirling defects. Introducing a small bias in each cell removes such defects - this has been an attractive argument for the involvement of large - scale gradients in orienting PCP. The graded distribution of Ds along the proximal-distal axis (orthogonal to the hinge-pouch interface) suggested a plausible candidate for such a signal. Strikingly, the Ds expression gradient gives rise to intracellular polarization of both Ft and Ds, and the recruitment of the atypical myosin Dachs to the distal side of each cell. Nevertheless, most of the PCP defects in ft mutants can be rescued by uniform overexpression of a truncated Ft version that cannot interact with Ds, and PCP defects in ds mutants can be rescued by uniform overexpression of Ds. Moreover, blocking overgrowth through removal of dachs also suppresses PCP phenotypes in both mutants. The remaining disturbances in PCP in each of these backgrounds are restricted to very proximal regions, both in adult wings and the wing disc. Thus, the graded distribution of Ds does not provide a direct cue to orient PCP over long distances; rather, it appears to be important only locally near the hinge. Furthermore, this study shows that the two other key signaling pathways that contribute to the global PCP pattern in the disc do not act directly through long-range gradients. How do these signals specify the PCP pattern, if not through gradients (Sagner, 2012)?

Simulations in the vertex model have suggested that long-range polarity can be established in the absence of global biasing cues if PCP is allowed to develop during growth. PCP easily aligns in a small system, and globally aligned polarity can then be maintained as the system grows. Such a model obviates the necessity of long-range biasing cues like gradients, at least to maintain long-range alignment of PCP domains. The finding that a global PCP pattern develops early during growth of the wing makes this idea plausible. It may be that a combination of local signals at the different organizer regions specifies the vector orientation of PCP when the disc is still small, and that the pattern is maintained during growth. This may explain why loss-of-function studies have failed to identify the signaling pathways at the AP and DV boundaries as important organizers of the PCP pattern (Sagner, 2012).

In addition to local signals, the orientation of growth may provide additional cues that help shape the PCP pattern. Simulating the interplay between PCP and growth in the vertex model showed that oriented cell divisions and cell rearrangements orient PCP either parallel or perpendicular to the axis of tissue elongation, depending on parameters. Interestingly, each of the signaling pathways that influence PCP in the disc also influences the disc growth pattern. Wg/Notch signaling at the DV boundary drives growth parallel to the DV boundary, consistent with the pattern of clone elongation at the DV boundary. Growth near the AP boundary, where Hh signaling is most active, is oriented parallel to the AP boundary. This behavior probably reflects oriented cell rearrangements rather than oriented cell divisions. Finally, Ft and Ds orient growth away from the hinge. Suppressing overgrowth in ft or ds mutant wings by altering downstream components of the Hippo pathway rescues normal PCP except in the most proximal regions of the wing. Thus, altered growth orientation may contribute to the PCP defects seen in ft and ds mutants (Sagner, 2012).

Growth orientation reflects the orientation of both cell divisions and neighbor exchanges, and these can each exert different effects on the axis of PCP. Understanding the influence of local growth patterns on PCP will require a quantitative description of the patterns of cell divisions and rearrangements in the disc. More refined simulations incorporating local differences in the orientation of cell divisions and rearrangements will allow exploration of how planar polarity patterns can be guided by different growth patterns (Sagner, 2012).

Serrate and the establishment of leg segments (part 1/2)

The possession of segmented appendages is a defining characteristic of the arthropods. By analyzing both loss-of-function and ectopic expression experiments, the Notch signaling pathway has been shown to play a fundamental role in the segmentation and growth of the Drosophila leg. Local activation of Notch is necessary and sufficient to promote the formation of joints between segments. This segmentation process requires the participation of the Notch ligands, Serrate and Delta, as well as Fringe. These three proteins are each expressed in the developing leg and antennal imaginal discs in a segmentally repeated pattern that is regulated downstream of the action of Wingless and Decapentaplegic. While Dl expression overlaps fngand Ser, in some cases, it appears to extend into regions of the disc where neither fng nor Ser is expressed (Rauskolb, 1999).

fng mutant clones also result in fused joints and shortened legs. fng is required with the formation of all joints except the tibia-tarsal (ta1: basitarsus) joint. In most cases, the formation of the joints appears to be an autonomous property of wild type cells, while the failure to form joint structures is an autonomous property of cells mutant for Notch, Dl, Ser or fng. However, some exceptions have been observed in which joint formation is inhibited within wild type cells that border mutant clones or mutant cells appear to contribute to joint structure (Rauskolb, 1999).

The four-jointed (fj) gene encodes a type 2 transmembrane protein and is also expressed in concentric rings within the developing leg imaginal disc. In fj mutants, growth of the femur, tibia, and first three tarsal segments is reduced, and the ta2-ta3 segment border is absent. The rings of fj expression in leg imaginal discs are complementary to the rings of Notch expression. Consistent with this complementarity, fj expression is inhibited in cells expressing activated Notch; in cells neighboring ectopically expressing Ser or Dl, and in cells along the borders of ectopic fng expression. By contrast, fj expression is activated within cells expressing Ser or Dl. These observations indicate that fj is negatively regulated downstream of Notch signaling in the leg. Thus, Notch signaling subdivides each leg segment into distinct domains of gene expression (Rauskolb, 1999).

Serrate and the establishment of leg segments (part 2/2)

Effects of mutation continued: part 2/2

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

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