Wingless shows a pattern of 16 regularly spaced bands in the extended germ band stage. These bands coincide with the posterior aspect of each parasegment (Rijsewijk, 1987)

wingless and DWnt4 are physically clustered and transcribed in overlapping embryonic territories under the control of the same regulatory molecules. Co-expression and co-regulation suggest that the close physical linkage results from the sharing of cis-control elements. Their proximity also suggests that these two Wnt signals cooperate in developmental patterning events. Antisense RNA experiments reveal that signaling by DWnt4 is essential for cells from the anterior compartment of each parasegment to adopt a denticled fate. It has been suggested that wingless and DWnt4 achieve opposite, but complementary functions in intrasegmental cell patterning of the embryonic ectoderm (Gieseler, 1996).

Wingless is also expressed in head segments. Based on the expression pattern of the segment polarity genes engrailed and wingless during the embryonic development of the larval head, it has been found that the head of Drosophila consists of remnants of seven segments (4 pregnathal and 3 gnathal) [Images] all of which contribute cells to neuromeres in the central nervous system (Schmidt-Ott, 1992)

The proneural genes achaete and scute and the segment polarity genes wingless and engrailed each have limited expression in only a few identifiable and stereotyped clusters of the head. For example, sc appears exclusively in a small part of the protocerebral domain, followed by transient expression in one to two protocerebral neuroblasts. wg is expressed in a total of three patches and engrailed is expressed in domains that are posterior and ventral to the adjacent wg domains. en is expressed in one patch in both the protocerebrum and the deuterocerebrum (Younossi-Hartenstein, 1996).

Wingless acts non-autonomously to specify the fate of a specific neuronal precursor, NB4-2. Gooseberry and Patched participate in the Wingless-mediated specification of NB4-2 by controlling the response to the wingless signal. Patched targets gooseberry distal and gooseberry-proximal in neuroblast determination. The RP2 neuron is a motoneuron and innervates muscle number 2 of the dorsal musculature. This neuron originates along with its sibling cell from the first ganglion mother cell derived from NB4-2, and occupies the anterior commissure along with several other RP2 neurons. NB4-2 itself is formed during the second wave of neuroblast delamination in stage 9. In gsb mutants, WG-positive NB5-3 is transformed to NB4-2 in a Wg-dependent manner, suggesting that GSB normally represses the capacity to respond to the wingless signal. In ptc mutants, gsb is ectopically expressed in normally Wg-reponsive cells, thus preventing the response to Wingless and consequently the correct specification of NB4-2 does not take place. The timing of the response to GSB suggests that the specification of neuroblast identities takes place within the neuroectoderm, prior to neuroblast delamination (Bhat, 1996).

During neurogenesis, the transmembrane protein Patched promotes a wingless-mediated specification of a neuronal precursor cell, NB4-2. Wg, secreted by row 5 cells promotes wingless expression in adjacent row 4 cells; Wg in turn represses gooseberry. Novel interactions of these genes with engrailed and invected during neurogenesis have been uncovered. While in row 4 cells Ptc represses gsb and wg, in row 5 cells en/inv relieve Ptc repression of gsb by a non-autonomous mechanism that does not involve hedgehog. The non-autonomous mechanism originates in Row 6/7 cells where en/inv engender hedgehog and another unknown secreted signal which acts in turn on adjacent row 5 cells to heighten wingless, and consequently, the expression of gooseberry. This differential regulation of gsb leads to the specification of NB5-3 and NB4-2 identities to two distinct neuroblasts. The row 5, NB5-3, neuroblasts are specified by high levels of gsb, expressed autonomously in row 5. The fate of row 4, NB4.2, requires an absence of gooseberry, assured by Patched repression and Wingless signaling from adjacent row 5 cells. The uncoupling of the ptc-gsb regulatory circuit by hedgehog and the unknown secreted signal from row 6/7 cells enables gsb to promote Wg expression in row 5 cells. These results suggest that the en/inv->ptc->gsb->wg pathway uncovered here and the hh->wg are distinct pathways that function to maintain the wild-type level of Wg. These results also indicate that Hh is not the only ligand for Ptc and similarly, that Ptc is not the only receptor for Hh (Bhat, 1997).

The progeny of wingless-expressing cells deliver the signal at a distance in Drosophila embryos

Pattern formation in developing animals requires that cells exchange signals mediated by secreted proteins. How these signals spread still remains unclear. It is generally assumed that they reach their target site either by diffusion or active transport. An alternative mode of transport for Wingless is described here. In embryos of Drosophila, the wingless gene is transcribed in narrow stripes of cells abutting the source of Hedgehog protein. These cells or their progeny are free to roam toward the anterior. As they do so, they no longer receive the Hedgehog signal and stop transcribing wg. However, the cells leaving the expression domain retain inherited Wg protein in secretory vesicles and carry it forward over a distance of up to four cell diameters. Experiments using a membrane-tethered form of Wg show that this mechanism is sufficient to account for the normal range of Wg. Nevertheless, evidence exists that Wg can also reach distant target cells independent of protein inheritance, possibly by restricted diffusion. It is suggested that both transport mechanisms operate in wild-type embryos (Pfeiffer, 2000).

In the ventral abdominal epidermis of Drosophila embryos, Wg signaling specifies bald cuticle by repressing the transcription of shavenbaby, a gene required for the formation of hair-like protrusions called denticles. As expected for a secreted product, the Wg protein is distributed more widely than its mRNA. Transcription of wg is in single-cell-wide stripes whereas the protein is detected over 3-4 cell diameters. Cuticle preparations from larvae carrying a lacZ reporter under the control of the wg promoter (wg-lacZ) show that bald cuticle is made by cells located as much as four cell diameters away from the wg expression domain (Pfeiffer, 2000).

Much of the Wg protein is detected in intracellular vesicles. Vesicles outside the domain of transcription are assumed to contain Wg that has been taken up by non-expressing cells. In fact, internalization of Wg has been proposed to be required for its transport: according to the model of planar transcytosis, internalized Wg is subsequently re-released into the extracellular space and hence presented to more distant cells. Of course, internalization of Wg could also constitute the first step towards degradation in lysosomes. An alternative to the planar transcytosis model of transport is that Wg diffuses in the extracellular space, possibly interacting with membrane-associated glycoproteins (Pfeiffer, 2000).

Irrespective of the transport mechanism, one would expect that, if the Wg protein were artificially tethered to the membrane of secreting cells and, hence, prevented from being released into the extracellular space, its range would be reduced and the area of bald cuticle would narrow. This was tested by expressing membrane-tethered Wg in a wg null mutant with a wg-GAL4 driver. Surprisingly, wg mutants rescued by membrane-tethered Wg could hardly be distinguished from wild-type embryos. In particular, the bands of naked cuticle were as wide as in the wild type, suggesting that membrane-tethered Wg can act as far as four cell diameters away. Two trivial explanations could account for the rescue. One is that membrane-tethered Wg might be leaky. Tethering Wg to the cell membrane was achieved by fusing it to the transmembrane protein Neurotactin (Nrt). Rescue could be explained if this fusion protein were cleaved, releasing active Wg into the extracellular space. There is, however, no indication of cleavage from Western blots. Moreover, functional assays confirm that Wg remains attached to expressing cells (Pfeiffer, 2000).

During normal development, en-expressing cells do not cross into the anterior compartment where wg is expressed. However, wg-expressing cells and their progeny may be free to roam in the anterior direction. This suggests an alternative explanation for the 'long-range action' of tethered Wingless: it could be carried toward the anterior by moving cells and their progeny. To test the feasibility of such a mechanism, the progeny of single cells were tracked, and marked at the time when wg expression commences. Single cells were marked by photoactivating caged rhodamine with the ultraviolet (UV) laser beam of a confocal microscope. To have a spatial landmark at later developmental times, this experiment was performed with embryos expressing the green fluorescent protein (GFP) in the posterior compartment. The progeny of marked cells were identified in live embryos at late stage 11 (after three mitoses) and mapped relative to the domain of en expression. Although no clones crossed the parasegment boundary, those located just in front of the en domain spanned several cell diameters (up to five) in the anteroposterior direction. Since the parasegment boundary is a clonal boundary, it imposes directionality to this spread, resulting in the net movement of wg-expressing cells towards the anterior. Importantly, clonal spread covers a broad area of the ectoderm and can account for the range of tethered Wg in these rescue experiments (Pfeiffer, 2000).

If, as proposed, Wg is carried by moving cells and their progeny, a stable non-secreted protein should also be transported towards the anterior. This prediction was tested using a Gal4-responsive transgene encoding nuclear-targeted beta-galactosidase (beta-gal), which is relatively stable. It was asked and determined whether this product was carried forward in the ventral epidermis. Indeed, in embryos carrying wg-GAL4 and tethered Wg, beta-gal is detected in stripes 3-4 cells wide in front of the parasegment boundary. This is substantially wider than the GAL4 RNA stripes. In hatched larvae of the same genotype, beta-gal activity is detected within similarly wide bands of cells occupying the middle of the bald regions. It is suggested that beta-gal made by wg-expressing cells is retained even when cells move away from the source of Hedgehog and therefore shut off the wg promoter. Since cells could move only towards the anterior of the parasegment boundary, beta-gal appears to spread in the anterior direction. Note that such spreading could not have occurred through cellular extensions such as cytonemes because the beta-gal product is nuclear in this experiment. Thus, a non-secreted protein can spread by being passed on to the progeny of expressing cells. As expected then, when driven by wg-GAL4, tethered Wg is detected in stripes that are similar in width to the stripes of Wg protein in wild-type embryos (3-4 cells wide). It is suggested that, like nuclear beta-gal, tethered Wg driven by wg-GAL4 is retained by cells as they spread toward the anterior and this accounts for its range of action (Pfeiffer, 2000).

The Wg-containing vesicles found at the anterior of the transcription domain are within cells that descended from wg-expressing cells. These vesicles are therefore not necessarily endocytic. They could equally contain unsecreted protein inherited from past expression. No good immunological markers are available in Drosophila to distinguish endocytic vesicles from secretory ones. To label the secretory pathway, flies were made that express GFP fused to the signal peptide of Wg (UAS-GFPsecr). When this fusion is expressed with the en-Gal4 driver, fluorescence is detected in bright intracellular dots within the en domain as well as weakly throughout the perivitelline space. The punctate fluorescence in expressing cells most probably represents GFP transiting through the secretory pathway and thus identifies secretory vesicles. In live embryos expressing GFPsecr under the control of wg-GAL4, vesicular staining is detected in stripes 3-4 cells wide at stage 11. Thus, secretory vesicles are present several cell diameters beyond the domain of expression (Pfeiffer, 2000).

These embryos were fixed and stained with anti-Wg antibody. Although much GFP fluorescence is lost upon fixation, extensive colocalization between Wg protein and the remaining GFP signal is detected, even outside the wg expression domain. This suggests that many Wg-containing vesicles are secretory as opposed to endocytic (although endocytic vesicles may exist as well). The presence of Wg-containing vesicles at the anterior of the Wg expression domain is often taken as evidence for transport from cell to cell. The results presented here show that this assumption must be revised. It also shows that, during normal development, cell spreading contributes significantly to the anterior movement of endogenous Wg protein. In conclusion, as cells proliferate and spread, they can retain the Wg signal and thus affect target cells some distance away from the site of wg transcription. It is important to note that Wg can also spread independent of cell movement, possibly by restricted diffusion. It has been possible to uncouple the two mechanisms of Wg movement and, thus, show that either is sufficient to ensure a normal range of action. Presumably, both contribute during wild-type development although their relative importance cannot yet be assessed. Interestingly, the parasegment boundary allows cells to carry Wg only toward the anterior and this adds to other mechanisms ensuring an asymmetric range of Wg in Drosophila embryos. Without this border, cells carrying Wg could wander towards the posterior and disrupt segment polarity (Pfeiffer, 2000).

Disruption of Drosophila melanogaster lipid metabolism genes causes tissue overgrowth associated with altered developmental signaling

Developmental patterning requires the precise interplay of numerous intercellular signaling pathways to ensure that cells are properly specified during tissue formation and organogenesis. The spatiotemporal function of many developmental pathways is strongly influenced by the biosynthesis and intracellular trafficking of signaling components. Receptors and ligands must be trafficked to the cell surface where they interact, and their subsequent endocytic internalization and endosomal trafficking is critical for both signal propagation and its down-modulation. In a forward genetic screen for mutations that alter intracellular Notch receptor trafficking in Drosophila melanogaster, mutants were recovered that disrupt genes encoding serine palmitoyltransferase and Acetyl-CoA Carboxylase (ACC). Both mutants cause Notch, Wingless, the Epidermal Growth Factor Receptor (EGFR), and Patched to accumulate abnormally in endosomal compartments. In mosaic animals, mutant tissues exhibit an unusual non-cell-autonomous effect whereby mutant cells are functionally rescued by secreted activities emanating from adjacent wildtype tissue. Strikingly, both mutants display prominent tissue overgrowth phenotypes that are partially attributable to altered Notch and Wnt signaling. This analysis of the mutants demonstrates genetic links between abnormal lipid metabolism, perturbations in developmental signaling, and aberrant cell proliferation (Sasamura, 2013).

The importance of lipid metabolism for the formation and maintenance of cell membranes is well established. Both serine palmitoyltransferase (SPT) and acetyl-CoA carboxylase (ACC) are critical enzymes that control different steps of lipid metabolism, and are highly conserved in diverse animal species. Genetic elimination of ACC1 or the SPT subunits Sptlc1 or Sptlc2 cause early embryonic lethality in mice, although the cellular basis for this lethality is unknown. In D. melanogaster, RNA-interfering disruption of ACC activity in the fat body results in reduced triglyceride storage and increased glycogen accumulation, and in oenocytes leads to loss of watertightness of the tracheal spiracles causing fluid entry into the respiratory system. This study demonstrates that D. melanogaster mutants lacking functional SPT or ACC exhibit endosomal trafficking defects, causing Notch, Wingless, EGFR, and Patched to accumulate abnormally in endosomes and lysosomes. These effects are accompanied by significant alterations in Notch and Wingless signaling, as revealed by changes in downstream target gene activation for both pathways. However, the mutants do not fully inactivate these developmental signaling pathways, and instead display phenotypes consistent with more complex, pleiotropic effects on Notch, Wingless, and potentially additional pathways in different tissues. These findings reinforce the importance of lipid metabolism for the maintenance of proper developmental signaling, a concept that has also emerged from studies demonstrating that: D. melanogaster mutants for phosphocholine cytidylyltransferase alter endosomal trafficking and signaling of Notch and EGFR; mutants for alpha-1,4-N-acetylgalactosaminyltransferase-1 affect endocytosis and activity of the Notch ligands Delta and Serrate; mutants for the ceramide synthase gene shlank disrupt Wingless endocytic trafficking and signaling, and mutants for the glycosphingolipid metabolism genes egghead and brainiac modify the extracellular gradient of the EGFR ligand Gurken (Sasamura, 2013).

Most strikingly, lace and ACC mutants also display prominent tissue overgrowth phenotypes. These tissue overgrowth effects are linked to changes in Notch and Wingless signaling outputs, and they involve gamma-secretase, Su(H), and Armadillo activities, suggesting that the overgrowth reflects an interplay of Wingless inactivation and Notch hyperactivation. Consistent with the findings, both Notch and Wingless regulate cell proliferation and imaginal disc size in D. melanogaster. Moreover, several observations indicate that Notch and Wingless are jointly regulated by endocytosis, with opposing effects on their respective downstream pathway activities, a dynamic process that might be especially sensitive to perturbations in membrane lipid constituents. Wingless itself exerts opposing effects on disc size that might depend on the particular developmental stage or disc territory. For example, hyperactivation of Wingless or inactivation of its negative regulators cause overproliferation, but Wingless activity can also constrain wing disc growth. Similar spatiotemporal effects might underlie the variability detected in studies with lace and ACC mutant clones, in which both tissue overgrowth and developmentally arrested discs were observed. Although no obvious changes were detected in downstream signaling for several other cell growth pathways that were examined, the trafficking abnormalities seen for other membrane proteins aside from Notch, Delta, and Wingless, as well as the incomplete suppression of the overgrowth phenotypes by blockage of Notch and Wingless signaling, suggest that other pathways might also be dysregulated in lace and ACC mutants, possibly contributing to the observed tissue overgrowth (Sasamura, 2013).

Wingless is modified by lipid addition, and lipoprotein vesicles have been suggested to control Wingless diffusion. In D. melanogaster embryos, endocytosis of Wingless limits its diffusion and ability to act as a long-range morphogen. Endocytosis can also affect Wingless signaling in receiving cells, where endocytosis both promotes signal downregulation and positively facilitates signaling. The apparently normal diffusion ranges for overaccumulated Wingless in lace and ACC mutant clones, yet reduced downstream target gene expression, is consistent with the idea that SPT and ACC act by promoting endocytic trafficking of Wingless in receiving cells rather than influencing the secretion and/or diffusion of Wingless from signal-sending cells (Sasamura, 2013).

The finding that lace and ACC mutant overgrowth phenotypes are also partially Notch-dependent is reminiscent of similar overproliferation phenotypes seen in certain D. melanogaster endocytic mutants, such as vps25, and tsg101. The overproliferation of disc tissue in these mutants is attributable to Notch hyperactivation, reflecting the fact that non-ligand-bound Notch receptors that are normally targeted for recycling or degradation are instead retained and signal from endosomes. Analogous effects are likely to contribute to the lace and ACC mutant overgrowth, where significant Notch overaccumulation was observed throughout the endosomal-lysosomal routing pathway. Some ectopic Notch signaling might emanate from the lysosomal compartment, which is enlarged and accumulates particularly high levels of Notch in lace and ACC mutant clones. Analysis of D. melanogaster HOPS and AP-3 mutants, which affect protein delivery to lysosomes, has identified a lysosomal pool of Notch that is able to signal in a ligand-independent, gamma-secretase-dependent manner (Sasamura, 2013).

How do SPT and ACC contribute to endosomal trafficking of Notch and other proteins? In the yeast SPT mutant lcb1, an early step of endocytosis is impaired due to defective actin attachment to endosomes, a phenotype that is suppressed by addition of sphingoid base. However, the trafficking abnormalities seen in lace and ACC mutants do not resemble those in the yeast lcb1 mutant, perhaps because endocytic vesicle fission is primarily dependent upon dynamin in D. melanogaster and mammals, instead of actin as in yeast. Nevertheless, the requirement for SPT and ACC in D. melanogaster endosomal compartments might reflect possible functions in endosome-cytoskeleton interactions. Another possibility is that the defective endosomal trafficking seen in lace and ACC mutants is caused by the inability to synthesize specific phospholipids needed for normal membrane homeostasis. Finally, lace and ACC might be important for the formation and/or function of lipid rafts, specialized membrane microdomains that have been implicated in both signaling and protein trafficking (Sasamura, 2013).

A remarkable feature of the lace and ACC mutant phenotypes that suggests an underlying defect in lipid biogenesis is the non-autonomous effect in mutant tissue clones, wherein nearby wildtype cells generate a secreted activity that diffuses several cell diameters into the mutant tissue and rescues the trafficking and signaling defects. One possibility is that these secreted activities are diffusible lipid biosynthetic products of SPT and ACC, which enter the mutant cells and serve as precursors for further biosynthetic steps that do not require SPT or ACC. An intriguing alternative is that the SPT and ACC enzymes are themselves secreted and taken up by the mutant cells. A precedent for this mechanism has recently been demonstrated for D. melanogaster ceramidase, a sphingolipid metabolic enzyme that is secreted extracellularly, delivered to photoreceptors, and internalized by endocytosis to regulate photoreceptor cell membrane turnover (Sasamura, 2013).

Recent work has highlighted the importance of lipid metabolism for oncogenic transformation, and ACC has been advanced as a promising target for cancer drug development. ACC is upregulated in some cancers, possibly as a result of high demands for lipid biosynthesis during rapid cell divisions. Sphingolipids and their derivatives are also thought to influence the balance of apoptosis and cell proliferation during tissue growth, and thus have also garnered attention as potential cancer therapy targets. The current findings regarding the requirements of SPT and ACC for proper trafficking and signaling of key developmental cell-surface signaling molecules, including Notch and Wingless, provide insights into how lipid metabolic enzymes might influence cell proliferation and tissue patterning in multicellular animals. Complex lipid biosynthesis is essential for the creation of the elaborate, interconnected, and highly specialized membrane compartments in which developmental pathways operate, and perturbations in lipid biosynthesis that are tolerated by the cell might nevertheless exert significant pleiotropic effects on developmental patterning, cell proliferation, and other cellular processes. Exploration of lipid metabolic enzymes as pharmacological targets must therefore take into account potentially unfavorable effects on critical signaling pathways controlling development and organogenesis (Sasamura, 2013).

Drosophila wingless and pair-rule transcripts localize apically by Dynein-mediated transport of RNA particles

Asymmetric mRNA localization targets proteins to their cytoplasmic site of function. The mechanism of apical localization of wingless and pair-rule transcripts in the Drosophila blastoderm embryo has been elucidated by directly visualizing intermediates along the entire path of transcript movement. After release from their site of transcription, mRNAs diffuse within the nucleus and are exported to all parts of the cytoplasm, regardless of their cytoplasmic destinations. Endogenous and injected apical RNAs assemble selectively into cytoplasmic particles that are transported apically along microtubules. Cytoplasmic dynein is required for correct localization of endogenous transcripts and apical movement of injected RNA particles. It is proposed that dynein-dependent movement of RNA particles is a widely deployed mechanism for mRNA localization (Wilkie, 2001).

To study the mechanism of apical localization, whether actin and/or MTs are necessary for localization of injected mRNA was tested by preinjecting cytoskeletal inhibitors 10 min before injecting the RNA. It was found that preinjection of Cytochalasin B, at concentrations that disrupt the organization of actin filaments, has no affect on Runt mRNA localization. However, a similar disruption of nuclear position has been observed in the cortical cytoplasm. In contrast, preinjection of colcemid, which destabilizes blastoderm MTs, disrupts runt, wingless, and fushi tarazu RNA localization almost entirely. It is concluded that an intact MT cytoskeleton is required for apical localization of injected RNA and that actin does not play a major role in the process. However, some minor role for actin in apical localization of RNA cannot be excluded (Wilkie, 2001).

Whether the localization of injected RNA occurs by minus end directed MT-dependent motor movement was tested by preinjecting embryos with antibodies against Drosophila cytoplasmic dynein heavy chain (dhc). Two independently raised monoclonal antibodies against dhc are each sufficient to inhibit RUN, FTZ, and WG mRNA apical localization in most, or all, embryos. Either one, the anti-dynein antibody or the colcemid injections, is sufficient to cause apical RNA to partly diffuse away from the site of injection in a similar manner to embryos injected with HB RNA alone. Injected apical RNA does not diffuse in the absence of anti-dynein antibodies or Colcemid preinjections. These results suggest that apical RNA is tethered to MTs by dynein and that dynein is required for the transport of RNA particles (Wilkie, 2001).

To further test the involvement of cytoplasmic dynein in apical transcript localization, RNA was injected into mutant cytoplasmic dynein heavy chain (Dhc64C) embryos. A marked reduction was found in the speed of movement of injected apical targeted RNAs in dynein mutants. Cytoplasmic dynein is essential for many cellular processes, so strong mutations in Dhc64C are homozygous lethal in Drosophila and cannot be studied at the blastoderm stage. Instead hypomorphic allelic combinations of Dhc64C, which are viable in trans due to intragenic complementation, were used. In two different allelic combinations of Dhc64C, injected RNA particles move at speeds 60% to 70% slower than they do in wild-type. Staining Dhc64C mutant embryos with anti-tubulin antibodies showsthat MT distribution is indistinguishable from wild-type, indicating that the reduced speed of localization is not due indirectly to a disruption of the MTs. Instead, the reduction in speed is likely to show a direct requirement for dynein in particle transport (Wilkie, 2001).

To test whether cytoplasmic dynein is also required for apical localization of endogenous transcripts, the effects of Dhc64C hypomorphic mutants and anti-dhc antibodies on the apical localization of endogenous FTZ transcripts was tested by in situ hybridization. As expected, hypomorphic Dhc64C mutants show no detectable effects on FTZ apical mRNA localization since injected RNA localizes correctly, but more slowly. In contrast, injection of anti-dhc antibody disrupts endogenous FTZ localization, leading to unlocalized stripes of ftz mRNA 20–30 min after injection. Given that FTZ mRNA has a half-life of 6 min in the blastoderm, the FTZ transcripts observed are likely to have been synthesized after the injection. It is concluded that endogenous apical mRNA localization is also dynein dependent (Wilkie, 2001).

Dynactin is a protein complex that is involved in coordinating the activities of cytoplasmic dynein, and is thought to be required for most forms of dynein-based transport. To test whether dynactin is also required for apical RNA localization, a large excess of p50/dynamitin is preinjected into embryos 10 min before injecting apically targeted RNA. p50/dynamitin causes a significant reduction in the speed of RNA particle movement. p50/dynamitin is a subunit of dynactin whose overexpression is a widely used method of disrupting the dynactin complex and demonstrating conclusively dynein-dependent motility. Dynactin is required for some cargo binding and for dynein processivity. It is concluded that apical transcript localization in the blastoderm embryo occurs by cytoplasmic dynein- and dynactin-mediated transport along MTs toward their minus ends (Wilkie, 2001).

It is thought that export and localization of apical mRNA in the blastoderm embryo can be divided into six distinct steps. (1) During or after completion of transcription and processing, transcripts are assembled into particles, which contain various hnRNPs and export factors, some of which may form part of the cytoplasmic localization machinery. (2) mRNA particles diffuse freely after release from the site of transcription and processing until they reach nuclear pore complexes (NPCs) on the nuclear periphery. (3) mRNA particles are exported through NPCs in all parts of the nuclear envelope. (4) The composition of the particles probably changes during export from the nucleus and in the cytoplasm to recruit dynein, dynactin, and associated proteins. (5) Particles attach to MTs and are actively transported to the apical cytoplasm. (6) Particle movement arrests in the apical cytoplasm, where they may associate with other particles and become anchored (Wilkie, 2001).

The first three steps of apical localization are thought to be common to most mRNAs, because they are essential universal processes in eukaryotic cells. However, the last three steps of the localization pathway are likely to vary among different kinds of transcripts, since the key determinant in sorting different mRNAs to their correct cytoplasmic destinations is presumably RNP particle composition in the cytoplasm. It is possible that some components required for cytoplasmic sorting are preassembled in the nucleus, as suggested by studies showing that the localization of injected FTZ mRNA depends on preincubation with the hnRNPA1 protein Squid. Indeed, a requirement for hnRNPs has also been shown for GRK mRNA localization in the oocyte, for myelin basic protein mRNA in rat oligodendrocytes, and for Vg1 transcripts in Xenopus oocytes. However, the data in this study show that injected protein-free apical RNA assembles in the cytoplasm into particles that localize correctly, arguing that all the factors needed to assemble competent localization particles can also be recruited in the cytoplasm (Wilkie, 2001).

Apical localization of wingless transcripts is required for Wingless signaling

Many developing and adult tissues are comprised of polarized epithelia. Proteins that are asymmetrically distributed in these cells are thought to be localized by protein trafficking. The distribution and function of the signaling protein Wingless is predetermined by the subcellular localization of its mRNA. High-resolution in situ hybridization reveals apical transcript localization in the majority of tissues examined. This localization is mediated by two independently acting elements in the 3' UTR. Replacement of these elements with either non-localizing or basolaterally localizing elements yields proteins with altered intracellular and extracellular distributions and reduced signaling activities. This novel aspect of the wingless signaling pathway is conserved and may prove to be a mechanism used commonly for establishing epithelial cell polarity (Simmonds, 2001).

Whereas wg transcripts are enriched apically, transcripts encoded by a lacZ reporter gene, expressed in the same cells under control of the wg promoter are distributed uniformly throughout the cytoplasm. It is concluded that this localization is a transcript-specific and not a cell-specific property. Indeed, apical localization of wg transcripts is also observed in most other polarized cells (Simmonds, 2001).

Different portions of the wg transcript were tested for their ability to confer apical localization in vivo to a nonlocalized lacZ transcript. Transgenic constructs with the wg 5' UTR and/or the wg ORF, fused either 5' (in-frame) or 3' to the lacZ sequence, yield uniformly distributed transcripts. However, fusion of the wg 3' UTR to lacZ results in apical transcript localization. Thus, the wg 3' UTR is both necessary and sufficient for apical transcript localization (Simmonds, 2001).

To map the specific sequences responsible for apical localization within the 1098 nt wg 3' UTR, deletions were introduced into the lacZ-wg 3' UTR reporter, and the deleted reporters were tested for their ability to confer apical transcript localization in transgenic embryos. These deletions defined two wg localization elements (WLEs), each of which is sufficient to confer apical transcript localization. WLE1 is located between nucleotides 60-178 and WLE2 is located between nucleotides 670-780. These elements may function differently as localization conferred by WLE2 is more closely associated with the apical cortex than that conferred by WLE1. Differences in function are also suggested by the lack of apparent similarity in sequence or predicted secondary structure (Simmonds, 2001).

To examine the effect of transcript localization on Wg signaling, constructs expressing wg transcripts that localize to different parts of the cell were made. The three constructs that were made differ only in their 3' UTRs. The first uses the endogenous wg 3' UTR, the second a 3' UTR derived from the SV40 small t antigen gene, and the third a 3' UTR derived from the partner of paired (ppa) gene. Each of the transgenes was placed under the control of a GAL4-dependent promoter and the vectors introduced into embryos to obtain transgenic flies. Transcripts containing the wg 3' UTR are localized apically while transcripts containing the SV40 3' UTR are uniformly distributed. In contrast, transcripts containing the ppa 3' UTR are localized basally. The distribution of the ppa-tagged transcript is not as tightly localized to the basal side of the cell as full-length wg transcripts are to the apical surface. Rather, the two distributions appear to be complementary (Simmonds, 2001).

Prior to comparing the signaling activities of the proteins made from these three transgenes, Western blot analysis was used to select transgenic lines that express equivalent levels of protein. Expression of the transgenes was induced by crossing the UAS-wg flies to ptc-GAL4 flies. These express GAL4 in the majority of ectodermal cells. Two matched sets of transgenic lines were selected: a 'low'-expressing set and a 'high'-expressing set. Quantitation of the protein levels expressed by each of the fly lines in these sets shows that, when the endogenous Wg contribution is subtracted, the high lines express about six times the levels of the low lines. Based on the spatial differences between endogenous wg and ptc-GAL4-driven wg expression patterns, it is estimated that the high lines express about half the levels of endogenous Wg protein on a per cell basis (Simmonds, 2001).

Semi-quantitative RT-PCR analysis of the transgenic transcripts shows that transcription levels are also equivalent for each of the three lines in each of the matched sets. Initial levels of protein and RNA were also observed to be approximately equal when visualized in situ by immunocytochemistry and in situ hybridization. It is concluded that the 3' UTR swaps have little effect on the synthesis and stability of wg transcripts and protein. Posttranslational modifications also appear to be the same for each protein, since each lane on the Western blot contains a similar set of bands equivalent in number, mobility, and relative intensity (Simmonds, 2001).

Wg facilitates its own expression via both autocrine and paracrine signaling pathways. To test whether the localization of wg transcripts affects these activities, pulses of wg construct expression were induced by crossing the high set of UAS-wg transgenic flies to a heat shock-Gal4 line and subjecting 3- to 5-hr-old embryos to a 30 min heat shock. Protein levels were assessed by Western blot analysis. Immediately following the heat pulse, each of the matched transgenic lines produced the same amount of protein. This was about three times the amount of endogenous Wg expressed in heat-shocked controls. In the apical transcript line, these levels rose about 5-fold higher during the next half hour, and subsequently remained at a high level. This increase in expression levels is due to the spatial expansion and intensification of endogenous Wg stripes. In the line with uniform wg transcript distribution, autoregulation also occurs but with slower kinetics. In contrast, the basal transcript line shows no further increase in Wg expression levels 30 min after the heat pulse, and by 60 min, expression levels are similar to those seen in the heat shock control. Transcript levels for each of the lines and each time point were also measured using RT-PCR and NIH image, and it was confirmed that, as with the ptc-GAL4-driven expression, each of the transgene mRNAs is expressed and turned over at equivalent rates. Thus, it is concluded that apical transcript localization is important for Wg autoregulation (Simmonds, 2001).

In order to test the signaling activities of the differentially localized transcripts in a more comprehensive fashion, each of the constructs of the high- and low-expressing matched sets was tested for their ability to rescue wg-dependent segmental patterning. This was accomplished by recombining the two sets of wg-expressing transgenic lines into a wg null mutant background and crossing these lines to flies that express GAL4 under wg promoter control. As expected, the apically localized transcript of the high apical line is capable of restoring much of the naked cuticle that is missing in wg mutant embryos. The incomplete nature of this rescue is most likely due to suboptimal levels of expression as compared to the endogenous wg gene (about 50%). In comparison, the high uniform construct yields significantly reduced rescuing activity and the high basolateral line very little rescuing activity. The low set of lines shows a similar trend, but with substantially lower degrees of rescue. It is concluded that transcript localization within apical cytoplasm is essential for robust signaling activity (Simmonds, 2001).

In order to help understand how transcript localization affects protein function, an examination was carried out to see whether differences in protein distribution, in and around wg-expressing cells, could be detected. Expression of the three transgenes in the high matched set was driven using a wg-GAL4 driver, and Wg distributions were observed in a wg null background. The single-cell-wide wg stripes serve as a point source from which diffusion of the protein, laterally and apically/basally, can be readily observed (Simmonds, 2001).

Most of the Wg protein detected in wild-type embryos is observed to be enriched in the apical cytoplasm of wg-expressing cells. Although the majority of this signal is diffuse, brightly staining punctate bodies are also observed. Similar punctae are also found in cells nearby. In the wg-expressing cells, these punctate bodies are thought to represent both endocytic and exocytic vesicles (Simmonds, 2001).

Wg expressed from the apically localized transgene transcript is distributed much the same as in the wild-type control. However, protein expressed from the uniformly distributed transcript shows clear differences in distribution. Although it still appears to be somewhat enriched in the apical cytoplasm of wg-expressing cells, there is less of the protein in these cells and more extending laterally into the middle of the segment. The difference in distribution of protein translated from the basal transcript is even more striking. There is little detectable enrichment within the wg-expressing cells, and more of the protein extends laterally across the segment. Interestingly, this extracellular protein still appears to be apically enriched. It is concluded that protein synthesized basally is secreted more efficiently, diffuses more rapidly within the extracellular matrix, or is less effectively endocytosed (Simmonds, 2001).

Transcripts encoded by the Drosophila virilis wg gene have been examined to see if apical transcript localization is conserved in this species. Despite an estimated evolutionary divergence of about 60 million years, and the tendency of 3' UTRs to diverge rapidly in sequence, this transcript is also localized apically. Indeed, the D. virilis wg 3' UTR is functional in D. melanogaster, and elements with sequence similarity to the two wg localization elements, WLE1 and WLE2, exist in similar positions within the 3' UTR. Although conserved between species, the sequences and predicted secondary structures of WLE1 and WLE2 bear no resemblance to one another. Their sequences also fail to show significant homology to other sequences in the database, including those of other localized transcripts. Taken together with the observation that wg transcripts colocalize to the same particles as other localized transcripts, it is surmised that transcript recognition is mediated either by transcript-specific adaptors or by common adaptors that recognize similar secondary structures (Simmonds, 2001).

This functional conservation of wg localization elements in Drosophila further substantiates the importance of wg transcript localization and suggests that this step in the pathway may be conserved in other organisms. Indeed, a number of vertebrate wnt transcripts have been shown to be localized within oocytes. For example, transcripts encoded by the Xwnt5 and Xwnt11 genes of Xenopus are localized in the oocyte vegetal pole while those of X-Wnt8b are localized to the animal pole. In Ascidians, the maternally expressed HrWnt-5 transcript is localized to the posterior of early embryos (Simmonds, 2001 and references therein).

Differential cytoplasmic mRNA localization of Wg

Establishment of segmental pattern in the Drosophila syncytial blastoderm embryo depends on pair-rule transcriptional regulators. mRNA transcripts of pair-rule genes localise to the apical cytoplasm of the blastoderm via a selective dynein-based transport system and signals within their 3'-untranslated regions. However, the functional and evolutionary significance of this process remains unknown. Subcellular localisation of mRNAs from multiple dipteran species has been analyzed both in situ and by injection into Drosophila embryos. Transcript localisation was assayed in four species that can be cultured in the laboratory. Two of them, Episyrphus (Syrphidae) and Megaselia (Phoridae), are cyclorrhaphan flies (i.e. higher dipterans) but, unlike Drosophila, belong to basal branches of this taxon; the other two, Coboldia (Scatopsidae) and Clogmia (Psychodidae), belong to different branches of lower Diptera. Although localisation of wingless transcripts is conserved in Diptera, localisation of even-skipped and hairy pair-rule transcripts is evolutionarily labile and correlates with taxon-specific changes in positioning of nuclei. In Drosophila localised pair-rule transcripts target their proteins in close proximity to the nuclei and increase the reliability of the segmentation process by augmenting gene activity. These data suggest that mRNA localisation signals in pair-rule transcripts affect nuclear protein uptake and thereby adjust gene activity to a variety of dipteran blastoderm cytoarchitectures (Bullock, 2004).

Apical localisation of pair-rule mRNAs in Drosophila syncytial blastoderm embryos was first noted 20 years ago, but the developmental and evolutionary significance of this process has remained unclear. Apical pair-rule mRNA localisation is conserved in cyclorrhaphan species that diverged over 145 million years ago, indicating that this process has a significant developmental role under natural conditions. Likewise, the widespread maintenance of wg transcript localisation in Diptera supports the importance of this process on a phylogenetic scale, even though, in Drosophila, wg appears to be less sensitive than pair-rule genes to a reduction in endogenous transcript localisation (Bullock, 2004).

Rho1 regulates Wg signaling during segmentation

The Rho small GTPase has been implicated in many cellular processes, including actin cytoskeletal regulation and transcriptional activation. The molecular mechanisms underlying Rho function in many of these processes are not yet clear. In Drosophila, reduction of maternal Rho1 compromises signaling pathways consistent with defects in membrane trafficking events. These mutants fail to maintain expression of the segment polarity genes engrailed (en), wingless (wg), and hedgehog (hh), contributing to a segmentation phenotype. Formation of the Wg protein gradient involves the internalization of Wg into vesicles. The number of these Wg-containing vesicles is reduced in maternal Rho1 mutants, suggesting a defect in endocytosis. Consistent with this, stripes of cytoplasmic β-catenin that accumulate in response to Wg signaling are narrower in these mutants relative to wild type. Additionally, the amount of extracellular Wg protein is reduced in maternal Rho1 mutants, indicating a defect in secretion. Signaling pathways downregulated by endocytosis, such as the epidermal growth factor receptor (EGFR) and Torso pathways, are hyperactivated in maternal Rho1 mutants, consistent with a general role for Rho1 in regulating signaling events governing proper patterning during Drosophila development (Magie, 2005).

The data indicate that a number of signaling pathways important during early development in Drosophila are compromised in maternal Rho1 mutants. The observation that secretion of Wg protein is aberrant in these mutants together with the endocytosis defects observed in S2R+ cells treated with Rho1 dsRNA and in maternal Rho1 embryos indicates that Rho1 plays a general role in membrane trafficking processes in the early embryo. The biochemical mechanisms through which Rho proteins affect membrane trafficking are currently unclear. One possibility is that the function of Rho1 in this process is a byproduct of its regulation of the actin cytoskeleton. In yeast, there is evidence that the actin cytoskeleton is important in endocytosis, as mutations in actin and some actin-binding proteins inhibit endocytosis. In addition, yeast Rho1 has been shown to be involved in endocytosis of the α-receptor. In mammalian cells, treatment with pharmacological agents that perturb actin structure can affect endocytosis in a cell-type-specific way. In polarized epithelial cells, for example, treatment with the actin-depolymerizing drug cytochalasin D inhibits endocytosis specifically at the apical, but not the basolateral surface. RhoA has also been implicated in endocytosis in polarized epithelial cells. In Drosophila, Rho1 has clear roles in actin cytoskeletal regulation during oogenesis and embryogenesis, consistent with the notion that Rho1 may be acting primarily through its effects on the actin cytoskeleton (Magie, 2005).

The observation that the segmentation phenotype in maternal Rho1 mutants is the result of general defects in membrane trafficking processes (both secretion and endocytosis) and not a primary effect on transcriptional activation has important implications for the interpretation of data linking Rho to disparate cellular processes. While current data cannot exclude the possibility that Rho directly acts in transcriptional activation or through many disparate mechanistic pathways, data are accumulating that suggest Rho may act primarily as a regulator of the actin cytoskeleton and other functions it has been linked to are indirect effects. For instance, the ability of Rho to influence transcriptional activation through the serum response factor (SRF), as well as affect cell cycle progression, is due to its direct effects on actin cytoskeletal regulation. Identifying the molecular mechanisms underlying each of Rho's activities will be crucial to determining whether Rho1 has direct effects on a number of pathways or has a small number of primary functions that indirectly affect other functions. Investigations of Rho GTPase function in genetically amenable model organisms are providing a diversity of developmental contexts in which to examine all aspects of Rho biology, and the ability to examine specific, loss-of-function phenotypes will continue to aid identification of the mechanisms underlying Rho function (Magie, 2005).

Temporal pattern of the posterior expression of Wingless in Drosophila blastoderm

In most animals, the antero-posterior (A-P) axis requires a gradient of Wnt signaling. Wnts are expressed posteriorly in many vertebrate and invertebrate embryos, forming a gradient of canonical Wnt/β-Catenin activity that is highest in the posterior and lowest in the anterior. One notable exception to this evolutionary conservation is in the Drosophila embryo, in which the A-P axis is established by early transcription factors of maternal origin. Despite this initial axial establishment, Drosophila still expresses Wingless (Wg), the main Drosophila Wnt homologue, in a strong posterior band early in embryogenesis. Since its discovery 30 years ago this posterior band of Wg has been largely ignored. This study re-examined the onset of expression of the Wg posterior band in relation to the expression of Wg in other segments, and compared the timing of its expression to that of axial regulators such as gap and pair-rule genes. It was found that the posterior band of Wg is first detected in blastoderm at mid nuclear cycle 14, before the segment-polarity stripes of Wg are formed in other segments. The onset of the posterior band of Wg expression was preceded by that of the gap gene products Hunchback (hb) and Krüppel (Kr), and the pair-rule protein Even-skipped (Eve). Although the function of the posterior band of Wg was not analyzed in this study, it is noted that in temperature-sensitive Wg mutants, in which Wg is not properly secreted, the posterior band of Wg expression is diminished in strength, indicating a positive feedback loop required for Wg robust expression at the cellular blastoderm stage. It is proposed that this early posterior expression could play a role in the refinement of A-P patterning (Vorwald-Denholtz, 2011).

Widespread rewiring of genetic networks upon Wnt cancer signaling pathway activation

Cellular signaling networks coordinate physiological processes in all multicellular organisms. Within networks, modules switch their function to control signaling activity in response to the cellular context. However, systematic approaches to map the interplay of such modules have been lacking. This study generated a context-dependent genetic interaction network of a metazoan's signaling pathway. Using Wnt signaling in Drosophila as a model, >290,000 double perturbations were measured of the pathway in a baseline state, after activation by Wnt ligand, or after loss of the tumor suppressor APC. Genetic interactions within the Wnt network were found to globally rewire after pathway activation. Between-state networks were derived that showed how genes changed their function between state-specific networks. This related pathway inhibitors across states and identified genes required for pathway activation. For instance, it was predicted and confirmed that the ER-resident protein Catsup is required for ligand-mediated Wnt signaling activation. Together, state-dependent and between-state genetic interaction networks identify responsive functional modules that control cellular pathways (Billmann, 2017).

Wnt, Hedgehog and junctional Armadillo/beta-catenin establish planar polarity in the Drosophila embryo

To generate specialized structures, cells must obtain positional and directional information. In multi-cellular organisms, cells use the non-canonical Wnt or planar cell polarity (PCP) signaling pathway to establish directionality within a cell. In vertebrates, several Wnt molecules have been proposed as permissible polarity signals, but none has been shown to provide a directional cue. While PCP signaling components are conserved from human to fly, no PCP ligands have been reported in Drosophila. This paper reports that in the epidermis of the Drosophila embryo two signaling molecules, Hedgehog (Hh) and Wingless, provide directional cues that induce the proper orientation of Actin-rich structures in the larval cuticle. Proper polarity in the late embryo also involves the asymmetric distribution and phosphorylation of Armadillo (Arm or β-catenin) at the membrane and that interference with this Arm phosphorylation leads to polarity defects. These results suggest new roles for Hh and Wg as instructive polarizing cues that help establish directionality within a cell sheet, and a new polarity-signaling role for the membrane fraction of the oncoprotein Arm (Colosimo, 2006).

These results indicate that Wg and Hh act as instructive cues in the Drosophila embryonic epidermis to establish planar cell polarity. Though the complete molecular mechanisms that control the complex system of PCP in the ventral epidermis remain to be determined, this process appears to occur in part through the asymmetric localization of Arm at the membrane. Further, proper polarity signaling is abolished if specific phosphorylation sites within the alpha-catenin binding domain of Arm are mutated. These sites were originally found to increase the affinity of β-catenin for alpha-catenin when phosphorylated by Casein Kinase II in vitro, suggesting one mechanism for stabilizing junctions. These findings provide in vivo support for this hypothesis, since low levels of ArmAA Arm in which two threonines were mutated to alanines) rescues cellular junction defects to a similar extent as expression of an alpha-catenin/E-cadherin fusion protein, a protein that makes overly stable junctions. Higher levels of ArmAA expression lead to apparent polarity defects. Since ArmAA does not localize asymmetrically the way that wild-type Arm does, it is inferred that CKII phosphorylation may be required for the accumulation of junctions in specific regions of cells implying that stable junctions at specific sites in a cell are required for proper planar cell polarity. Further, these findings revealed that when all signaling activity is abolished through null mutations in the Wg or Hh signaling pathways, both cell identity and polarity determination was disrupted. It remains to be determined how Wg and Arm proteins function in polarity signaling, specifically whether they work through known PCP components, function similarly to their role in dorsal closure, or perhaps through novel signaling mechanisms like the interaction with Notch or Axin (Colosimo, 2006).

The wg and hh genes are required for the proper establishment of cell identities within segments. Uniform expression of Wg in the embryo leads to a completely naked cuticle, but short early bursts of expression establish what appears to be relatively normal patterning. Upon closer inspection, however, the denticle orientations of these early expression rescue experiments do not entirely resemble the wild-type patterning. This suggests that early expression of Wg can rescue several aspects of cell identity, including development of naked cuticle, but Wg is also required in the later stages when denticles form to specify proper orientations. Expression of ectopic Wg has been observed to correlate with denticles pointing toward the source of Wg, and expression of ectopic Hh also leads to denticles pointing away from its source. These previous studies, however could not distinguish between cell fate transformation and changes in cell polarity since the sources of both ligands were in the normal orientation. The current observations argue that Hh and Wg can have direct effects on cell polarity since denticles and their precursors (the Actin foci) are rotated 90° away from the anterior-posterior axis corresponding to the direction of ligand expression (Colosimo, 2006).

In the early embryo, expression of Wg and Hh is determined by pair-rule genes, but this effect is transient and requires mutually reinforcing positive activation loops to form between cells expressing Wg and En/Hh. This is the early signaling event that establishes an organizer region in each parasegment. Therefore, if either Hh or Wg is missing, expression of both is lost. The early effects of Hh and Wg expression are important for the establishment of segment boundaries, and these boundaries function in limiting Wg function, giving this morphogen an asymmetric range. The current findings agree with these observations, because it was observed that the Wg effect is best observed when hh is absent, suggesting that when the hh gene is present a boundary may be formed, thus preventing Wg from orienting the denticles to the same extent. It also appears that the distance over which Wg can act is longer in the absence of hh as expected from previous observations. According to the proposed boundary model, the extent of Wg influence is to the first denticle-secreting cell, but not beyond. This finding, along with the discovery that denticles orient toward the source of Wg, may explain why the first row of denticles in wild-type larvae points toward the anterior of the embryo. Only this row of cells receives Wg signal as the segment boundary blocks further action by Wg to the next row of cells. In contrast, Hh can and does affect the next two rows of cells. It was found that expression of Hh causes a rotation away from the source, and could explain why the next two rows of denticles point toward the posterior of the embryo. These results do not explain the final orientation of all rows of denticles, and one likely complication is that in late embryonic stages the Notch and EGFR signaling pathways affect the identities of cells within the denticle belt. It will be interesting to test what effects these signals have on the final orientation of the orientation of denticles, and whether the Notch pathway functions in polarity as well (Colosimo, 2006).

The PCP signaling pathway determines planar polarity in a variety of tissues. In vertebrate and C. elegans studies, Wnts have been implicated in the establishment of polarity, but only one study in Drosophila suggested a role for Wg in PCP (Price, 2006). In fact, the present model excludes the known morphogens, and suggests that PCP is established through cell-cell interactions involving atypical cadherins like Flamingo or through an as yet unidentified factor X. Though this study does not address the function of the known components of PCP signaling in the embryo, it is interesting that mutants in PCP signaling pathway components affect the polarity of the first two rows of denticles. The current findings support the possibility that Wg and Hh lead to the expression of an unknown factor affecting the polarization of denticles, because blocking the transcriptional readout of either Wg or Hh with tcf or ci mutations respectively prevents the polarizing activity of both pathways. This is similar to the PCP disruptions found in the Drosophila eye model for Wg signaling components. The current observations do, however, offer a further possibility, namely that by blocking all Wg signaling with null mutations the underlying polarity organizing function of Wg may be obscured. In the weak armF1A mutant the orientation of denticles can be affected by the expression of Wg without affecting the cell-fates, suggesting that perhaps Wg can affect polarity directly. This effect of Wg was not observed in stronger arm mutant embryos suggesting that Arm protein is required for the Wg effect on denticle orientation. Interestingly, cell culture work has recently implicated Wg in controlling adherens junction strength (Colosimo, 2006).

The use of the embryonic epidermis led to the the interesting possibility that Arm functions in cell polarity. Since some of the molecules involved in the PCP signaling pathway are similar to Cadherins, it seems logical that adhesion is involved in the establishment of polarity. However, adherens junctions have not been implicated so far. This is likely due to the difficulty of working with adherens junction component mutations that are often cell-lethal in the systems that have been used to study PCP. Use of the embryo allows relatively simple perturbation of arm function, and efficient ubiquitous or directional ectopic expression. Unfortunately, the major limitation of the ventral midline expression assay is that it only works for secreted, diffusible ligands. Thus, cell-autonomous activation of Hh or Wg pathway components (such as with activated Arm or Smo) along the ventral midline cannot be observed, since these cells invaginate and do not become a part of the external epidermis (Colosimo, 2006).

The fact that β-catenin is both an oncogene and a component of adherens junctions has led to many studies attempting to link the phosphorylation state of β-catenin in adherens junctions to the epithelial to mesenchymal transition (EMT) in cancer cells and during development. Phosphorylation of tyrosine residues in β-catenin is thought to lead to disassembly of adherens junctions, but recent studies both in vivo and in vitro have challenged this. Certainly these discrepancies will have to be resolved, but this study provides evidence for a different mechanism for regulating junctions, and perhaps EMT, through threonine phosphorylation-based stabilization or dephosphorylation-based destabilization of junctions. It will be crucial to establish which is the regulated step, and whether there are any phosphatases involved in this process in addition to the known kinase CKII (Colosimo, 2006).

Interestingly, the recent findings that alpha-catenin and ß-catenin do not form a stable complex in junctions, suggests a possible explanation for these findings. It is speculated that expression of ArmAA can rescue the basic activity of junctions lost in strong arm mutant embryos, which is to hold a tissue together. However, its reduced affinity for alpha-catenin does not cause a local increase in alpha-catenin levels and therefore Actin levels do not become asymmetric. This leads to a skewing of the normal polarization of the Actin cytoskeleton. It will be crucial to determine how junctions are localized asymmetrically in the first place, and whether this is dependent on extracellular signaling. These findings, and the effects of alpha-catenin mutations on inflammation and tumor progression in the mouse epidermis make analysis of the interaction between alpha- and β-catenin particularly important (Colosimo, 2006).

These experiments provide some of the first evidence that the Hh signaling pathway is involved in polarity. It is particularly interesting that Hh expression leads to the reorganization of Actin structures within epithelial cells, since this suggests that Hh can affect the polarity of the Actin cytoskeleton. This finding is also relevant to cancer biology, because during metastasis, cancer cells lose polarity and essentially ignore their environment. These results show that Wnts and Hh can affect cell polarity, in addition to their well-known effects on cell proliferation. Along with the recent report that TGFβ signaling affects polarity and EMT, these findings imply that this dual role may be a general feature of oncogenic signaling pathways (Colosimo, 2006).

The Drosophila HMG-domain proteins SoxNeuro and Dichaete direct trichome formation via the activation of shavenbaby and the restriction of Wingless pathway activity

Trichomes are cytoplasmic extrusions of epidermal cells. The molecular mechanisms that govern the differentiation of trichome-producing cells are conserved across species as distantly related as mice and flies. Several signaling pathways converge onto the regulation of a conserved target gene, shavenbaby (svb, ovo), which, in turn, stimulates trichome formation. The Drosophila ventral epidermis consists of the segmental alternation of two cell types that produce either naked cuticle or trichomes called denticles. The binary choice to produce naked cuticle or denticles is affected by the transcriptional regulation of svb, which is sufficient to cell-autonomously direct denticle formation. The expression of svb is regulated by the opposing gradients of two signaling molecules - the epidermal growth factor receptor (Egfr) ligand Spitz (Spi), which activates svb expression, and Wingless (Wg), which represses it. It has remained unclear how these opposing signals are integrated to establish a distinct domain of svb expression. This study shows that the expression of the high mobility group (HMG)-domain protein SoxNeuro (SoxN) is activated by Spi, and repressed by Wg, signaling. SoxN is necessary and sufficient to cell-autonomously direct the expression of svb. The closely related protein Dichaete is co-regulated with SoxN and has a partially redundant function in the activation of svb expression. In addition, SoxN and Dichaete function upstream of Wg and antagonize Wg pathway activity. This suggests that the expression of svb in a discreet domain is resolved at the level of SoxN and Dichaete (Overton, 2007).

In the embryonic ventral epidermis of Drosophila, two alternative cell fates are specified: smooth cells and trichome-producing cells. These binary cell fates are distinguished by the expression of svb, the most-downstream effector of epidermal morphogenesis. svb is necessary and sufficient to cell-autonomously direct trichome formation. The expression of svb is regulated by the opposing gradients of two signaling molecules: Spi, which activates, and Wg, which represses, svb expression. svb is expressed in segmentally reiterated, epidermal stripes, which invariantly encompass six rows of cells. This raises the question of how is opposing extrinsic information integrated to establish a distinct domain of svb expression with a sharp posterior border (Overton, 2007)?

This study demonstrates that the HMG-domain proteins SoxN and Dichaete represent a molecular link between the expression of svb and the upstream Der- and Wg-signaling cascades. SoxN and Dichaete are expressed in the ventral epidermis at the time when epidermal cell fates are specified. The late phase of SoxN and Dichaete expression is stimulated by Der- and repressed by Wg-pathway activity. These regulatory mechanisms result in the expression of SoxN and Dichaete in those six rows of cells within each abdominal segment that differentiate to produce trichomes. SoxN and, to a lesser extent, Dichaete, are necessary and sufficient to activate the expression of svb. Furthermore, these results show that the well-described repression of svb by Wg is due to the repression of SoxN, which, in turn, results in the loss of svb activation. Likewise, the Spi-mediated activation of svb expression relies on the activation of SoxN, which, in turn, activates svb. This indicates that the competition of Der- and Wg-pathway activities for the specification of trichome-producing versus smooth cell fates is resolved at the level of SoxN and Dichaete (Overton, 2007).

These results do not provide much insight into the issue of how opposing extrinsic information is integrated such that a sharp posterior border of svb expression is achieved. Instead, they raise the question of how is a sharp posterior border of SoxN and Dichaete expression established/maintained? The findings suggest that this is achieved by a combination of negative- and positive-feedback loops. (1) Evidence is provided that SoxN and Dichaete negatively regulate Wg pathway activity. This negative-feedback loop provides a likely mechanism for the establishment and maintenance of a sharp posterior border of SoxN and Dichaete expression. The issue arises of how robust this system might be in the face of fluctuating levels of Wg pathway activity. The efficiency with which SoxN and Dichaete restrict Wg pathway activity will crucially rely on the levels of SoxN and Dichaete protein. In this context, it is noteworthy that the levels of SoxN protein, but not Dichaete, are several-fold higher in the two posterior-most rows of the SoxN stripe compared with the anterior four rows. The regulatory mechanisms that underlie the different levels of SoxN expression are currently unclear. (2) Evidence is provided that the maintenance of SoxN and Dichaete expression is supported by a positive-feedback loop: svb, the expression of which is activated by SoxN and Dichaete, is itself required for the maintenance of SoxN and Dichaete expression. Together, these mechanisms contribute to an invariant read-out of cell identity from opposing Der- and Wg-pathway activities (Overton, 2007).

In Drosophila, SoxN and Dichaete are necessary and sufficient to activate the expression of svb, which in turn directly regulates the expression of genes involved in trichome morphogenesis. Is a function in hair formation of the Sox proteins conserved in other species, including vertebrates? A previous study has shown that the mouse Sox9 protein is required for the differentiation of hair-producing epidermal cells and acts genetically downstream of sonic hedgehog pathway activity (Vidal, 2005). This study did not address whether Sox9 regulates the expression of movo1 (Ovol1), the mouse ortholog of svb. Nevertheless, the demonstrated roles of SoxN, Dichaete and Sox9 raise the exciting question of do Sox proteins have an essential function in the activation of an epidermal differentiation program that is conserved across species as distantly related as mice and flies (Overton, 2007).

Wingless and Engrailed expression in the brain

The insect brain is traditionally subdivided into the trito-, deuto- and protocerebrum. However, both the neuromeric status and the course of the borders between these regions are unclear. The Drosophila embryonic brain develops from the procephalic neurogenic region of the ectoderm, which gives rise to a bilaterally symmetrical array of about 100 neuronal precursor cells, called neuroblasts. Based on a detailed description of the spatiotemporal development of the entire population of embryonic brain neuroblasts, a comprehensive analysis was carried out of the expression of segment polarity genes (engrailed, wingless, hedgehog, gooseberry distal, mirror) and DV patterning genes (muscle segment homeobox, intermediate neuroblast defective, ventral nervous system defective) in the procephalic neuroectoderm and the neuroblast layer (until stage 11, when all neuroblasts are formed). The data provide new insight into the segmental organization of the procephalic neuroectodem and evolving brain. The expression patterns allow the drawing of clear demarcations between trito-, deuto- and protocerebrum at the level of identified neuroblasts. Furthermore, evidence is provided indicating that the protocerebrum (most anterior part of the brain) is composed of two neuromeres that belong to the ocular and labral segment, respectively. These protocerebral neuromeres are much more derived compared with the trito- and deuto-cerebrum. The labral neuromere is confined to the posterior segmental compartment. Finally, similarities in the expression of DV patterning genes between the Drosophila and vertebrate brains are discussed (Urbach, 2003).

In the trunk neuroectoderm, segment-polarity genes are expressed in stereotypical segmental stripes, and in NBs that delaminate from these domains, subdividing each neuromere along the AP axis. In the pregnathal head region the expression domains of segment polarity genes are less obvious, but analysis of engrailed and wingless expression in the head peripheral ectoderm, and of PNS mutant phenotypes, support the existence of four pregnathal segments in Drosophila: the intercalary, antennal, ocular and labral segments (from posterior to anterior). However, the identity and organization of brain structures deriving from these segments is still obscure. In order to obtain evidence concerning the number and extent of the brain neuromeres, and to map the position of their boundaries, the expression of segment polarity genes, including wingless, hedgehog, gooseberry-distal, engrailed, invected and mirror, was analyzed. The spatiotemporal pattern of their expression was traced in the neuroectoderm and in the NB-layer until stage 11, when all brain NBs are formed. The data show that segmental expression is retained for most of the investigated segment polarity genes in both the developing head ectoderm (procephalon) and brain NBs, providing landmarks for the definition of segmental domains within the developing brain NB pattern (Urbach, 2003).

engrailed (en) expression domains in the trunk define the posterior segmental compartments, from which NBs of row 6 and 7 and NB1-2 derive. In the pregnathal head en expression was found as follows: from late stage 8 in the posterior ectoderm of the antennal segment (en antennal stripe; en as) from which four deutocerebral NBs (Dv8, Dd5, Dd9, Dd13) delaminate; from stage 9 in a small ectodermal domain in the posterior part of the ocular segment, the en head spot (en hs), from which two protocerebral NBs (Ppd5, Ppd8) evolve; and from stage 10 in an ectodermal stripe in the posterior intercalary segment (en intercalary stripe; en is), which gives rise to three to four tritocerebral NBs (Tv4, Tv5, Td3, Td5). Furthermore, from stage 11 onwards, En is weakly detected in the anteriormost ectoderm of the procephalon corresponding to the region of the 'anterior dorsal hemispheres' (en dh). About 10 weakly En-positive NBs were identified that delaminate from the en dh. Thus all four pregnathal head segments contribute to the early embryonic brain. The spatial distribution of the En-positive NBs closely corresponds to the en domains of their origin in the ectoderm. This suggests they demarcate the posterior borders of the respective brain neuromeres (Urbach, 2003).

In the trunk, hedgehog (hh) matches en expression. This is also the case for the intercalary segment in the pregnathal head ectoderm. By contrast, the En-positive antennal stripe and head spot are only subfractions of the large hh-lacZ domain, which, between stages 9 and 10, encompasses the antennal segment and the posterior part of the ocular segment. All NBs delaminating from this domain express hh-lacZ. From stage 10 onwards, en expressing NBs maintain a strong hh-lacZ signal, whereas hh-lacZ subsequently diminishes in the neuroectoderm and in NBs between the en antennal stripe and head spot. Additionally, hh-lacZ-expressing NBs positioned dorsally to the en/hh-lacZ-co-expressing Ppd5 and Ppd8 (both NBs demarcating part of the posterior border of the ocular neuromere), appear to prolong the boundary between the deuto- and proto-cerebrum in the dorsal direction (Urbach, 2003).

From late stage 8 onwards, Wingless (Wg) protein is expressed in a neuroectodermal domain spanning a broad area of the ocular and the anterior antennal segment (and in the invaginating foregut). This becomes clearer in En/Wg double labelling at stage 9, revealing that the en hs is localized within this Wg domain. At that stage, Wg is already detectable in about 4-5 protocerebral NBs (Pcd6, Pcd15, Pcd7, Ppd3), derived from the region with strongest Wg expression (which later corresponds to the wg head blob). Furthermore, Wg is faintly expressed in the deutocerebral Dd7 emerging from the antennal part of the Wg domain, which corresponds to the later wg antennal stripe. By stage 10, when the wg head blob is clearly distinguishable from the wg antennal stripe, about 10-12 Wg-positive NBs have emerged from this domain. In addition, a small, spot-like wg domain was found in the intercalary segment from which a single NB (Td4) delaminates. Thus, all three wg domains, the intercalary, antennal and ocular (head blob), contribute to the anlage of the brain. From late stage 9 an additional wg domain is visible in the ectodermal anlage of the clypeolabrum, which is the wg counterpart to the En/Inv-positive region in the 'dorsal hemispheres'. Upon double labelling for either asense or deadpan (both are general markers for neural precursor cells) and wg, in embryos between stage 9 and 11 no NB emerging from the wg labral spot could be detected. By stage 11 the number of wg expressing NBs originating from the ocular head blob has increased to about 16-20, which is more than 25% of the total number of identified protocerebral NBs. Three Wg-positive NBs are identified in the deutocerebrum and one in the tritocerebrum (Urbach, 2003).

Wnt controls the medial-lateral subdivision of the Drosophila head

In insects, the subdivision of the head into a lateral region, harbouring the compound eyes (CEs), and a dorsal (medial) region, where the ocelli localize, is conserved. This organization might have been already present in the insects' euarthropodan ancestors. In Drosophila, the Wnt-1 homologue wingless (wg) plays a major role in the genetic subdivision of the head. To analyse specifically the role of wg signalling in the development of the dorsal head, this pathway was attenuated specifically in this region by genetic means. Loss of wg signalling transforms the dorsal/medial head into lateral head structures, including the development of ectopic CEs. This genetic analysis further suggests that wg signalling organizes the dorsal head medial-lateral axis by controlling, at least in part, the expression domains of the transcription factors Otd and Ey/Pax6 (Magri, 2018).

Wingless function in heart development

wingless function is specifically required for heart development. A temperature-sensitive mutation of wg has been used to inactivate wg function during precise developmental time periods. Elimination of wg function for a short time period after gastrulation results in the selective loss of heart precursors, without significantly affecting the formation of the body wall or visceral muscles, although some pattern defects are observed. This developmental requirement of wg for cardiac organogenesis is distinct from its function in segmentation and neurogenesis (Wu, 1995).

The embryonic dorsal vessel in Drosophila possesses anteroposterior polarity and is subdivided into two chamber-like portions, the aorta in the anterior and the heart in the posterior. The heart portion features a wider bore as compared with the aorta and develops inflow valves (ostia) that allow the pumping of hemolymph from posterior toward the anterior. Homeotic selector genes provide positional information that determines the anteroposterior subdivision of the dorsal vessel. Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) are expressed in distinct domains along the anteroposterior axis within the dorsal vessel, and, in particular, the domain of abd-A expression in cardioblasts and pericardial cells coincides with the heart portion. Evidence is provided that loss of abd-A function causes a transformation of the heart into aorta, whereas ectopic expression of abd-A in more anterior cardioblasts causes the aorta to assume heart-like features. These observations suggest that the spatially restricted expression and activity of abd-A determine heart identities in cells of the posterior portion of the dorsal vessel. Abd-B, which at earlier stages is expressed posteriorly to the cardiogenic mesoderm, represses cardiogenesis. In light of the developmental and morphological similarities between the Drosophila dorsal vessel and the primitive heart tube in early vertebrate embryos, these data suggest that Hox genes may also provide important anteroposterior cues during chamber specification in the developing vertebrate heart (Lo, 2002).

Since abd-A expression coincides with the heart portion of the dorsal vessel, tests were made to see whether it acts to specify the cardioblasts in which it is expressed to eventually form the heart. In order to distinguish aorta cardioblasts from heart cardioblasts, two different molecular markers were utilized. The first marker was the pattern of ß-Gal derived from the tinCdelta5-lacZ transgene, where the expression of a lacZ gene is controlled by an internally deleted tinman cardiac enhancer element, tinCdelta5. This element drives ß-Gal expression in all the cardioblasts of the aorta, whereas in the heart it is only expressed in three segmentally-spaced double pairs of cardioblasts. These particular cardioblasts correspond to the svp cardioblasts of the heart. The second marker is wingless (wg), which is expressed in these same three double pairs of svp cardioblasts within the heart of the late embryonic dorsal vessel (Lo, 2002).

In abd-A null mutant embryos, the pattern of tinCdelta5-lacZ-derived ß-Gal is continuous in the heart as well as in the aorta of the dorsal vessel. In addition, it appears that the width of the heart is now the same as that of the aorta when compared with a wildtype embryonic dorsal vessel. Similarly, the late expression of Wg in the svp cardioblasts of the heart is not detectable in these mutant embryos. The alterations in the pattern of these two markers strongly suggest that heart cardioblasts have not been specified in the posterior of the dorsal vessel of abd-A null mutant embryos and that these posterior cardioblasts have been transformed instead into aorta cardioblasts. This would indicate that abd-A is necessary for the specification of heart cardioblasts in the posterior portion of the dorsal vessel where it is normally expressed (Lo, 2002).

The pattern of Wg expression in three segmentally repeated double pairs of cardioblasts within the late stage heart is strongly reminiscent of the pattern of svp expression, which suggests that these are the heart svp cardioblasts. Double antibody staining for Wg and ß-Gal in the dorsal vessel of svp-lacZ embryos clearly confirmed that the heart svp cardioblasts express the Wg protein. Since the heart svp cardioblasts eventually form the ostia (inflow valves) of the larval heart and since Wg is a developmentally significant signaling molecule, the regulation of Wg expression in the heart svp cardioblasts during late embryogenesis was more closely examined (Lo, 2002).

Wg expression in heart cardioblasts is dependent on abd-A. Since these Wg-expressing cardioblasts correspond to svp cardioblasts, whether Wg expression is also dependent on svp function was also tested. In homozygous null svpAE127 mutant embryos, there is no detectable Wg expression in the heart cardioblasts that are marked by svp-lacZ. Therefore, the Wg expression seen in the heart svp cardioblasts of late embryonic dorsal vessels requires both abd-A and svp function. Accordingly, ectopic expression of SvpI in the cardioblasts of the entire dorsal vessel results in wg expression in all cardioblasts of the heart, and ectopic expression of both SvpI and Abd-A in the whole dorsal vessel causes Wg expression in the majority of the cardioblasts of the entire dorsal vessel. These results demonstrate that the combination of abd-A and svp is both necessary and sufficient to activate wg expression in cardioblasts during late dorsal vessel development (Lo, 2002).

Since Wg expression in heart svp cardioblasts initiates toward the end of embryogenesis (stage 16), tests were made to see whether this expression could also be detected in the dorsal vessel during later larval stages when the corresponding cells have formed the ostia. Because of high levels of unspecific background staining with Wg antibodies in larval preparations, wg expression in the dorsal vessel of third instar larvae was indirectly monitored by anti-ß-Gal staining of dissected wg-lacZ animals. Moderate levels of wg-lacZ-derived ß-Gal can indeed be detected in the ostia of the heart, although stronger levels are now present in four separated patches in the aorta that correspond to Tin-negative svp cardioblasts. While this pattern of expression differs from that seen in the late embryonic dorsal vessel, it is clear that wg is expressed differentially and in a temporally regulated manner within the heart and aorta, respectively, of late stage embryos and third instar larvae. These observations suggest a yet undefined role for the signaling molecule in larval dorsal vessel development and/or functioning (Lo, 2002).

The target genes of abd-A that are required for generating functional ostia and for the other heart cells to adopt their characteristic morphology are not yet known. Based on its ostia-specific expression in late stage embryos, wg is a candidate target of abd-A that may function either in an autocrine fashion during ostia differentiation or in a paracrine fashion during the differentiation of the adjacent heart cardioblasts. The activation of the wg gene in the svp cells of the aorta during third instar also precedes ostia formation, in this case of the adult ostia, from these cells. Hence, there is a strong correlation between the initiation of wg expression in svp cardioblasts and their subsequent differentiation into functional ostia (Lo, 2002).

Evidence for a fruit fly hemangioblast and similarities between lymph-gland hematopoiesis in fruit fly and mammal aorta-gonadal-mesonephros mesoderm

The Drosophila lymph gland is a hematopoietic organ and, together with prospective vascular cells (cardioblasts) and excretory cells (pericardial nephrocytes), arises from the cardiogenic mesoderm. Clonal analysis provided evidence for a hemangioblast that can give rise to two daughter cells: one that differentiates into heart or aorta and another that differentiates into blood. In addition, the GATA factor gene pannier (pnr) and the homeobox gene tinman (tin), which are controlled by the convergence of Decapentaplegic (Dpp), fibroblast growth factor (FGF), Wingless (Wg) and Notch signaling, are required for the development of all cardiogenic mesoderm, including the lymph gland. An essential genetic switch differentiates between the blood or nephrocyte and vascular lineages involves the Notch pathway. Further specification occurs through specific expression of the GATA factor Serpent (Srp) in the lymph-gland primordium. These findings suggest that there is a close parallel between the molecular mechanisms functioning in the Drosophila cardiogenic mesoderm and those functioning in the mammalian aorta-gonadal-mesonephros mesoderm (Mandal, 2004).

Blood and vascular cells in the vertebrate embryo are thought to derive from oligopotent progenitor cells, called hemangioblasts, that arise in the yolk sac and in the aorta-gonadal-mesonephros (AGM) mesenchyme. A close relationship between blood and vascular progenitors is well established, but in vivo evidence that a single cell can divide to produce a blood cell and an endothelial cell is lacking in vertebrate systems. Similarly, the molecular mechanism that distinguishes between the two lineages is not well understood. To address these issues in a simple, genetically amenable system, the genetic control of hematopoiesis was analyzed in Drosophila. The results show that there are close lineage relationships between hematopoietic and vascular cells, similar to those present in the AGM of mammalian systems. Evidence is provided for conserved cassettes of transcription factors and signaling cascades that limit the pool of hemangioblastic cells and promote the blood versus vascular fate (Mandal, 2004).

In the mature Drosophila embryo, the lymph gland is formed by a paired cluster of ~20 cells flanking the aorta. The aorta and heart represent a contractile tube lined by a layer of myoepithelial vascular cells called cardioblasts. The cells flanking the aorta and heart posterior to the lymph gland are the pericardial cells, which function as excretory cells (nephrocytes). Lymph gland progenitors express the prohemocyte marker Srp and ultrastructurally resemble prohemocytes that develop at an earlier stage from the head mesoderm. Monitoring expression of the zinc-finger protein Odd-skipped (Odd) shows that the lymph gland originates from the dorsal thoracic mesoderm. Odd is expressed in segmental clusters in the dorsal mesoderm of segments T1-A6. The three thoracic Odd-positive clusters coalesce to form the lymph gland, whereas the abdominal clusters formed the pericardial nephrocytes (Mandal, 2004).

Lymph-gland progenitors, cardioblasts and pericardial cells are closely related by lineage. Labeled 'flipout' (FLP/FRT) clones were induced in embryos aged 3-4 h such that the clones contained only 2-4 cells. Of the two-cell clones, ~50% contained cardioblast and lymph-gland cells; the other clones comprised either cardioblasts or lymph-gland cells alone. Mixed clones were recovered at the late third larval stage. The finding of mixed clones indicates that the cardiogenic mesoderm of D. melanogaster contains oligopotent progenitors that, up to the final division, can give rise both to Srp-positive blood-cell progenitors that form the lymph gland and to vascular cells (Mandal, 2004).

The cardiogenic mesoderm forms part of the dorsal mesoderm, which requires the homeobox protein Tin and the GATA factor Pnr. In embryos with mutations in tin or pnr, the lymph gland was absent. Maintenance of Tin expression in the dorsal mesoderm requires the activity of at least two signaling pathways regulated by Dpp (the Drosophila homolog of transforming growth factor-ß) and Heartless (Htl; one of the D. melanogaster homologs of the FGF receptor); the dependence of cardioblast and pericardial nephrocyte development on these signaling pathways has been documented. Lymph-gland progenitors did not develop in loss-of-function dpp and htl mutants (Mandal, 2004).

Between 6 h and 8 h of development, the dorsal mesoderm splits into the cardiogenic mesoderm and the visceral mesoderm. The cardiogenic mesoderm is regulated positively by Wg and negatively by Notch. Lack of Wg signaling results in the absence of all cardiogenic lineages including lymph gland. Notch signaling has the opposite effect and restricts cardiogenic mesodermal fate. Notch is active in the dorsal mesoderm from 6 h to 10 h of development. Eliminating Notch during the first half of this interval by raising embryos homozygous with respect to the temperature-sensitive allele Nts1 at the restrictive temperature resulted in substantially more cardioblasts, pericardial cells and lymph-gland progenitors (Mandal, 2004).

Lymph-gland progenitors, cardioblasts and pericardial nephrocytes are specified in the cardiogenic mesoderm around the phase of germband retraction 8-10 h after fertilization. At this stage, Tin, which was initially expressed in the whole cardiogenic mesoderm, becomes restricted to a narrow medial compartment containing the cardioblasts. Pnr follows the same restriction. Cells located at a more lateral level in the cardiogenic mesoderm give rise to lymph-gland progenitors (in the thoracic domain) and pericardial nephrocytes (in the abdominal domain) and activate the gene odd. Slightly later, Srp is expressed in lymph-gland progenitors. As reported for the early hemocytes derived from the embryonic head, srp is centrally involved in lymph-gland specification. In srp-null embryos, Odd-expressing cells still formed a lymph gland-shaped cluster flanking the aorta, but these cells also express the pericardial marker pericardin (Prc), suggesting that they lose some aspects of hemocyte precursor identity or gain properties of nephrocytes. As a countercorrelate, ectopic expression of Srp in the whole cardiogenic mesoderm directed by mef2-Gal4 induces pericardial cells to adopt lymph-gland fate (Mandal, 2004).

Downregulation of tin and pnr in cells in the lateral domain of the cardiogenic mesoderm is essential for lymph-gland specification. Ectopic expression of tin or pnr by twist-Gal4 (or mef2-Gal4) causes a marked reduction in the number of lymph-gland and pericardial cells. The antagonistic effect of tin on lymph-gland progenitors resembles its earlier role in the head mesoderm that gives rise to the larval blood cells; here too, ectopic expression of tin causes a reduction in the number of hemocytes (Mandal, 2004).

Inhibiting tin and upregulating odd and srp requires input from the Notch signaling pathway. A function of Notch at 6-8 h in specification of the cardiogenic mesoderm is described. Reducing Notch function between 8 h and 10 h causes an increase in the number of cardioblasts and a concomitant loss of pericardial and lymph-gland cells. Overexpressing an activated Notch construct causes a marked increase in lymph-gland size. This late requirement for Notch signaling is separable from the earlier role of Notch in restricting the overall size of the cardiogenic mesoderm. Thus, the sum total of cardioblasts and pericardial or lymph-gland cells in Nts1 embryos shifts between 8 h and 10 h and does not differ substantially from that in wild type, whereas a combined effect on cell number and cell fate is seen in embryos with a Notch deletion. In these embryos, the cardiogenic mesoderm is hyperplasic and develops as cardioblasts at the expense of lymph-gland progenitors and pericardial nephrocytes. The dual role of Notch in restricting the numbers of a pluripotent progenitor pool and in distinguishing between the progeny of these progenitors is reminiscent of the function of Notch in sense-organ development (Mandal, 2004).

Lymph-gland formation is restricted to the thoracic region by positional cues that are provided by expression of the homeobox proteins of the Antennapedia and Bithorax complex. Specifically, Ultrabithorax (Ubx), which is expressed in segments A2-A5 of the cardiogenic mesoderm, inhibits lymph-gland formation. Loss of Ubx results in the expansion of the lymph-gland fate into the abdominal segments. Conversely, overexpression of Ubx driven by mef2-Gal4 causes the transformation of lymph-gland progenitors into pericardial nephrocytes (Mandal, 2004).

These findings are suggestive of a model of lymph-gland development in Drosophila that is similar to mammalian hematopoiesis. Lymph-gland progenitors develop as part of the cardiogenic mesoderm that also gives rise to the vascular cells (aorta and heart) and to excretory cells. Similarly, progenitor cells of the blood, aorta and excretory system are closely related both molecularly and developmentally in mammals, where they form part of the AGM. Specification of the cardiogenic mesoderm requires the input of FGF and Wg signaling, as in vertebrate hematopoiesis, where the AGM region is induced in response to several converging signaling pathways including FGF, BMP and Wnt (Mandal, 2004).

The cardiogenic mesoderm in Drosophila evolves from the dorsal mesoderm and requires input from the Htl, Dpp, Wg and Notch (N) signaling pathways. The cardiogenic mesoderm then differentiates into lymph gland, vascular cells (cardioblasts) and excretory cells (pericardial nephrocytes). A subpopulation of cardioblasts and lymph-gland cells is derived from one progenitor (hemangioblast; HB). Essential for the differentiation of the cardiogenic mesoderm is the Notch-Delta (Dl)-dependent restriction of Tin and Pnr to cardioblasts and the expression of Srp in the lymph gland. In vertebrates, similar cell types are derived from a mesodermal domain called the AGM, which also requires the input of FGF, BMP and Wnt signaling. A subset of AGM-derived cells has been proposed to constitute hemangioblasts, which produce blood progenitors and endothelial cells (Mandal, 2004).

These findings show that in Drosophila, the cardiovascular and blood-cell lineages are differentiated by an antagonistic relationship between Tin or Pnr expression in the cardioblasts and Srp expression in the lymph-gland progenitors. In vertebrates, GATA factors also have a pivotal role in specifying different lineages among blood-cell progenitors, although not much is known about what differentiates between blood progenitors as a group and endothelial progenitors. The results indicate that this step is driven by input from the Notch signaling pathway. In the thoracic cardiogenic mesoderm, Notch antagonizes tin and pnr expression and aortic cardioblast formation, and promotes srp expression and the development of lymph-gland progenitors. In vertebrates, Notch signaling is also involved in both blood and vascular development. The role of Notch during AGM morphogenesis remains to be investigated (Mandal, 2004).

Cardioblasts and lymph-gland cells can arise from the division of a single cardiogenic mesodermal cell, which should be called a hemangioblast. A previous study induced clones in the cardiogenic mesoderm but used only Tin as a marker. This study also yielded mixed two-cell clones comprising a cardioblast and a nonlabeled cell, which, in light of the current findings, must be interpreted as a lymph-gland cell. Hemangioblasts have been proposed in vertebrates, although the definitive experiment in which a precursor is marked and its lineage is tracked has not been done. Blast colony-forming cells that give rise to both lineages in vitro and common markers that belong to both cell types in vivo have been identified, but direct evidence for the existence of a common precursor has not yet been found. This study, using genetic analysis of two-cell clones, establishes the existence of such a population in Drosophila. On the basis of these results, and given the conservation of the signaling and transcriptional components described here, the prediction is that many cells of the AGM in vertebrates may give rise to only blood or only vascular cells, but a number of intermixed hemangioblasts may give rise to mixed lineages. Future genetic screens aimed at finding components in early lymph-gland development will probably identify additional pathways and strategies important for vertebrate hematopoiesis (Mandal, 2004).

Direct sensing of systemic and nutritional signals by haematopoietic progenitors in Drosophila

The Drosophila lymph gland is a haematopoietic organ in which progenitor cells, which are most akin to the common myeloid progenitor in mammals, proliferate and differentiate into three types of mature cell -- plasmatocytes, crystal cells and lamellocytes -- the functions of which are reminiscent of mammalian myeloid cells. During the first and early second instars of larval development, the lymph gland contains only progenitors, whereas in the third instar, a medial region of the primary lobe of the lymph gland called the medullary zone contains these progenitors, and maturing blood cells are found juxtaposed in a peripheral region designated the cortical zone. A third group of cells referred to as the posterior signalling centre functions as a haematopoietic niche. Similarly to mammalian myeloid cells, Drosophila blood cells respond to multiple stresses including hypoxia, infection and oxidative stress. However, how systemic signals are sensed by myeloid progenitors to regulate cell-fate determination has not been well described. This study shows that the haematopoietic progenitors of Drosophila are direct targets of systemic (insulin) and nutritional (essential amino acid) signals, and that these systemic signals maintain the progenitors by promoting Wingless (WNT in mammals) signalling. It is expected that this study will promote investigation of such possible direct signal sensing mechanisms by mammalian myeloid progenitors (Shim, 2012).

It is known that metabolic dysfunction in mammals causes abnormal inflammatory responses in the blood system. However, how metabolic stresses impinge on haematopoiesis is still unclear. This study found that starvation of Drosophila larvae leads to blood cell phenotypes. The most striking effect is acceleration of blood cell differentiation both in time and number of cells affected in the lymph gland. Following 24h of starvation, cells occupying the medullary zone begin to express differentiation markers such as Peroxidasin (Hml) normally restricted to the cortical zone. Corresponding to this increase, a substantial reduction of Domeless (Dome) marking the progenitor population is also evident. The protein Eater, normally expressed at very low levels in the progenitors and at high levels in differentiated cells, is expressed at high levels in all cells on starvation (Shim, 2012).

The starvation experiments were carried out on either PBS-soaked Whatman paper or a 1% agar plate. Aseptic conditions to control against indirect effects due to bacterial infection were also used. In all controlled experimental conditions, starvation reduced the progenitor population and caused an increase in the number of differentiating cells, without an obvious alteration in the size of the haematopoietic organ, or the apoptotic profile of its cells (Shim, 2012).

Similarly to metabolically induced inflammation in mammals, starvation in Drosophila larvae activates NFkappaB-like transcription factors, assessed by the expression of the reporter D4–LacZ and antimicrobial peptides in circulating haemocytes and within the lymph gland. Starvation also causes an increase in the number of circulating blood cells arising from the embryonic head mesoderm, infiltration of Pxn+ plasmatocytes into the fat body, the Drosophila equivalent of the mammalian liver and adipose tissue, and differentiation of lamellocytes, another hallmark of inflammatory response, in both the lymph gland and in the circulating blood cell population. Finally, starvation induces the rupture of crystal cells, a process known to release coagulation and melanization enzymes. This rupture depends on JNK signalling. Thus, starvation alters the homeostatic balance between progenitors and differentiating blood cells through extensive progenitor differentiation, and also activates mature blood cells in a manner that is reminiscent of mammalian inflammatory response (Shim, 2012).

In Drosophila, the systemic level of glucose is regulated by insulin-like peptides (Dilps) that are produced and secreted by neuroendocrine cells in the brain, much like insulin production by pancreatic β-cells of mammals. As in mammals, insulin signalling in Drosophila plays a conserved role in regulating metabolism and growth, and the levels of nutrients, such as amino acids, regulate secretion of Dilps. This study finds the effects of starvation on Drosophila blood particularly interesting given the connection between myeloid cell function and insulin signalling in human metabolic diseases. The mechanisms are delineated by which a systemic signal, namely insulin signalling, controls maintenance and differentiation of progenitors in the haematopoietic organ (Shim, 2012).

The insulin-producing cells (IPCs) were specifically ablated by inducing cell death with the expression of the pro-apoptotic genes hid and rpr, and robust differentiation of blood cells was found in the lymph gland similar to that seen on starvation. Although several larval dilp genes, including dilp2, 3 and 5, are produced by IPCs, further analysis showed that deficiency mutants containing a dilp2 lesion or a specific deletion of the dilp2 gene using the null dilp2 mutant allele cause blood cell differentiation. Depletion of any of the other dilp genes, including dilp6 or 7, does not cause this phenotype. No Dilp2 expression was detected in the lymph gland cells, and it is proposed that the ligand source is the IPC neurons in the brain. Consistent with previous findings, it was found that starvation blocks Dilp2 release from the IPCs. Furthermore, forced depolarization of the IPCs by expressing the bacterially derived voltage-gated sodium channel (NaChBac), which will cause an increase in Dilp secretion, suppresses blood cell differentiation under both well-fed and starved conditions. Finally, overexpression of Dilp2 using the neuronal driver elav–Gal4 causes suppression of differentiation of mature blood cells. Taken together, it is concluded that Dilp2 expression from the IPC neurons is essential for progenitor maintenance and loss of Dilp2 release during starvation results in excessive differentiation of blood cells (Shim, 2012).

The loss-of-function, heteroallelic combination InRE19/InRGC25 for the Drosophila insulin receptor (InR) is viable and larvae from this genotype also exhibit extensive differentiation of the progenitor population. As Dilp2 is a secreted protein, its target receptor could, in principle, be functional in any tissue that then, in turn, signals to the haematopoietic organ using a secondary pathway. However, disrupting InR function directly in the lymph gland with the use of the lymph-gland-specific driverHHLT-Gal4 was found to cause precocious differentiation of the progenitors from an earlier stage in development than is seen in wild type. As HHLT–Gal4 is also expressed in the heart (dorsal vessel), the possible involvement of cardiogenic cells was examined by disrupting InR using the heart-specific driver Mef2–Gal4; this does not induce abnormal differentiation in the lymph gland, indicating that the HHLT–Gal4-driven phenotype is due to its expression in the haematopoietic system. Within the lymph gland, downregulation of InR in the progenitor cell population (using dome–Gal4) causes their robust differentiation, whereas loss of InR in the already differentiating cells of the cortical zone (using hml–Gal4) or the niche cells of the posterior signalling centre (using antp–Gal4) does not affect the progenitor population. Consistent with these findings, high levels of InR transcript were detected in the progenitors, and mutant clones of InR (InRE19/InRE19) within the medullary zone region induce precocious differentiation. Furthermore, downregulation of chico, a downstream effector of insulin receptor signalling, in the progenitor population recapitulates the InRRNAi phenotype, whereas activation of PI(3)K kinase inhibits differentiation of blood cells. These results establish that the haematopoietic progenitor directly responds to brain IPC-derived Dilp2 by activating InR signalling, which serves to maintain the progenitor cell population within the lymph gland (Shim, 2012).

Next the function of AKT, which acts downstream of InR as a protein kinase, was examined by downregulating its expression in the progenitors, and this too promotes progenitor differentiation, identical to loss of InR and dilp2. Likewise, loss of the TORC1 components dTOR (using the dominant-negative mutant protein dTORDN or Raptor (using RNA-mediated interference (RNAi), which together function downstream of AKT, causes loss of progenitors due to their differentiation. Feeding rapamycin, which blocks dTOR function also phenocopies this effect. Consistent with a role for this pathway in progenitor maintenance, overexpression of rheb, which activates dTOR, strikingly inhibits differentiation of blood cells under both normally fed and starved conditions. Interestingly, mammalian haematopoietic stem cells also respond to mTOR signalling. Overall, it is evident that the canonical Dilp–InR and Rheb–dTOR signalling pathways play a critical role in the maintenance of haematopoietic progenitors, and this maintenance role is overridden during metabolic stress caused by starvation (Shim, 2012).

Dilp2 levels rise during early instars and then gradually decrease during the third instar of larval development, indicating a possible mechanism for maintaining InR signalling through the third instar in well-fed larvae. To determine whether InR signalling is modulated during normal development, levels of phospho-AKT (pAKT) were assessed at different developmental stages in the lymph gland. Using this approach, two distinct phenomena were found. First, pAKT expression in progenitor cells is high during the second instar and gradually decreases in these cells during the third instar. Second, pAKT is low, relative to progenitor levels, in differentiating cells at all stages when they are present. These observations indicate that during the course of normal development, InR signalling is modulated in progenitors, thereby differentially promoting maintenance at different stages. It is also apparent that once cells are committed to differentiate, little, if any, InR signalling occurs, consistent with lower levels of InR expression and the lack of a phenotype associated with InR loss-of-function in these cells (Shim, 2012).

In mammals, glucose levels control insulin secretion. This is less clear in Drosophila, but it is well established that amino-acid levels are sensed by the fat body through the mediation of the amino-acid transporter protein Slimfast (Slif) and that the fat body indirectly controls insulin secretion from the brain IPCs. As expected, it was found that slifAnti expressed in the fat body mimics the starvation phenotype in the lymph gland, probably owing to decreased Dilp2 secretion from the brain. More interestingly, however, knocking down slif expression directly in the lymph gland, but not in the dorsal vessel, and specifically in the progenitor population within the lymph gland, accelerates differentiation of mature cells similar to that seen with starvation. As with insulin signalling, this result shows that the haematopoietic progenitors themselves directly sense amino-acid levels to maintain their stem-like fate. Taken together, these findings indicate a dual control of haematopoietic homeostasis by systemic levels of insulin and amino acids. Amino acids are sensed by the fat body, which then controls insulin secretion from the brain. Insulin is then directly sensed by the blood progenitors. Amino acids are also directly sensed by the blood progenitors to maintain their undifferentiated state (Shim, 2012).

Supplementation of essential amino acids (EAAs) partially restores the progenitor population during an otherwise starved condition, whereas neither sucrose nor non-essential amino acid (NEAA) supplementation rescues the progenitors from differentiation. Loss of slif in the lymph gland prevents progenitor maintenance despite EAA supplementation, further establishing that the progenitors directly sense EAA and use the signal to promote their maintenance (Shim, 2012).

In wild-type lymph glands, the progenitors (expressing Dome; Dome+) rarely overlap with the maturing cells (expressing Pxn; Pxn+); however, downregulation of InR or expression of dominant-negative TOR (TORDN) in the progenitors causes a significant increase in the number of double-positive cells (Dome+ and Pxn+) that are in transition towards differentiation. An increase in this particular cell type is reminiscent of the phenotype seen on downregulation of the wingless (wg) signalling pathway, which has previously been linked to the process of progenitor maintenance. Wingless (Wg) is dynamically expressed in the lymph gland with higher levels at earlier stages that then decrease during the third instar. Wg is expressed at high levels by progenitors at these stages and is withdrawn from differentiating cells, which is reminiscent of pAKT staining patterns and the expression of InR in the third instar. Downregulation of InR or Slif in the progenitors was found to causes a significant decrease in Wg expression, whereas Rheb overexpression significantly increases Wg levels when compared with that seen in wild type, indicating that Dilp2–dTOR and Slif–dTOR activities positively regulate the expression of Wg within the progenitors. Importantly, overexpression of Wg restores the progenitor population in both starvation conditions and in the presence of reduced InR levels, demonstrating that Wg is likely to be the most direct downstream target of Dilp2–InR signalling, which maintains progenitors within the progenitors. However, the studies do not rule out either direct or indirect involvement of additional pathways downstream of InR–dTOR in this process (Shim, 2012).

A model describing the systemic and nutritional control of myeloid-like progenitors by insulin and amino acids is presented. The results demonstrate that metabolic changes are perceived by blood progenitors and this causes alteration of their cell-fate determination program. A major consequence of reduced InR and amino acid levels is the reduction of Wg expression in the lymph gland, which functions to promote progenitor maintenance. In addition to accelerated differentiation of myeloid progenitors, starvation also causes a response similar to the inflammatory response typically associated with metabolic disorders. These responses indicate that metabolically induced inflammatory responses in mammals have an ancestral origin that arose to balance an organism's ability to withstand an unfavourable environment and the normal development of myeloid cells. Nutrient/insulin signalling has been linked to the homeostatic control of various Drosophila stem cell populations. Given the highly conserved nature of the blood system in flies and mammals, and the known functional role of metabolism and insulin signalling in myeloid cells, it will be important to determine whether the direct metabolic and nutritional regulation mechanisms uncovered in these studies might also be relevant for the mammalian common myeloid progenitors. Such studies will probably yield insights into chronic inflammation and the myeloid cell accumulation seen in patients with type II diabetes, and other metabolic disorders (Shim, 2012).

The canonical Wingless signaling pathway is required but not sufficient for inflow tract formation in the Drosophila melanogaster heart

The inflow tracts of the embryonic Drosophila cardiac tube, termed ostia, arise in its posterior three segments from cardiac cells that co-express the homeotic transcription factor Abdominal-A (abdA), the orphan nuclear receptor Seven-up (Svp), and the signaling molecule Wingless (Wg). To define the roles of these factors in inflow tract development, this study assessed their function in inflow tract formation. Using several criteria, it was demonstrated that abdA, svp, and wg are each critical for normal inflow tract formation. Wg acts in an autocrine manner to impact ostia fate, and it mediates this effect at least partially through the canonical Wg signaling pathway. By contrast, neither wg expression nor Wg signaling are sufficient for inflow tract formation when expressed in anterior Svp cells that do not normally form inflow tracts in the embryo. Instead, ectopic abd-A expression throughout the cardiac tube is required for the formation of ectopic inflow tracts, indicating that autocrine Wg signaling must be supplemented by additional Hox-dependent factors to effect inflow tract formation. Taken together, these studies define important cellular and molecular events that contribute to cardiac inflow tract development in Drosophila. Given the broad conservation of the cardiac regulatory network through evolution, these studies provide insight into mechanisms of cardiac development in higher animals (Trujillo, 2016).

Wingless and the stomatogastric nervous system

The stomatogastric nervous system (SNS) of Drosophila is a simply organized neural circuitry that innervates the anterior enteric system. Unlike the central and the peripheral nervous systems, the SNS derives from a compact epithelial anlage in which three invagination centers, each giving rise to an invagination fold headed by a tip cell, are generated. Tip cell selection involves lateral inhibition, a process in which Wingless (Wg) activity adjusts the range of Notch signaling. RTK signaling mediated by the Epidermal growth factor receptor plays a key role in two consecutive steps during early SNS development. Like Wg, Egfr signaling participates in adjusting the range of Notch-dependent lateral inhibition during tip cell selection. Subsequently, tip cells secrete the Egfr ligand Spitz and trigger local RTK signaling, which initiates morphogenetic movements resulting in the tip cell-directed invaginations within the SNS anlage (González-Gaitán, 2000).

In order to investigate the role of RTK signaling in SNS development, lack-of-function mutants of the Egfr ligand Spitz were examined. In spitz mutants, the formation of the four SNS ganglia is strongly impaired. The SNS anlage, however, forms normally. In addition, the expression domain of wg and proneural AS-C genes is indistinguishable from a wild-type SNS anlage. At the stage when the three ac-expressing cells were singled-out within the wild-type SNS anlage, only one ac positive cell is found in spitz mutants. The same phenotype has been observed in wg mutants or mutants lacking an integral component of the wg pathway. Since no altered wg pattern was found in the spitz mutant SNS anlage, Spitz-dependent RTK signaling may act in parallel or in combination with wg to adjust the proper range of Notch-dependent lateral inhibition. In contrast to wg mutants, however, no invagination fold is observed. This observation indicates that the singled-out ac-expressing cell of spitz mutants has lost the ability to function as a tip cell and possibly fails to induce morphogenetic movements within the SNS anlage (González-Gaitán, 2000).

spitz, like other genes encoding components of the Egfr signaling pathway such as Egfr, Ras, Raf and the cascade of MAP kinases, is ubiquitously expressed. Local activation of Egfr signaling requires the transmembrane protein Star, which is necessary for the secretion of Spitz. Star is expressed in restricted patterns corresponding to the Spitz secreting cells. In the SNS anlage, it was noted that Star becomes restricted to the three tip cells and is maintained in these cells when invagination takes place. As in spitz mutants, the Star mutant SNS anlage is established normally; only one ac-expressing cell is selected and no invagination occurs. Consistently, Star mutants fail to develop the proper set of SNS ganglia and the associated nerves. These observations suggest that tip cells are a Star-dependent source of Spitz activity that triggers Egfr-dependent RTK signaling in the neighboring cells within the SNS anlage. This conclusion is supported by the finding that phosphorylated MAPK, a cellular marker for RTK signaling activity, is indeed activated in cells of the invagination folds, whereas phosphorylated MAPK does not appear in the Star mutant or in the spitz mutant SNS anlage (González-Gaitán, 2000).

To examine whether activated Spitz is sufficient to induce cell movements within the SNS anlage, use was made of the GAL4/UAS system to misexpress secreted Spitz in an ectopic pattern. This was achieved through the expression of activated Spitz from a UAS promotor driven transgenethat was activated by Gal4 under the control of the actin promotor. Under the conditions applied, scattered UAS-dependent transgene expression is observed throughout the early embryo, including the SNS anlage. When activated Spitz is expressed in such a pattern, a variable number of supernumerary infoldings within the SNS anlagen are observed, indicating that activated Spitz is sufficient to initiate cell movements. This result, in conjunction with the observation that the invaginated cells express phosphorylated MAPK, provides evidence that tip cell-derived activated Spitz triggers RTK signaling to initiate the invagination process. This proposal was tested by blocking Egfr signaling in the anterior most region of the SNS anlage that gives rise to the first invagination fold. For this, a GAL4 driver (SNS1-Gal4) was used that causes UAS-dependent gene expression in the corresponding region of the SNS anlage. SNS1-Gal4-mediated expression of a dominant-negative Egfr mutant form from a UAS-controlled transgene causes a specific suppression of the anterior most invagination fold without affecting the others (González-Gaitán, 2000).

The results demonstrate that RTK signaling participates in the selection of tip-cell-dependent invagination centers in the SNS anlage and is subsequently required to initiate morphogenetic movements resulting in invagination folds. This study does not focus on how RTK signaling ties into the wg-modulated Notch signaling process previously shown to be necessary for the selection of the three SNS invagination centers. The data indicate, however, that RTK signaling acts either in parallel or in combination with wg signaling to adjust the proper range of Notch-dependent lateral inhibition. Although in both wg and Egfr signaling mutants, only one ac-expressing cell is singled-out, the selected cells differ with respect to whether they function as tip cells or not. In wg mutants, the single cell causes an invagination, whereas in Egfr signaling mutants, the selected cell fails to provide this feature of SNS invagination centers. The results, therefore, consistently argue that tip cell-derived Spitz triggers local RTK signaling and thereby initiates the formation of invagination folds each headed by the Spitz-secreting tip cell. Thus, Egfr-dependent RTK signaling in Drosophila does not only participate in cell fate decisions and cell proliferation, but also triggers morphogenetic movements within an epithelium, as has been recently demonstrated for fibroblast growth factor (FGF) signaling. It will be interesting to see whether the role of the EGF pathway in cell migration differs at the cellular level from cell migration events triggered by activated FGF receptors (González-Gaitán, 2000).

The role of Wingless signaling in the patterning of embryonic leg primordium

Cellular interaction between the proximal and distal domains of the limb plays key roles in proximal-distal patterning. In Drosophila, these domains are established in the embryonic leg imaginal disc as a proximal domain expressing escargot, surrounding the Distal-less expressing distal domain in a circular pattern. The leg imaginal disc is derived from the limb primordium that also gives rise to the wing imaginal disc. Essential roles of Wingless in patterning the leg imaginal disc are described. (1) Wingless signaling is essential for the recruitment of dorsal-proximal, distal, and ventral-proximal leg cells. Wingless requirement in the proximal leg domain appears to be unique to the embryo, since it has previously been shown that Wingless signal transduction is not active in the proximal leg domain in larvae. (2) Downregulation of Wingless signaling in wing disc is essential for its development, suggesting that Wg activity must be downregulated to separate wing and leg discs. In addition, evidence is provided that Dll restricts expression of a proximal leg-specific gene expression. It is proposed that those embryo-specific functions of Wingless signaling reflect its multiple roles in restricting competence of ectodermal cells to adopt the fate of thoracic appendages (Kubota, 2003).

At embryonic stage 11, the early expression of D11 expression marks the entire limb primordium that gives rise to both wing and leg discs. After separation of wing and leg discs at stage 12, Dll expression becomes restricted to the center of the leg disc. Double labeling of stage 15 leg discs reveals that there is still a significant number of cells that coexpress Dll and the proximal leg marker Esg, suggesting that expression of Dll and Esg is not a strictly exclusive event. Rather, the result suggests that those marker genes respond differentially to inductive signals in the leg primordium (Kubota, 2003).

In the leg disc, Hth defines the trunk and proximal cell identities, and its expression is excluded in the distal leg domain in the larval stage. Double labeling with antibodies against Esg and Hth reveal that the Esg expression overlaps with Hth expression. Esg is used as a marker uniquely labeling the distinct cell identity of the proximal leg domain in the trunk region (Kubota, 2003).

The expression domain of wg and the position of wing and leg primordia were compared. Wg expression in the trunk ectoderm starts as stripes along the anterior side of the compartment boundaries. At early stage 11, most of the limb primordia marked with Dll protein expression overlap with wg stripes, as revealed by the wg-lacZ reporter. At late stage 11, wg-lacZ stripes break up into dorsal patches and ventromedial stripes. By late stage 12, expression of Dll protein becomes limited to a group of cells partially overlapping the dorsal edge of ventromedial wg stripes. The ventromedial wg stripe also overlaps with proximal leg cells that are labeled with anti-Esg at stage 15. The ventral half of proximal leg cells is nearly completely included within the ventral wg stripes. The dorsal half of leg cells is also located adjacent to, but not included in, the dorsal edge of the wg stripes. On the other hand, a reciprocal relationship between wg expression and wing primordia was observed. When wing primordia are first recognizable at stage 12 as cells expressing Vestigial (Vg), they do not overlap with the stripe of wg. Dorsal cell migration further separates wing primordia from the source of Wg at stage 15. The absence of Wg expression near wing primordia suggests that Wg does not play a positive role in wing disc development (Kubota, 2003).

Wg has been shown to be required for the induction of the thoracic limb primordium and other imaginal discs. To investigate a late role of Wg signaling, the functions of intracellular signal transducers of Wg were studied. The Drosophila homolog of ß-catenin encoded by armadillo (arm) plays dual roles, one as a mediator of Wg signaling by regulating transcription of various target genes, and the other as a component of Cadherin-dependent cell adhesion. Two alleles of arm were analyzed, one being null allele armYD35, and the other armH8.6, which is specifically defective in Wg signaling. Since both armYD35 and armH8.6 show the same phenotype, the function of Arm in Wg signaling, but not in cell adhesion, is required for leg disc development (Kubota, 2003).

To confirm whether Wg signaling is required cell autonomously for leg disc development, the dominant-negative forms of Drosophila TCF (DTCFdeltaN) or Drosophila axin (Daxin) were expressed in the limb primordia. The Dll-Gal4 driver, which is turned on in the limb primordium at stage 11 and continues to be active in leg and wing discs, was used. In Dll-GAL4 embryos carrying UAS-DTCFdeltaN or UAS-Daxin, the overall size of leg discs was reduced. Expression of Esg was preferentially reduced in the dorsal side. The drastic reduction of Dll mRNA and protein in distal leg cells in the armH8.6 mutants as well as in DTCFdeltaN- and Daxin-expressing embryos demonstrate that Wg signaling is required for both proximal and distal leg cells. In arm mutants, Hth-expressing cells expand to the distal domain. This observation suggests that, upon loss of Wg signaling, prospective leg disc cells lose their identity and adopt the fate of trunk ectoderm. However, disc-specific reduction of Wg signaling does not affect wing disc formation, although the Dll-Gal4 driver is active in the wing primordium. It is concluded that late function of Wg signaling promotes formation of the leg disc with a higher requirement in the proximal domain, but is dispensable for wing disc formation (Kubota, 2003).

Cell fate maintenance of proximal leg requires continuous signaling by Wg. The requirement for arm and wg is higher in the proximal leg domain. arm mutations nearly eliminate all Esg expression, but leave some Dll-positive cells. wgts is a hypomorph at the restrictive temperature and leaves distal leg cells nearly intact while significantly affecting proximal leg cells, especially those at the dorsal side of the disc. Dorsal cells are far from the source of Wg and are first to lose identity upon reduction of Wg activity. Since Esg expression in ventral proximal cells overlaps with the wg stripe, it is proposed that the localized expression of Wg and its range of diffusion are major determinants of the site of proximal cell formation. It is likely that dorsal-proximal cells require a higher level of Wg to be produced to reach their position (Kubota, 2003).

Dll expression is initially found in the entire limb primordia and becomes restricted to the edge of the Wg stripe that becomes the center of the leg disc. One candidate for an additional factor that places Dll in this position is Dpp, is expressed in stripes abutting the Wg stripe; Dpp is known to be required for distal leg development (Kubota, 2003).

Finally, the center of the embryonic leg disc is devoid of the expression of proximal cell markers Esg and Hth, marking the distal leg domain. Separation of the proximal and distal leg domains is a slow process, taking several hours to complete. One model for the mechanism regulating this separation process is that proximal gene expression is downregulated by a distal gene, as shown by ectopic Dll expression repressing Esg expression. Since expression of Esg is also regulated by positive input from Wg signaling, Esg expression does not necessary mirror the absence of Dll. In support of this idea, Dll is known to repress proximal genes in larval leg discs. The second possibility is a restriction of proximal cell movement into the distal domain. Cells in the Hth-expressing proximal domain in the larval leg disc have distinct cell-adhesive properties from those in the Dll-expressing distal domain, and by extension, cells with high levels of Dll or Hth may not mix well in the embryo as well. Since Hth is widely expressed in the embryonic ectoderm, Dll-expressing cells may be forced to localize at the center of the leg disc (Kubota, 2003).

This study tested a distal organizer model. The model proposes that a distal organizer placed in the field of a developing appendage signals surrounding cells to acquire proximal cell fate. If this signal acts in all directions, circular arrangement of proximal cells can be achieved. This study demonstrates that dorsal and ventral halves of proximal leg cells have different requirements for Wg. The way Wg acts to organize proximal cell differentiation is not consistent with the distal organizer model. Rather, the results support a second model where dorsoproximal, distal, and ventral-proximal cells are specified separately and assembled to form a circular pattern (Kubota, 2003).

The preferential loss of proximal leg cells upon partial loss of Wg signaling is very similar to the phenotype of EGFR mutant embryos, suggesting that both Wg and EGFR contribute to differentiation of proximal leg cells. A difference in the temporal requirement for the two signals was noted, however. EGFR signaling is activated transiently at the time of disc specification, and the requirement of its activity is limited to a short period around this stage. It has been proposed that EGFR acts within limb primordial cells to promote leg development. This study shows that Wg is persistently expressed at the ventral part of the leg disc and maintains the fate of proximal and distal leg cells. These findings indicate that the leg disc development is initiated by transient activation of EGFR, and its cell fate is maintained by the persistent activity of Wg (Kubota, 2003).

Wg and EGFR signaling are not active in wing discs and are not required for wing formation after stage 11. However, ectopic activation of either of the signals in the limb primordium suppresses the wing disc development, suggesting that downregulation of Wg and EGFR signals have a permissive role in the wing development. These signals are reactivated in postembryonic stages to organize the wing disc. The downregulation of Wg and EGFR signals in a prospective wing disc is accomplished by two mechanisms, one by limiting activation of the two signals to the ventral side of the embryo, and the other by allowing wing primordium to migrate to the dorsal direction away from the source of the inhibitory signals. Thus, cell migration serves as a novel mechanism to restrict the effect of diffusible signaling molecules (Kubota, 2003).

Although the analogous pattern of Wg and Dpp expression plays essential roles in PD patterning in embryonic and larval leg development, significant differences are noted. In embryonic leg discs, expression of both proximal and distal leg markers is lost in mutants of Wg signaling or Dpp signaling. Therefore, Wg and Dpp contribute to both proximal and distal leg development in the embryo. In the larvae, reduction of Wg and Dpp expression due to the loss of hh function causes a loss of the distal domain, but no effect on the proximal gene expression was observed, suggesting that Wg and Dpp play little or no role in the development of proximal domain. The inability of Wg or Dpp to participate in the proximal leg patterning in the larvae is due to, at least in part, the function of Hth to block activation of target genes for Wg and Dpp. In the embryo, however, Hth does not block expression of esg, a target gene for Wg, as demonstrated by coexpression of Esg and Hth. Therefore, proximal domains of embryonic and larval leg discs are different in the way Hth regulates target genes for Wg. This difference may reflect distinct stages of leg development in the embryo, where proximal leg and epidermal cells are continuous, as defined by Hth expression, and in the larvae, where they are separated by the peripodial membrane (Kubota, 2003).

The complementary pattern of Wg and Dpp expression in the larval leg disc is maintained by mutual repression. No evidence for mutual repression of Wg and Dpp was observed in embryonic leg discs. Perhaps the complementary expression pattern of Wg and Dpp in the embryonic leg disc is under the control of the mechanism regulating the global dorsoventral pattern of the embryo (Kubota, 2003).

Thus, in Drosophila, specific mechanisms are involved in embryonic development as opposed to larval leg development. This finding gives rise to the question as to which of the mechanisms is used in other primitive hemimetabolous insects, where the specification and growth of the leg occur simultaneously (Kubota, 2003).

Association of tracheal placodes with leg primordia in Drosophila and implications for the origin of insect tracheal systems

Adaptation to diverse habitats has prompted the development of distinct organs in different animals to better exploit their living conditions. This is the case for the respiratory organs of arthropods, ranging from tracheae in terrestrial insects to gills in aquatic crustaceans. Although Drosophila tracheal development has been studied extensively, the origin of the tracheal system has been a long-standing mystery. Tracheal placodes and leg primordia arise from a common pool of cells in Drosophila, with differences in their fate controlled by the activation state of the wingless signalling pathway. Early events that trigger leg specification have been elucidated and it is shown that cryptic appendage primordia are associated with the tracheal placodes even in abdominal segments. The association between tracheal and appendage primordia in Drosophila is reminiscent of the association between gills and appendages in crustaceans. This similarity is strengthened by the finding that homologues of tracheal inducer genes are specifically expressed in the gills of crustaceans. It is concluded that crustacean gills and insect tracheae share a number of features that raise the possibility of an evolutionary relationship between these structures. An evolutionary scenario is proposed that accommodates the available data (Franch-Marro, 2006).

The Drosophila tracheal system has a clearly metameric origin, arising from clusters of cells, on either side of each thoracic and abdominal segment, that express the tracheal inducer genes trachealess (trh) and ventral veinless (vvl). Conversely, the leg precursors can be recognized as clusters of cells that express the Distal-less (Dll) gene, on either side of each thoracic segment; these will give rise both to the Keilin's Organs (KOs, the rudimentary legs of the larvae) and to the three pairs of imaginal discs that will give rise to the legs of the adult fly (Franch-Marro, 2006).

To investigate whether there is a direct physical association between the leg and tracheal primordia, Drosophila embryos co-stained for the expression of trh and early markers of leg primordia were examined. Although Dll is one of the most commonly used markers for the leg primordia, it is not the earliest gene required for their specification. Instead, a couple of related and apparently redundant genes, buttonhead (btd) and Sp1, act upstream of Dll in the specification of these primordia (Estella, 2003). Examining the specification of tracheal cells with respect to btd expression, tracheal cells were observed to appear in close apposition to btd-expressing cells, from the earliest stages of their appearance (by stage 9/early stage 10). Interestingly, unlike Dll, btd is initially expressed both in the thoracic and abdominal segments, and its expression is restricted to the thoracic segments later, under the influence of the BX-C. Thus, the cells of the respiratory system in Drosophila always arise in close proximity to the cells that are fated to give rise to the legs (Franch-Marro, 2006).

To fully endorse this conclusion it is necessary to show that the btd-expressing cells in the abdomen correspond to cryptic leg primordia. This may be a key point because, although many of the genes required for leg development are already known, it has not yet been possible to induce leg development in abdominal segments (except by transforming these segments into thoracic ones). In particular, although the Dll promoter contains BX-C binding sites that repress its expression in the abdominal segments, no ectopic appendage has been reported by misexpressing Dll in the abdomen. These observations have lead to some doubts as to whether a leg developmental program is at all compatible with abdominal segmental identity (Franch-Marro, 2006).

Since the initial expression of btd in the abdominal segments is downregulated by the BX-C genes, it was reasoned that sustained expression of btd might overcome the repressive effect of the BX-C genes and force the induction of leg structures in the abdomen. To test this, a btd-GAL4 driver was used to drive btd expression, expecting that the perdurance of the GAL4/UAS system would ensure a more persistent expression of btd in its endogenous expression domain. No sign was ever obtained of ectopic Dll expression or KOs in the abdominal segments, but the increased expression of btd had an effect on the KOs of the thoracic segments, which had more sensory hairs than the three normally found in wild-type KOs. Thus, on its own, btd seems unable to overcome BX-C repression of leg development (Franch-Marro, 2006).

One possibility would be that the BX-C genes could suppress appendage development in the abdomen by independently repressing both btd and Dll in this region. To assess this possibility, the same btd-GAL4 driver was used to simultaneously induce the expression of both btd and Dll. Under these circumstances, it was observed that KOs develop in otherwise normal abdominal segments; as in the previous experiment, the newly formed KOs have more than three sensory hairs. These results suggest that expression of btd and Dll in the btd-expressing abdominal primordia is sufficient to induce the development of leg structures in the abdomen, overcoming the repressive effect of the BX-C genes. Furthermore, these results demonstrate that these clusters of btd-expressing cells in the abdomen are indeed cryptic leg primordia. These results clearly show that tracheal cells are specified in close proximity to the leg primordia, in both thoracic and abdominal segments (Franch-Marro, 2006).

Previous results have shown that the leg primordia are specified straddling the segmental stripes of wingless (wg) expression in the early embryonic ectoderm, whereas tracheal cells are specified in between these stripes. To investigate whether wg might play a role in determining the fate of these primordia, what happens when the normal pattern of wg expression is disrupted was studied. In wg mutant embryos, trh and vvl from the earliest stages of their expression are no longer restricted to separate clusters of cells; instead larger patches of expression add up to a continuous band of cells running along the anteroposterior axis of the embryo, while btd expression is suppressed in this part of the embryonic ectoderm. Conversely, ubiquitous expression of wg suppresses trh expression, while causing an expansion of btd expression along the embryo. Restricted activation or inactivation of the wg pathway by the expression of a constitutive form of armadillo or a dominant-negative form of dTCF, respectively, are also able to specifically induce or repress trh and btd expression. trh/vvl and btd seem to respond independently to wg signalling and there is no sign of cross-regulation among them, since btd expression is normal in trh vvl double mutants, and trh and vvl expression is normal in mutants for a deficiency uncovering btd and Sp1 (Franch-Marro, 2006).

The role of wg as a repressor of the tracheal fate is further illustrated by looking at the behaviour of transformed cells: the clusters of cells that have lost btd expression and gained trh and vvl expression in wg mutant embryos begin a process of invagination that is characteristic of tracheal cells. Furthermore, these cells also express the dof (stumps) gene, a target gene of both trh and vvl in the tracheal cells. Although further development of these cells is hard to ascertain because of gross abnormalities in wg- embryos, these results indicate that they have been specified as tracheal cells. Thus, wg appears to act as a genetic switch that decides between two mutually exclusive fates in this part of the embryonic ectoderm: the tracheal fate, which is followed in the absence of wg signalling; and the leg fate, which is followed upon activation of the wg pathway. Given that there are no cell lineage restrictions setting apart the cells of the tracheal and leg primordia, these two cell populations could be considered as a single equivalence group, with the differences in their fate controlled by the activation state of the wg signalling pathway (Franch-Marro, 2006).

A link between respiratory organs and appendages is also found in many primitively aquatic arthropods, like crustaceans, where gills typically develop as distinct dorsal branches (or lobes) of appendages called epipods. Following the current observations, which suggest a link between respiratory organs and appendages in Drosophila, whether further similarities could be found between insect tracheal cells and crustacean gills was examined. Specifically, whether homologues of the tracheal inducing genes might have a role in the development of appendage-associated gills in crustaceans was considered (Franch-Marro, 2006).

RT-PCR was used to clone fragments of the vvl and trh homologues from Artemia franciscana and from Parhyale hawaiensis, representing two major divergent groups of crustaceans (members of the branchiopod and malacostracan crustaceans, respectively). In the case of Artemia vvl, a fragment was cloned that corresponds to the APH-1 gene and an antibody was generated for immunochemical staining in developing Artemia larvae. It was observed that Artemia Vvl is initially absent from early limb buds; it becomes weakly and uniformly expressed while the limb is developing its characteristic branching morphology, and becomes strongly upregulated in one of the epipods as its cells begin to differentiate. Uniform weak expression persists in mature limbs, but expression levels in the epipod are always significantly higher. Expression of the trh homologue from Artemia appears to be restricted to the same epipod as Vvl. Similarly, homologues of vvl and trh were cloned from Parhyale hawaiensis and their expression was studied by in situ hybridization. Both genes are specifically expressed in the epipods of developing thoracic appendages. Besides epipods, the Artemia trh and vvl homologues are also expressed in the larval salt gland, an organ with osmoregulatory functions during early larval stages of Artemia development (Franch-Marro, 2006).

What is the significance of the two Drosophila tracheal inducer genes being specifically expressed in crustacean epipods/gills? One possibility is that the expression of these two genes was acquired independently in insect tracheae and in crustacean gills. Alternatively, tracheal systems and gills may have inherited these expression patterns from a common evolutionary precursor, perhaps a respiratory/osmoregulatory structure that was already present in the common ancestors of crustaceans and insects (Franch-Marro, 2006).

The latter possibility is considered unlikely by conventional views, because of the structural differences between gills and tracheae (external versus internal organs, discrete segmental organs versus fused network of tubes), and the difficulty to conceive a smooth transition between these structures. Yet, analogous transformations have occurred during arthropod evolution: tracheae can be organized as large interconnected networks or as isolated entities in each segment (as in some apterygote insects), invagination of external respiratory structures is well documented among groups that have made the transition from aquatic to terrestrial environments (terrestrial crustaceans, spiders and scorpions), and conversely evagination of respiratory surfaces is common in animals that have returned to an aquatic environment (tracheal gills or blood gills in aquatic insect larvae). A very similar (but independent) evolutionary transition is, in fact, thought to have occurred in arachnids, where gills have been internalised to give rise to book lungs, and these in turn have been modified to give rise to tracheae in some groups of spiders. Thus, a relationship between insect tracheae and crustacean gills is plausible (Franch-Marro, 2006).

A particular type of epipod/gill has also been proposed as the origin of insect wings, a hypothesis that has received support from the specific expression in a crustacean epipod of the pdm/nubbin (nub) and apterous (ap) genes - that have wing-specific functions in Drosophila. In fact, the Artemia nub and ap homologues are expressed in the same epipod as trh and vvl, raising questions as to the specific relationship of this epipod with either tracheae or wings. A resolution to this conundrum becomes apparent when one considers the different types of epipods/gills found in aquatic arthropods, and their relative positions with respect to other parts of the appendage (Franch-Marro, 2006).

The primary branches of arthropod appendages, the endopod/leg and exopod, develop straddling the anteroposterior (AP) compartment boundary, which corresponds to a widely conserved patterning landmark in all arthropods. Different types of epipods/gills, however, differ in their position with respect to this boundary. For example, in the thoracic appendages of the crayfish, some epipods develop spanning the AP boundary [visualized by engrailed (en) expression running across the epipod], whereas others develop exclusively from anterior cells (with no en expression). Given that wing primordia comprise cells from both the anterior and posterior compartments, wings probably derived from structures that were straddling the AP boundary. Conversely, given that tracheal primordia arise exclusively from cells of the anterior compartment (anterior to en and even wg-expressing cells), it seems probable that tracheal cells evolved from a population of cells that was located in the anterior compartment. In this respect, it is interesting to note that the former type of epipods express nub, whereas the latter do not (Franch-Marro, 2006).

In summary, it is suggested that the ancestors of arthropods had specific areas on the surface of their body that were specialized for osmoregulation and gas exchange. Homologues of trh and vvl were probably expressed in all of these cells and played a role in their specification, differentiation or function. Some of these structures were probably associated with appendages, in the form of epipods/gills or other types of respiratory surfaces. A particular type of gill, straddling the AP compartment boundary, is likely to have given rise to wings, whereas respiratory surfaces arising from anterior cells only may have given rise to the tracheal system of insects. Confirmation of this hypothetical scenario may ultimately come from the discovery of new fossils, capturing intermediate states in the transition of insects from an aquatic to a terrestrial lifestyle (Franch-Marro, 2006).

Hox-controlled reorganisation of intrasegmental patterning cues underlies Drosophila posterior spiracle organogenesis: Hh, Wg and Egfr pathways provide specific inputs for posterior spiracle morphogenesis

Hox proteins provide axial positional information and control segment morphology in development and evolution. Yet how they specify morphological traits that confer segment identity and how axial positional information interferes with intrasegmental patterning cues during organogenesis remains poorly understood. This study investigates the control of Drosophila posterior spiracle morphogenesis, a segment-specific structure that forms under Abdominal-B (AbdB) Hox control in the eighth abdominal segment (A8). The Hedgehog (Hh), Wingless (Wg) and Epidermal growth factor receptor (Egfr) pathways provide specific inputs for posterior spiracle morphogenesis and act in a genetic network made of multiple and rapidly evolving Hox/signalling interplays. A major function of AbdB during posterior spiracle organogenesis is to reset A8 intrasegmental patterning cues, first by reshaping wg and rhomboid expression patterns, then by reallocating the Hh signal and later by initiating de novo expression of the posterior compartment gene engrailed in anterior compartment cells. These changes in expression patterns confer axial specificity to otherwise reiteratively used segmental patterning cues, linking intrasegmental polarity and acquisition of segment identity (Merabet, 2005).

In the dorsal ectoderm of stage 10 embryos, hh and wg follow the same striped expression patterns in A8 as in other abdominal segments. rho expression, which marks cells secreting an active form of the Egf ligand, occurs in all primordia of tracheal pits, in A8 as in more anterior segments (Merabet, 2005).

Specification of posterior spiracle primordia occurs at early stage 11. The primordia can then be recognised by Cut expression in spiracular chamber cells and by Sal, the homogenous expression of which in A8 becomes restricted dorsally to stigmatophore cells (forming the external structure of the posterior spiracle) that form a crescent surrounding Cut-positive cells. From mid-stage 11, wg and rho adopt in the dorsal ectoderm expression patterns specific to A8, with wg transcribed in two cells only and rho in a second cell cluster, dorsal and posterior to the tracheal placode. To localise wg- and rho-expressing cells with regard to stigmatophore and spiracular chamber cells, co-labelling experiments for wg or rho transcripts and for Cut or Sal proteins were performed: the two wg cells lie between Cut- and Sal-positive cells; the second cell cluster expressing rho in A8 also expresses Cut but not Sal. This cluster is likely to produce the Egf ligand required for posterior spiracle development, since mutations that alleviate rho expression in the tracheal placodes do not abolish spiracles formation. At mid-stage 11, the hh pattern in A8, along a stripe lying posterior and adjacent to the spiracular chamber and overlapping stigmatophore presumptive cells, resembles expression in other abdominal segments. Analyses at later stages indicate that the relationships between posterior spiracle cells and hh, wg and rho patterns are maintained (Merabet, 2005).

Null mutations of wg, hh or Egfr result in the absence of posterior spiracles. The strong cuticular defects observed raise the possibility that the phenotypes result indirectly from early loss of segment polarity. Removing the Wg, Hh or Egfr signals from 5-8 hours of development using thermosensitive alleles causes strong segment polarity defects but allows filzkörpers, stigmatophores or even complete posterior spiracles to form. Thus, spiracular chamber and stigmatophore can develop in embryos that have pronounced segment polarity defects (Merabet, 2005).

It was next asked whether defects in primordia specification could account for posterior spiracle loss, and Cut and Sal expression was examined in the dorsal A8 ectoderm of hh, wg and Egfr mutant embryos. Expression of Cut and Sal is initiated at stage 11 in all of these mutants, although the somewhat disorganised patterns, especially from late stage 11, may reveal roles for these genes in signalling in sizing or shaping the posterior spiracle primordia. Alternatively, these defects may result from altered morphology of mutant embryos. In any case, the induction of the early markers Sal and Cut in A8 dorsal ectoderm of mutant embryos indicates that posterior spiracle primordia specification does occur in the absence of signalling by Wg, Hh or Egfr. Transcription of ems, another AbdB target that is activated slightly later than Cut, although not affected in hh mutants, is lost in wg or Egfr mutants. Thus, proper regulation of AbdB downstream targets activated following primordia specification appears dependent on signalling activities (Merabet, 2005).

The role was examined of Wg, Hh and Egfr signalling pathways in posterior spiracle organogenesis (i.e., after the specification of presumptive territories). Co-labelling experiments performed on embryos expressing GFP driven by ems-Gal4 or by sal-Gal4 indicate that whereas Cut and Sal are already expressed at early stage 11, GFP is detected from late stage 11 only. These two drivers, which promote expression approximately 1 hour after primordia specification, were used to express DN molecules for each pathway, counteracting Wg (DN-TCF), Egfr (DN-Egfr) or Hh [DN-Cubitus interuptus (Ci)] signalling from that time on. Blocking either pathway in spiracular chamber cells does not perturb stigmatophore morphogenesis, but specifically leads to the loss of differentiated filzkörpers. Conversely, blockade in stigmatophore cells provokes in each case its flattening, while differentiated filzkörpers do form (Merabet, 2005).

To ask how signalling inhibition interferes with the genetic modules initiated downstream of AbdB, expression of Sal and Cut was examined from stages 11 to 13. No major defects are seen until late stage 12. Strong deviation from the wild-type patterns is, however, observed slightly later, from stage 13 onwards: Sal expression in basal cells of the stigmatophore is lost and Cut expression remains in only a few scattered cells. The 2-hour delay seen between the onset of DN molecules expression and the detection of Sal and Cut could reflect the time required for shutting down the pathways. Alternatively, Sal and Cut expression may not require signalling activities before stage 13. To discriminate between these possibilities, an earlier expression of the DN molecules was forced, using the 69B-Gal4, known to promote protein accumulation by the onset of stage 11 (i.e., slightly before posterior spiracle primordia specification). Strong defects in Sal and Cut expression were again seen only in stage 13 embryos, supporting the notion that signalling activities are dispensable before the end of stage 12, but are required from stage 13 onwards to maintain Sal in basal stigmatophore cells and Cut in the spiracle chamber (Merabet, 2005).

A8-specific modulation of rho and wg patterns at mid-stage 11 suggests a regulation by AbdB. In AbdB mutants, rho expression in the spiracle-specific cell cluster is lost, and wg transcription does not evolve towards an A8-specific pattern. In embryos expressing AbdB ubiquitously, ectopic posterior spiracle formation in the trunk can be identified as ectopic sites of Cut accumulation. In such embryos, rho and wg are induced in trunk segments following patterns that resemble their expression in A8: rho in a cluster that overlaps the Cut domain, and wg in few cells abutting ectopic Cut-positive cells. These transcriptional responses to loss and gain of function of AbdB indicate that the Hox protein controls the A8-specific expression patterns of wg and rho. The lines gene (lin), which is known to be required for Cut and Sal activation by AbdB, also controls wg and rho patterns respecification (Merabet, 2005).

In contrast to wg and rho, hh does not adopt an A8-specific expression pattern at mid-stage 11. At that stage, hh expression pattern is not affected upon AbdB mutation. The hh stripe in A8 lies posterior and adjacent to spiracular chamber cells and overlaps stigmatophore cells, suggesting that Hh signalling may participate in the regulation of rho and wg transcription by AbdB. In support of this, it was found that the AbdB-dependent aspects of rho and wg transcription patterns are missing in hh mutant embryos. Thus, inputs from both Hh and AbdB are required to remodel Wg and Egfr signalling in A8 (Merabet, 2005).

The dependence of wg and rho A8 expression patterns on Hh, and the loss of ems expression in wg and rho but not in hh mutants, suggest that transcription of ems requires Wg and Egfr signalling prior to wg and rho pattern respecification by AbdB and Hh. To explore this point further, the time course of ems, wg and rho expression was comparatively analyzed. Embryos bearing an ems-lacZ construct stained for ß-Gal and for wg or rho transcripts show that ems expression precedes wg pattern respecification, and occurs at the same time as rho acquires an A8-specific pattern. Importantly, A8-specific rho clusters were never observed before the onset of ems expression. Thus, ems transcription starts before wg and at the same time as rho pattern respecification, supporting that signalling by Wg and Egfr is required prior to mid-stage 11. These observations also indicate that respecification of the wg pattern occurs slightly later than that of rho, which could not been concluded from changes in embryo morphology (Merabet, 2005).

To determine whether signalling by Wg and Egfr from local sources is important for posterior spiracle organogenesis, the production of Wg and SpiS (the mature form of Spi) ligands was forced from domains broader than normal in A8 dorsal ectoderm. This was performed after posterior spiracle specification, using the ems-Gal4 and sal-Gal4 drivers. Ectopic signalling results in abnormally shaped posterior spiracles: stigmatophores are reduced in size and filzkörpers do not elongate properly. Ectopic signalling from all presumptive stigmatophore cells results in stronger defects than those produced when ectopic signals emanate from all spiracular chamber cells. This can be correlated to the fact that sal-Gal4 drives expression in a pattern that more strongly diverges from the wild-type situation than ems-Gal4 does. Thus, restricted delivery of Wg and SpiS signals is required for accurate posterior spiracle organogenesis (Merabet, 2005).

It was next asked whether, downstream of Hh, the Wg and Egfr pathways provide separate inputs for posterior spiracle organogenesis. Two sets of experiments were conducted and it was found that: (1) in embryos respectively mutant for Egfr or wg, wg and rho acquire A8-specific patterns; (2) epistasis experiments performed by forcing in spiracular or stigmatophores cells the activity of one pathway while inhibiting the other indicate that loss of one pathway could not be rescued by the other. Thus, Egfr and Wg pathways do not act as hierarchically organised modules, but provide independent inputs for posterior spiracle organogenesis (Merabet, 2005).

The expression of the posterior compartment selector gene engrailed (en) until stage 12 follows a striped pattern identical in all trunk segments. Later on, En adopts a pattern that is specific to A8: it is no longer detected in the ventral part of the segment; dorsally, the En stripe has turned to a circle of cells that surround the future posterior spiracle opening and express the stigmatophore marker Sal. The transition from a striped to a circular pattern depends on AbdB. This transition could result either from a migration of en posterior cells towards the anterior, or from transcriptional initiation in cells that were not expressing en before stage 12, and that can therefore be defined as anterior compartment cells (Merabet, 2005).

To distinguish between the two possibilities, en-Gal4/UAS-lacZ embryos were simultaneously stained with anti ß-Gal and anti-En antibodies. If circle formation results from cell migration, one would expect ß-Gal and En to be simultaneously detected in all cells of the circle since the two proteins are already co-expressed in the posterior compartment stripe earlier on. Conversely, if the circle results from de novo expression, one would expect anterior cells in the circle to express En before ß-Gal, since ß-Gal production requires two rounds of transcription/translation compared with one for En. It was found that cells from the anterior part of the circle express En but not ß-Gal in stage 13 embryos, which demonstrates that de novo expression of En occurs in anterior compartment cells. Further supporting En expression in anterior compartment cells, it was found that precursors of anterior spiracle hairs that do not express En at stage 12 do so at stage 13. Engrailed function in A8 is essential for posterior spiracle development, since stigmatophores do not form in en mutants, and are restored if En is provided in stigmatophore cells (Merabet, 2005).

It was also found that although identical in all abdominal segments at stage 11, hh transcription adopts an A8-specific pattern from stage 12 onwards: transcripts are then localised only at the anterior border of the En stripe. This expression of hh is lost in AbdB mutants and still occurs in en mutant. The uncoupling of hh transcription from En activity in the dorsal A8 ectoderm correlates with the distinct phenotypes seen for en mutants, which do differentiate filzkörper like structures, and for hh mutants, which do not (Merabet, 2005).

Data in this paper allow the distinguishing of four phases in functional interactions between AbdB and signalling by Wg, Hh and Egfr during posterior spiracle formation. The first phase corresponds to the specification of presumptive territories of the organ. The signalling activities are not involved in this AbdB-dependent process, since they are not required for the induction of the earliest markers of spiracular chamber and stigmatophore cells, Cut and Sal, in the dorsal ectoderm of A8. The second phase, which immediately follows primordia specification, concerns the regulation of AbdB target genes activated slightly later. Inputs from the Hox protein and the Wg and Egfr pathways are then simultaneously needed, as seen for transcriptional initiation of the ems downstream target. This function of Wg and Egfr signalling precedes and does not require the reallocation of signalling sources in A8-specific patterns; impairing A8-specific expression of wg and rho by loss of hh signalling does not affect ems expression. Within the third phase, AbdB and Hh activities converge to reset wg and rho expression patterns. The three phases take place in a narrow time window, less than 1 hour during stage 11, and could only be distinguished by studying the functional requirements of Wg, Hh and Egfr for transcriptional regulation in the posterior spiracle (Merabet, 2005).

The fourth phase is referred to as an organogenetic phase. Data obtained using DN variants to inhibit the pathways in cells already committed to stigmatophore or filzkörper fates, indicate that Wg, Egfr and Hh pathways are required for organ formation after specification and early patterning of the primordia. Their roles are then to maintain the AbdB downstream targets' expression in posterior spiracle cells as development proceeds, as shown for Cut and Sal at stage 13 (Merabet, 2005).

A salient feature of AbdB function during posterior spiracle development is to relocate Wg and Egfr signalling sources in the dorsal ectoderm at mid-stage 11. wg and rho then adopt expression patterns that differ from expressions in other abdominal segments, conferring axial properties unique to A8 to otherwise segmentally reiterated patterning cues. Resetting Wg and Egfr signalling sources into restricted territories is of functional importance for organogenesis, as revealed by the morphological defects that result from the delivery of Wg or SpiS signals in all spiracular chamber or stigmatophore cells after the specification phase. During stage 12, AbdB also relocates the Hh signalling source by inducing En-independent expression of hh in the dorsal ectoderm. Thus, later than Wg and Egfr signalling, the Hh signal also acquires properties unique to A8. In generating this pattern, AbdB plays a fundamental role in uncoupling hh transcription from En activity, providing a context that prevents anterior compartment En-positive cells to turn on hh transcription, and that allows hh expression in the absence of En in other cells. Slightly later, at stage 13, AbdB modifies the expression of the posterior selector gene en, initiating de novo transcription in anterior compartment cells. In these cells, En fulfils different regulatory functions than in posterior cells, as discussed above for hh regulation. Changes in En expression and function can be interpreted as a requisite to loosen AP polarity in A8 and gain circular coordinates required for stigmatophore formation (Merabet, 2005).

The ETS domain transcriptional repressor Anterior open inhibits MAP kinase and Wingless signaling to couple tracheal cell fate with branch identity

Cells at the tips of budding branches in the Drosophila tracheal system generate two morphologically different types of seamless tubes. Terminal cells (TCs) form branched lumenized extensions that mediate gas exchange at target tissues, whereas fusion cells (FCs) form ring-like connections between adjacent tracheal metameres. Each tracheal branch contains a specific set of TCs, FCs, or both, but the mechanisms that select between the two tip cell types in a branch-specific fashion are not clear. This study shows that the ETS domain transcriptional repressor anterior open (aop) is dispensable for directed tracheal cell migration, but plays a key role in tracheal tip cell fate specification. Whereas aop globally inhibits TC and FC specification, MAPK signaling overcomes this inhibition by triggering degradation of Aop in tip cells. Loss of aop function causes excessive FC and TC specification, indicating that without Aop-mediated inhibition, all tracheal cells are competent to adopt a specialized fate. Aop plays a dual role by inhibiting both MAPK and Wingless signaling, which induce TC and FC fate, respectively. In addition, the branch-specific choice between the two seamless tube types depends on the tracheal branch identity gene spalt major, which is sufficient to inhibit TC specification. Thus, a single repressor, Aop, integrates two different signals to couple tip cell fate selection with branch identity. The switch from a branching towards an anastomosing tip cell type may have evolved with the acquisition of a main tube that connects separate tracheal primordia to generate a tubular network (Caviglia, 2013).

This work has investigated how the choice between the two types of specialized tip cells in the tracheal system is controlled. The transcriptional repressor Aop plays a key role in linking tracheal tip cell fate selection with branch identity. First, a novel tube morphogenesis phenotype is described in aop mutants, which is due to the massive mis-specification of regular epithelial cells into specialized tracheal tip cells. aop is specifically required for controlling tracheal cell fate, whereas aop, like pnt, is dispensable for primary tracheal branching, thus uncoupling roles of RTK signaling in cell fate specification and cell motility. The finding that tracheal branching morphogenesis proceeds normally in the presence of excess tip cell-like cells suggests that collective cell migration is surprisingly robust and that mis-specified cells apparently do not impede the guided migration of the tracheal primordium. Second, it was demonstrated that in the absence of inhibitors of MAPK signaling (aop and sty), all tracheal cells are competent to assume either TC or FC fate. The transcriptional repressor Aop globally blocks both TC and FC differentiation, but high-levels of MAPK signaling in tip cells relieve Aop-mediated inhibition, thus permitting differentiation. Third, the results suggest that in the DT region Aop limits FC induction through a distinct mechanism by antagonizing Wg signaling in addition to MAPK signaling. Conversely, in the other branches, Aop limits TC differentiation by blocking MAPK-dependent activation of Pnt. Fourth, it was shown that the region-specific choice between the two cell fates in the DT is determined by Wg signaling and by the selector gene salm. Based on these results, a model is proposed in which a single repressor, Aop, integrates MAPK and Wg signals to couple tip cell fate selection with branch identity. High levels of Bnl signaling trigger Pnt activation and Aop degradation in tracheal tip cells. It is proposed that in the DT, unlike in other tracheal cells, MAPK-induced degradation of Aop releases inhibition of Wg signaling. This is consistent with recent work showing an inhibitory effect of Aop on Wg signaling, possibly through direct interaction of Aop and Arm, or through Aop-mediated transcriptional repression of Wg pathway component. The current work extends the evidence for this unexpected intersection between two major conserved signaling pathways, suggesting that this function of Aop is likely to be more widespread than previously appreciated. The findings also provide an explanation for the puzzling observation that, in pnt mutants, TCs are lost, while FCs become ectopically specified. As pnt is required for expression of the feedback inhibitor sty, loss of pnt is expected to lead to MAPK pathway activation and consequently to increased Aop degradation. This would release Aop-mediated repression of Wg signaling, resulting in extra FCs, whereas TCs are absent because of the lack of pnt-dependent induction. This suggests that excessive FC specification in the DT of aop and sty mutants is mainly due to deregulated Wg signaling, rather than to de-repression of pnt-dependent MAPK target genes. Consistent with this notion, it was shown that pnt is not required for Delta and Dys expression in tracheal cells, although constitutively active AopACT represses their expression (Caviglia, 2013).

The results further show that salm function constrains the fate that is chosen by cells when released from the Aop inhibitory block. MAPK signaling triggers Aop degradation in all tip cells, but only in the absence of salm does this signal lead to TC induction. In salm-expressing cells, degradation of Aop releases Wg signaling, resulting in FC specification. Thus, salm biases the choice between two morphologically different types of seamless tubes. This is reminiscent of the role of salm in switching between different cell types in the peripheral nervous system and in muscles. salm expression is sufficient to repress TC formation. The genetic results, consistent with biochemical data showing that Salm acts as a transcriptional repressor, suggest that salm promotes FC fate by repressing genes involved in TC development. However, salm is not sufficient to overcome the requirement for Wg signaling in FC induction, indicating that Wg does not act solely via salm to induce FC fate. Indeed, FC induction requires genes whose expression is independent of salm (esg, dys). In addition, it is proposed that a feedback loop between Wg signaling and salm expression maintains levels of Wg signaling in the DT sufficiently high to induce FC fate. Taken together, these results suggest that the default specialized tip cell fate, and possibly an ancestral tracheal cell state, is TC fate. Although FCs and TCs differ in their morphology, they share a unique topology as seamless unicellular tubes. FCs and TCs might therefore represent variations of a prototypical seamless tube cell type. Salm might modify cellular morphology by repressing TC genes, including DSRF, which mediates cell elongation and shape change. Intriguingly, Wg-dependent salm expression in the DT of dipterans correlates with a shift towards FC as the specialized fate adopted by the tip cells of this branch. This study has shown that salm expression inhibits TC fate, while promoting the formation of a multicellular main tracheal tube by inhibiting cell intercalation. It is therefore tempting to speculate that the salm-dependent switch from a branching towards an anastomosing tip cell type in the DT may have evolved with the acquisition in higher insects of a main tube that connects separate tracheal primordia to generate a tubular network. It will be of great interest to identify the relevant target genes that mediate the effect of Salm on tube morphology and tip cell fate (Caviglia, 2013).

The mechanisms of tip cell selection during angiogenesis in vertebrates are beginning to be understood at the molecular level. However, the signals that control the formation of vascular anastomoses by a particular set of tip cells are not known. Intriguingly, the development of secondary lumina in aop mutants is reminiscent of transluminal pillar formation during intussusceptive angiogenesis, which is thought to subdivide an existing vessel without sprouting. Although the cellular basis for this process is not understood, it is conceivable that specialized endothelial cells are involved in transluminal pillar formation. This work provides a paradigm for deciphering how two major signaling pathways crosstalk and are integrated to control cell fate in a developing tubular organ. It will be interesting to see whether similar principles govern tip cell fate choice during tube morphogenesis in vertebrates and invertebrates (Caviglia, 2013).

DWnt4 and wingless elicit similar cellular responses during imaginal development

Wnt genes encode evolutionarily conserved secreted proteins that provide critical functions during development. Although Wnt proteins share highly conserved features, they also show sequence divergence, which almost certainly contributes to the variety of their signaling activities. Wnt4 and wingless, two divergent clustered Wnt genes, can have either antagonist or distinct functions during Drosophila embryogenesis. Both genes can elicit similar cellular responses during imaginal development. Ectopic expression of Wnt4 along the anterior/posterior (A/P) boundary of imaginal discs alters morphogenesis of adult appendages. In the wing disc, Wnt4 phenocopies ectopic Wg activity by inducing notum to wing transformation, suggesting similar signaling capabilities of both molecules. In support of this, it has been demonstrated that Wnt4 can rescue wg loss-of-function phenotypes in the antenna and haltere and is able to substitute for Wg in wing field specification. In addition to effects on the axial pattern of haltere and wing, ectopic Wnt4 produces supernumerary bristles in specific regions. Wnt4, like Wg, induces extra sternopleural bristles. This may reflect an endogenous activity for Wnt4: unlike wg, it is transcribed in a dorsal domain of the leg disc that roughly corresponds to the sternopleura. Both genes are transcribed in overlapping domains in imaginal discs, suggesting that Wnt4 may cooperate with wg during limb patterning (Gieseler, 2001).

wg and Wnt4 are transcribed in overlapping patterns during embryogenesis. They also show largely similar expression profiles in third instar imaginal discs. In the wing disc, transcripts of the two genes are synthesized at the D/V compartment border, the future wing margin, and the presumptive wing hinge region. Wnt4 expression, however, is weak compared to that of wg and appears more spatially restricted. It proceeds following a cell stripe comparable in width to the wg stripe in the distal part of the future wing margin, but is more faintly detected in more proximal parts of anterior and posterior compartments. The patterns are different in the presumptive notum, where WG mRNA is found in a broad D/V stripe, whereas WNT4 transcripts faintly label a central cell domain. These cells are located beneath the columnar epithelium and, therefore, likely correspond to adepithelial cells. In antenna and leg discs, the wg domain corresponds to a ventral/anterior sector, whereas Wnt4 is expressed only in a subset of these cells. Whereas WG mRNA is detected along the entire proximal-distal axis in the leg disc, Wnt4 transcription is restricted to two segments that correspond to primordia of the tibia and a more distal segment and in a dorsally located cell cluster that presumably corresponds to the sternopleural region. The wg pattern in the haltere disc is reminiscent of that observed in the wing disc. Wnt4 is transcribed in two large stripes, which give rise to the pedicel and/or the scabellum. However, in contrast to wg, Wnt4 is not expressed in the notum and in the most distal part of the disc, which is homologous to the margin of the wing. A clear difference in expression of the two genes is also seen in the larval central nervous system, where wg is not expressed, while Wnt4 transcripts follow a segmentally repeated pattern of small clusters in the central cortex and in the optic anlagen. In summary, the transcription profile of Wnt4 in third instar imaginal discs partially overlaps that of wg but is not restricted to wg-positive cells. For example, in the wing blade, the two genes are expressed along the future margin and in the presumptive hinge region, suggesting that the encoded Wnt products may be required together for the differentiation of these structures. In the central nervous system or in the dorsal part of the leg disc, Wnt4 is expressed where wg is not, suggesting it may also function in cells that do not depend on wg (Gieseler, 2001).

The ability of Wnt4 to induce, as Wg does, additional wings indicates that the two molecules can elicit similar cellular responses. Strong support for this conclusion is provided by rescue experiments of wg loss-of-function phenotypes. Wnt4 can restore normal wing development in the absence of a functional Wg protein at the second instar. Eliminating Wg signaling during the third instar, allows wing field specification but not subsequent wing patterning and growth. Wnt4 therefore appears unable to substitute for Wg signaling during late wing development. This failure to replace Wg might be the result of ectopic expression in inappropriate domains or of distinct signaling abilities during wing growth and patterning. In support of the latter possibility, the two Wnt molecules produce different effects, depending on the developmental context. Ectopic expression of Wg, but not of DWnt4, perturbs leg, eye, or antenna development, which reveals competency to respond to Wg but not to Wnt4. In addition, Wnt4 is able to antagonize late embryonic Wg signaling in the Drosophila ventral epidermis and to block the Wg-induced body axis duplication in Xenopus. Furthermore, the two genes induce different phenotypes in the dorsal embryonic epidermis. Taken together these results strongly support a context dependence for Wnt4 activity (Gieseler, 2001).

The molecular basis underlying the ability of Wg and Wnt4 to perform antagonistic, distinct, or similar signaling activities remains to be explored. It has been proposed that the Wnt4/Wg antagonistic relationship in Drosophila embryonic ventral ectoderm and in Xenopus axis induction assay results from strong sequence divergences in the C-terminal parts of the two proteins. This has been supported by reports that C-terminal truncations in Wg and XWnt8 result in proteins with dominant negative or antagonistic functions. If this interpretation is correct, domains other than the divergent C-terminal ends in the Wnt proteins would be critical for function in other developmental contexts. Although interactions of the C-terminus with specific partners dictate activity in the ventral embryonic epidermis, the recruitment of imaginal disc-specific factors by other domains in Wg and Wnt4 would allow the proteins to exhibit similar activities during wing field specification. The existence of separated functional domains in Wnt proteins is also supported by the nature and effect of the wgNEI mutation, where a single amino acid change affects a subset of Wg functions only (Gieseler, 2001).

An alternative model to explain antagonism versus similarity is that Wnt4 acts as a low-activity agonist of Wg. In this model, tissue-specific differences in receptor concentrations and/or differences in receptor-binding affinities would determine whether Wnt4 mimics or antagonizes Wg signaling. If, for example, receptor concentrations are limiting in the ventral epidermis, Wnt4 may act as a competitive inhibitor of Wg for receptor binding but would provide less stimulation of the pathway than would Wg itself, therefore lowering the level of Wg signaling. However, if receptor concentrations are high, Wnt4 may be able to increase signaling by binding receptors without competing with endogenous Wg. If Wnt4 interacts with Wg receptors with lower affinity than that of Wg, the tissue-specific differences in Wnt4's ability to engage the Wg signaling pathway might be explained. In particular, this may explain the inability of Wnt4 to stimulate the Wg pathway in wild type legs while stimulating the pathway, and rescuing leg defects, in the absence of Wg (Gieseler, 2001).

Proximodistal subdivision of Drosophila legs and wings: the elbow-no ocelli gene complex serves as a mediator of the function of the Wg and Dpp signaling systems

Appendages are thought to have arisen during evolution as outgrowths from the body wall of primitive bilateria. In Drosophila, subsets of body wall cells are set aside as appendage precursors through the action of secreted signaling proteins that direct localized expression of transcription factors. The Drosophila homeodomain protein Distal-less is expressed in the leg primordia and required for formation of legs, but not wings. The homeodomain protein Nubbin is expressed in the wing primordia and required for formation of wings, but not legs. Given that insect legs and wings have a common developmental and evolutionary origin, attempts were made to identify genes that underlie the specification of all appendage primordia. Evidence is presented that the zinc-finger proteins encoded by the elbow and no ocelli genes act in leg and wing primordia to repress body wall-specifying genes and thereby direct appendage formation (Weihe, 2004).

Evidence suggests that the el and noc genes serve as mediators of the function of the Wg and Dpp signaling systems in specification of the appendage field within the imaginal discs. El and Noc are induced by Wg and Dpp and are required to repress the proximally expressed proteins Hth and Tsh. Previous work had identified Dll as a gene required for appendage formation in leg and antenna, and nub as a gene required for wing. This report identifies El and Noc as a pair of zinc-finger proteins that function in both ventral and dorsal appendages. However, there are interesting differences in the way that they do so, when examined in detail (Weihe, 2004).

Dll expression is required for the formation of all leg and antenna elements in the ventral (leg) discs, and until this work Dll was the earliest known marker for the distal region leg disc. Previous work has shown that repression of Hth and Tsh by Dpp and Wg was not required for expression of Dll in the leg, nor could Dll repress Hth and Tsh. Thus an essential mediator of the effects of Wg and Dpp was missing. The current results present evidence that El and Noc serve this function, since their removal leads to ectopic expression of Hth and Tsh. Removal of El and Noc does not cause loss of Dll expression, so it is concluded that Wg and Dpp act independently to induce El and Noc expression and Dll to define the distal region of the leg disc (Weihe, 2004).

The situation differs slightly in the wing. Repression of Tsh is the earliest marker for specification of the distal wing region, preceding the onset of Hth repression or of Nub induction. Loss of Tsh and Hth are required to allow Nub expression. Ectopic expression of Hth and Tsh and loss of Nub is observed in clones lacking El and Noc activity. Thus in the wing, expression of the distal marker Nub cannot be demonstrated to be independent of El and Noc (because ectopic Hth can repress Nub, but not Dll). The vestigial gene is also important for wing development and has been proposed to be a wing specifying gene. However, Vestigial is expressed all along the DV boundary of the wing, both in the wing primordium and in the body wall. This led to the suggestion that while Vestigial is essential for wing development, its expression cannot be taken as a molecular marker for wing identity per se, particularly at early stages. For this reason analysis of the relationship between El, Noc and Vestigial was not performed in this study (Weihe, 2004).

Is the repression of trunk genes needed to specify appendage, as opposed to body wall, in wing and leg discs? In the wing disc the answer appears to be yes; repression of 'trunk genes' like hth is necessary to make the remaining part of the disc competent to form the appendage. However, in the leg the situation is more complex. Coexpression of Dll and Hth does not disrupt proximal-distal axis formation, but leads to homeotic transformation of leg tissue into antennal tissue. Hth is not repressed and limited to proximal areas in the antenna. However, loss of el and noc activities in the leg disc leads to loss of distal leg tissue without any evident transformation into antennal tissue. Thus, El and Noc may regulate the expression of other 'trunk genes', whose restricted expression is required to make the remaining leg and antenna disc competent to form the appendage (Weihe, 2004).

The regional requirements for El and Noc highlight another interesting difference between leg and wing disc development. el noc double mutant cells are excluded from contributing to the tarsal region of the leg but not from contributing to the femur and tibia. Lineage tracing has shown a considerable net flux of cells from the proximal (Tsh-expressing domain) into femur and tibia. While there is no boundary of lineage restriction separating these domains, cells must be able to change from expressing the proximal marker Hth to expressing the distal marker Dll in order to move from one territory to the other. The wing in contrast does not appear to normally exhibit this large net flux of cells from proximal to distal and the el noc double mutant cells are excluded from contributing to the entire wing region. Clonal analysis has suggested that el noc double mutant cells attempt to sort out toward proximal territory, or if that fails, they can be lost from the disc, apparently by sorting out perpendicular to the epithelium. These observations suggest that El and Noc activity may contribute to the production of proximal-distal differences in cell affinities and thereby may help to maintain segregation of these cell populations during development (Weihe, 2004).

Doublesex, Wingless and Engrailed and the development of the genital disc

Each Drosophila genital imaginal disc contains primordia for both male and female genitalia and analia. The sexually dimorphic development of this disc is governed by the sex-specific expression of doublesex. Data is presented that substantially revises understanding of how dsx controls growth and differentiation in the genital disc. The classical view of genital disc development is that in each sex, dsx autonomously 'represses' the development of the inappropriate genital primordium while allowing the development of the appropriate primordium. Instead, dsx is shown to regulate the A/P organizer to control growth of each genital primordium, and then dsx directs each genital primordium to differentiate defined adult structures in both sexes (Keisman, 2001b).

Recent findings concerning the growth of clones of genital disc cells whose sex was altered genetically suggest that the growth of each genital primordium is controlled by the sex of a subset of its cells. Such clones were expected to develop according to their genetic sex, because sex determination is cell autonomous. For instance, female clones in the male primordium should adopt the 'repressed' state characteristic of that primordium in females. Consistent with this prediction, female clones cannot contribute normally to adult male genital structures. However, such clones frequently grow substantially and contribute to a morphologically normal male genital primordium in the larval genital disc, suggesting that growth and the capacity to differentiate are under separate control. Yet occasional female clones in the male primordium are associated with severe reductions in the size of the corresponding genital primordium in the disc. That some clones in the male primordium disrupt growth while others do not led to a proposal that growth in the genital primordia is controlled nonautonomously from within an unidentified organizing region. Clones that grow normally would lie outside of this organizing region, while those that cause reductions would intersect it. An obvious candidate for this organizing region is the strip of anterior compartment cells along the A/P border that express wg and dpp, which is referred to as the A/P organizer (Keisman, 2001b).

Therefore, it was hypothesized that the sex of the A/P organizer region nonautonomously controls the sex-specific patterns of proliferation in the genital disc. To test this hypothesis, advantage was taken of the fact that the A/P organizer coincides with high levels of expression of the patched (ptc) gene, while the posterior compartment is defined by engrailed (en) expression. Thus, gene expression can be targeted to these regions using ptc-GAL4 and en-GAL4 drivers, respectively. Chromosomally male cells were feminized by expressing a female tra cDNA, while chromosomally female cells were masculinized by expression of a tra-2 inverted repeat construct (tra2IR) that blocks the function of tra-2 through the mechanism of dsRNA-mediated interference. If the hypothesis is correct, changing the sex of cells in the A/P organizer region would cause each primordium to develop as it does in the corresponding sex. Conversely, changing the sex of the posterior compartment cells should have no effect on genital disc morphology (Keisman, 2001b).

When cells of the A/P organizers in chromosomally male genital discs are feminized, a radical change in the morphology of both the male and female genital primordia is observed. The chromosomally male genital discs resemble female genital discs: the female primordium grows to dominate the disc epithelium, while the male primordium is substantially reduced. Feminization of the posterior compartment of chromosomally male genital discs, in contrast, has no discernable effect on disc morphology. As expected, the morphology of chromosomally female genital discs is unaffected by the expression of tra. The transformation produced by ptc-GAL4-driven tra expression in XY animals is not perfect, as the female primordium overgrows and is thrown into folds. Occasionally, these discs have male primordia with vestiges of male morphology. This pattern of growth is usually only on one side of the disc, and it is attributed to variability in tra expression produced by the ptc-GAL4 driver. To confirm that the intended transformation had been produced, the adult phenotypes of the feminized flies were examined. The expected correlation exists between the domain of tra expression and the affected elements of the male and female adult structures (Keisman, 2001b).

The reciprocal transformation, masculinization of the A/P organizer cells in a chromosomally female disc, also produces a striking transformation of disc morphology. Many of these discs are morphologically indistinguishable from those of their male siblings. The male primordium is wild-type or near wild-type in size, while the female primordium is reduced in size. This transformation is not completely penetrant. While the majority of the chromosomally female discs (11/17) had predominantly or completely male morphology, there were a few discs in which the female primordia grew slightly. Nevertheless, for a significant fraction of the masculinized female discs, it would have been impossible to determine their chromosomal sex without anti-Sxl staining to identify them. When the posterior compartment of female discs is masculinized, there are only minor changes in the morphology of these discs. The female primordium overgrows slightly, deepening a normally shallow groove that runs between its left and right halves and occasionally causing extra folds. The male primordium of these discs is also slightly thickened. Taken together, these experiments demonstrate that the primary determinant of disc growth and morphology is the sex of the cells of the A/P organizer, although the sex of other cells makes a minor contribution to morphology (Keisman, 2001b).

Tracing the fate of the male primordium in the female genital disc has revealed that its cells persist throughout metamorphosis and give rise to the parovaria, the internal female accessory glands. The male primordium of the female disc was tracked during metamorphosis by following the expression of reporter genes that reveal the arrangement of the three primordia in the disc. The parovaria bud forms from the female genital disc in the first 12 hr of metamorphosis, during which there is a radical rearrangement of the epithelium's geometry. The major element of this rearrangement is an elongation of the disc along the A/P axis. This elongation is driven by an apparent convergent extension, most pronounced in the thickened ventral epithelium. This convergent extension drives the primordia of the spermathecae, which originate ventrally in the female primordium, onto the dorsal side of the disc. Cells on the lateral edges of the disc are also driven dorsally and medially. Almost immediately after this rearrangement, the emerging parovaria become evident just posterior to the emerging spermathecae. By 12 hr after puparium formation (hAPF), the protrusion of the parovaria and spermathecae becomes more pronounced and the identification of these structures can be made unequivocally (Keisman, 2001b).

That the parovaria arise from the male genital primordium can be seen by following the expression patterns of wg and en. In the third instar female genital disc, wg is expressed in a thin band of cells in the male primordium just anterior to the en-expressing domain. These two domains of gene expression define the male primordium. During the first 4 hr of metamorphosis, the en and wg bands from the male primordium are joined on the dorsal surface by additional, more anterior bands of en and wg that derive from the ventral female primordium and are driven dorsally by the convergent extension of the disc. At 4, 8, and 12 hAPF, it is evident that the parovaria are emerging from within the domain of en expression that, at third instar, defines the posterior compartment of the male primordium (Keisman, 2001b).

Previous cell lineage analysis and gynandromorph fate mapping studies assigned the parovaria to the anal (A10) primordium. Although the anal primordium is physically distant from where the parovaria originate, the data were corroborated by tracking the anal primordium during metamorphosis. Since the anal primordium (A10) is defined by the expression of caudal (cad), a GAL4 enhancer trap insertion in cad was used to drive expression of GFP in the anal primordium and this expression was followed in the female genital disc during the first 12 hr of metamorphosis. In the third instar female disc, cad expression extends from the posterior edge of the disc anteriorly, approximately two-thirds of the way across the disc. This anterior border correlates with the posterior edge of the male primordium as defined by en expression. It is clear that the parovaria bud from a region of the disc well anterior to the domain of cad expression. Thus, the parovaria do not derive from the anal primordium (Keisman, 2001b).

Tracing the cells of the female primordium in male genital discs shows that these cells persist throughout metamorphosis and produce a miniature eighth tergite at the anterior edge of the male genital arch. The topology of the three primordia in the male genital disc epithelium is similar to that in the female. However, the morphogenesis of the male genitalia is substantially more complex than that of the female, and determining the fate of the female primordium requires following its metamorphosis until 48 hAPF (Keisman, 2001b).

The posterior compartment of the female primordium in males corresponds to the long patch of en expression at the posterior edge of the disc. During metamorphosis, the male genital disc opens at its posterior edge and turns partially inside-out to expose the apical surface of the genital disc. If the disc is viewed from the posterior, the female primordium is at the leading edge of the ventral 'lip' when the disc everts. The en domain is toward the back of this lip, preceded by the anterior compartment of the female primordium. Following this group of cells until 24 hAPF reveals that it persists and proceeds to completely encircle the differentiating male genitalia. Importantly, this band can be distinguished from the thick band of en expression in the male genital arch, which corresponds to segment A9. Intermediate time points (at 8, 30, and 36 hr) were used to confirm that these cells are continually present and not lost and then replaced by other cells. By 48 hr the A8 en band can be seen as a tight collar that rings the male genitalia. This band is easily distinguished from the A6 band of en expression and persists in later pupae. This band is also present in the adult, where it labels the anterior rim of the genital arch. The border of the A8 en band in the adult correlates roughly with a seam in the anterior cuticle of the genital arch; it is concluded that the region of the genital arch anterior to this seam is a vestigial male eighth tergite (T8) (Keisman, 2001b).

The analysis was complicated by the presence of en expression in the larval epidermal cuticle (LEC), which persists until it is replaced by the expanding histoblast nests. The male genital disc integrates into the LEC as it everts, making it necessary to confirm that the en expression, which is inferred to derive from the female primordium, is indeed of imaginal origin. Advantage was taken of a GAL4-expressing enhancer trap insertion in escargot (esg) was used to confirm the identity of these cells, since esg is expressed in imaginal cells but not in the larval cuticle. esg is expressed strongly in a thick epithelial mantle just ventral to the male genitalia. Comparison with the expression of en in a separate 24 hr male genitalia shows that the band of en expression that defines the female primordium is well within this same mantle of cells. The imaginal origin of the A8 en band is also supported by the size of the nuclei in these cells: the LEC has large polyploid nuclei, while the imaginal nuclei of the presumptive female primordium at 24 hAPF are diploid and much smaller. At 24 hr the expanding diploid histoblast nests have only partially completed the replacement of the LEC. As a result, bands of en in the LEC consist of a mix of small diploid nuclei and large polyploid nuclei. In contrast, the entire circumference of the en ring in the presumptive female primordium consists of small diploid nuclei. The simplest interpretation of this observation is that this ring of en-expressing cells derives from the diploid genital disc and identifies the female primordium (Keisman, 2001b).

There appears to be expression of GFP in the polyploid cells of the LEC, casting doubt on the reliability of the esg-GAL4 as an imaginal marker at this stage. However, these animals do not express GFP in the LEC at larval stages. Moreover, many enhancer traps become ubiquitously activated in the LEC after 10-12 hr APF. Even though there is some GFP expression in the LEC, the intensity of GFP expression in the everting genitalia is stronger than in the surrounding cells. In whole mounts of esg-GAL4/UAS-GFP abdomens, the genitalia stand out dramatically and there is a perceptible change in the intensity of GFP expression that correlates with where the thick epithelial mantle meets a much thinner epithelium. It is inferred that this mantle is the female primordium, based on its location, the relative intensity of esg-driven GFP expression, and its contiguity with the male genitalia (Keisman, 2001b).

Because the sex determination pathway acts cell autonomously to determine sex, the reduced growth in the 'repressed' primordium has long been thought to reflect a cell autonomously regulated quiescent state. However, the results show that the major factor controlling the growth of the genital primordia is the sex of the cells at the A/P border, not the sex of individual cells. When the cells of the A/P organizer are feminized in a male disc or masculinized in a female disc, both genital primordia respond by switching to growth patterns that reflect the sex of the cells at the organizer. When the sex of posterior compartment cells is genetically altered, there is no major change in disc morphology. It is inferred that these posterior compartment cells continue to grow normally under the influence of the unaffected A/P organizer (Keisman, 2001b).

It is thought that the primary activity of the sex determination hierarchy in the A/P organizer is to regulate wg and dpp signaling. It has been suggested that cell growth in the genital disc is controlled by dsx acting either directly or indirectly through the expression of dpp and wg. In the genital disc, wg and dpp are expressed along the A/P border in the same cells that express the ptc-GAL4 driver and the activity of wg and dpp is the primary determinant of disc size and shape in the thoracic imaginal discs, and the reduced male primordium of a female genital disc does not express dpp. However, the female primordium expresses wg and dpp in both sexes yet grows to different sizes and shapes in each. Thus, it remained a distinct possibility that this difference in growth was attributable to the response of individual cells to wg and dpp. The current results argue otherwise, suggesting that the sex determination pathway produces different patterns of growth by regulating the absolute levels and/or timing of wg and dpp expression (Keisman, 2001b).

The results also suggest that while the A/P organizer is the primary determinant of growth in the two genital primordia, the sex of other cells is not completely irrelevant. ptc-GAL4 driven feminization of the A/P organizer in chromosomally male discs is not perfect, as the female primordia of these discs overgrow and are thrown into folds. Masculinization of the posterior compartment in chromosomally female discs also cause slight overgrowth and subtle alterations in the morphology of the female primordia. The most important nontrivial possibility raised by these results is that the shape that the female primordium adopts remains partially dependent on the sex of its constituent cells (Keisman, 2001b).

The results add to evidence indicating that dsx plays an active role in directing the differentiation of the genital primordia and that dsx acts instructively at multiple steps during development to direct sex-specific differentiation. Specifically, the control of growth and differentiation by dsx are separable processes: dsx controls growth primarily by regulating the activity of the A/P organizer, while differentiation is controlled by dsx cell autonomously (Keisman, 2001b).

The control of growth and the establishment of pattern in imaginal discs are mediated by the same molecules, the morphogens encoded by wg and dpp. This conservation implies that in directing the correct sex-specific differentiation of a given genital primordium, dsx acts on wg and dpp signaling twice: at the A/P organizer, dsx acts to direct the correct patterns of growth via wg and dpp expression; dsx must then act again in individual cells, probably throughout the disc, to direct the correct sex-specific interpretation of the positional identities specified by wg and dpp. This prediction is borne out by recent findings that the expression of individual genes in the genital primordia is under the cell-autonomous control of dsx. For instance, dsx determines whether cells in the male (A9) primordium will express dachshund in response to wg, as in female discs, or in response to dpp, as in male discs (Keisman, 2001b).

Since the homeotic genes specify the identity of segments A8 and A9, they must provide the context for the differential action gggvyof dsx on the two genital primordia, both at the A/P organizer (to regulate growth) and in individual cells (to control differentiation). The segmental identities of A8 and A9 are specified by the homeotic genes abd-A and the two genetically distinct functions of the Abd-B gene, Abd-BI, and Abd-BII. The exact division of labor in this respect is not clear, but most evidence suggests that abd-A and Abd-BI specify different parts of segment A8, while Abd-BII specifies segment A9. Removal of Abd-B from the genital disc causes it to switch to a leg-like mode of differentiation in which, for instance, the expression of dac reverts to a broader domain of expression. Thus, sex-specific dac expression requires not only dsx, but also Abd-B, confirming that differentiation in the genital disc requires the collaboration of these two types of genetic inputs. It is proposed that the sex-specific growth and differentiation of A8 and A9 are specified jointly by the homeotic genes and the sex-specific functions of dsx (Keisman, 2001b).

teashirt (tsh) encodes a Drosophila zinc-finger protein. Misexpression of tsh has been shown to induce ectopic eye formation in the antenna. tsh can also suppress eye development. This novel function of tsh is due to the induction of homothorax (hth), a known repressor of eye development, and requires Wingless (Wg) signaling. Interestingly, tsh has different functions in the dorsal and ventral eye, suppressing eye development close to the ventral margin, while promoting eye development near the dorsal margin. It affects both growth of eye disc and retinal cell differentiation (Singh, 2002).

Interestingly, although tsh is expressed symmetrically in the dorsal and ventral halves of the eye disc, overexpressing tsh in these regions suppresses eye development in the ventral region, while it promotes eye development in the dorsal region. Why would overexpressing tsh in a region where it is normally expressed cause phenotype reciprocal to the loss-of-function tsh mutant phenotype? It is possibly a dose effect, since the ectopic expression of two copies of tsh transgene causes a stronger effect. The normal level of Tsh may be balanced with some opposing forces for proper development, thus too little and too much of Tsh will cause reciprocal effects. A similar case is Wg, which is normally expressed in both dorsal and ventral margins. Reducing Wg level causes ectopic MF formation, while raising Wg level blocks MF initiation (Singh, 2002).

The eye-suppression function of tsh requires Wg signaling, since blocking Wg signaling by co-expressing dTCFDeltaN or sgg with tsh, or overexpressing tsh in a wgts mutant at the non-permissive temperature blocks the suppression effect. The critical time for wg involvement is 48-72 hours AEL, corresponding to the second instar larval stage. At this stage, the expression patterns of tsh, hth and wg in the eye disc overlap considerably, consistent with their functional interaction (Singh, 2002).

Tsh can induce Hth and suppress eye development only in the ventral margin of the eye disc. Internal clonal induction of tsh expression (Act>tsh) clones has no eye-suppression effects. The restriction of eye suppression to the eye disc margin, where wg is expressed, suggests that tsh does not induce wg but requires high level Wg signaling. Indeed, clonal expression of tsh internal in the eye disc does not induce Wg expression. When Tsh is co-expressed with Wg or an activated Arm, eye suppression can occur away from the margin, possibly because higher levels of Wg signaling are provided by the ectopic expression. Tsh also requires a high level of Wg to repress Ubx transcription in the embryonic midgut (Singh, 2002).

Ectopic expression of Wg in the region ahead of MF induces Hth, while blocking Wg signaling (by clonal expression of dTCFDeltaN) reduced Hth in the presumptive head region of the eye disc. These locations correspond to tsh expression domain, consistent with the Tsh-Wg collaboration. Act>hth clones can block MF initiation without inducing ectopic wg expression, also suggesting that hth acts downstream of Wg. Thus, these results suggest that Tsh collaborates with Wg signaling to induce Hth to suppress eye development (Singh, 2002).

Tsh and Wg signaling also collaborate during embryonic development. Tsh acts in the late phase of Wg signaling to promote the naked cuticle cell fate of larvae. Tsh phosphorylation and nuclear accumulation is partially promoted by Wg signaling. Hypophosphorylated Tsh can bind directly to the intracellular Arm. The effect of Tsh overexpression on embryo development is dependent on the interaction with Arm. Tsh can also associate with Sgg, an inhibitory component of Wg signaling that promotes Arm degradation and acts downstream of Sgg. Whether the same molecular interaction operates in the eye disc awaits further study (Singh, 2002).

Based on the loss-of-function phenotype and overexpression phenotype, tsh suppresses eye development only in the ventral eye, while promoting eye development in the dorsal eye. The DV difference in Tsh function is not likely to be due to wg, since wg is expressed in both dorsal and ventral margins, with even higher levels in dorsal parts. In a wg temperature-sensitive mutant, an ectopic MF initiates more on the dorsal side. Wg signaling upregulates hth in both dorsal and ventral regions of the eye disc. Thus, wg can induce hth and suppress eye development in both ventral and dorsal margins, but through different mechanisms. Tsh collaborates with Wg signaling for eye suppression only in the ventral margin, but not in the dorsal margin. Whether Wg requires other co-factors in the dorsal margin is not known (Singh, 2002).

In both sexes, the Drosophila genital disc comprises three segmental primordia: the female genital primordium derived from segment A8, the male genital primordium derived from segment A9 and the anal primordium derived from segments A10-11. Each segmental primordium has an anterior (A) and a posterior (P) compartment, the P cells of the three segments being contiguous at the lateral edges of the disc. Hedgehog (Hh) expressed in the P compartment differentially signals A cells at the AP compartment border and A cells at the segmental border. As in the wing imaginal disc, cell lineage restriction of the AP compartment border is defined by Hh signalling. There is also a lineage restriction barrier at the segmental borders, even though the P compartment cells of the three segments converge in the lateral areas of the disc. Lineage restriction between segments A9 and A10-11 depends on factors other than the Hh, En and Hox genes. The segmental borders, however, can be permeable to some morphogenetic signals. Furthermore, cell ablation experiments show that the presence of all primordia (either the anal or the genital primordium) during development are required for normal development of genital disc. Collectively, these findings suggest that interaction between segmental primordia is required for the normal development of the genital disc (Gorfinkiel, 2003).

The three segmental primordia of the genital disc are contiguous. This means that the P compartment of one primordium is adjacent to A cells of the corresponding primordium and to A cells of the following primordium. In addition, the P compartment cells of the three segments converge in lateral areas of the genital disc. Hh activates target genes in the receiving cells both behind and in front. These target genes are different on each side of its expression domain. Particularly, Hh at the posterior compartment of the male genital primordium (A9 segment) signals anteriorly, inducing Wg and/or Dpp expression in anterior cells of this primordium, and posteriorly, inducing Ptc expression. Hh also posteriorly signals anterior cells of the anal primordium (segments A10-11), inducing En expression in a narrow band of cells. Interestingly, Cad expression is reduced in these cells. A similar situation has been described in embryonic segments in which Hh activates wg at the AP border and rhomboid at the segmental border. Hh controls Wg and EGF signalling pathways on each side of its expression domain in embryos (Gorfinkiel, 2003).

Hh has a pivotal role in the morphogenesis of all imaginal discs. Ectopic Hh gives rise to duplications of parts of, or whole, appendages in the imaginal discs of the fly. In wild-type genital discs, such as in the leg and antenna, Hh induces the expression of wg and dpp in A compartment cells close to the AP border of each of the three primordia. The ectopic expression of hh in the anal primordium induces complete duplication of the genital disc with the corresponding expression of these genes in their normal expression domains. The repressed male and female primordia also seemed to be duplicated in the female and male genital discs, respectively. These results indicate again that Hh diffuses across the border between the genitalia and analia, although this border acts as a cell lineage restriction barrier. It should be noted that Hh also diffuses across the border between the embryonic segments and between the abdominal segments of adult flies. The results presented here also show that the ectopic expression of either dpp or wg in the analia also affects the development of the male and female genitalia (Gorfinkiel, 2003).

The non-autonomous effect that ectopic Dpp in the analia has on the development of the genitalia is due to diffusion of Dpp itself from the analia to the genitalia, and not to the non-autonomous effect of Dpp downstream genes. By contrast, the same effect on the development of the genitalia is observed when Wg itself or any of the downstream components of the Wg-pathway, Tcf or Arm, are ectopically expressed in the analia. Ectopic expression of Wg in the analia can either recover structures of the genitalia or can prevent the development of both genitalia and analia. These results together with the observation that no Wg protein is detected in the genitalia when it is ectopically expressed in the analia indicate that the non-autonomous effect of ectopic Wg is due to an unknown signal activated by the Wg-pathway (Gorfinkiel, 2003).

Wg spreads and acts within the embryonic epidermis of Drosophila in different ranges in anterior and posterior directions. Transport or stability is reduced in engrailed-expressing cells, and further posterior Wg movement is blocked at the presumptive segmental boundary. hh function is involved in the formation of this barrier. If Wg diffusion across the genitalia-analia border is limited, it might be established very early in development by a similar mechanism to that observed in the embryo (Gorfinkiel, 2003).

EGFR, Wingless and JAK/STAT signaling cooperatively maintain Drosophila intestinal stem cells

Tissue-specific adult stem cells are commonly associated with local niche for their maintenance and function. In the adult Drosophila midgut, the surrounding visceral muscle maintains intestinal stem cells (ISCs) by stimulating Wingless (Wg) and JAK/STAT pathway activities, whereas cytokine production in mature enterocytes also induces ISC division and epithelial regeneration, especially in response to stress. This study shows that EGFR/Ras/ERK signaling is another important participant in promoting ISC maintenance and division in healthy intestine. The EGFR ligand Vein is specifically expressed in muscle cells and is important for ISC maintenance and proliferation. Two additional EGFR ligands, Spitz and Keren, function redundantly as possible autocrine signals to promote ISC maintenance and proliferation. Notably, over-activated EGFR signaling could partially replace Wg or JAK/STAT signaling for ISC maintenance and division, and vice versa. Moreover, although disrupting any single one of the three signaling pathways shows mild and progressive ISC loss over time, simultaneous disruption of them all leads to rapid and complete ISC elimination. Taken together, these data suggest that Drosophila midgut ISCs are maintained cooperatively by multiple signaling pathway activities and reinforce the notion that visceral muscle is a critical component of the ISC niche (Xu, 2011).

Adult stem cells commonly interact with special microenvironment for their maintenance and function. Many adult stem cells, best represented by germline stem cells in Drosophila and C. elegans, require one primary maintenance signal from the niche while additional signals may contribute to niche integrity. ISCs in the Drosophila midgut do not seem to fit into this model. Instead, they require cooperative interactions of three major signaling pathways, including EGFR, Wg and JAK/STAT signaling, for long-term maintenance. Importantly, Wg or JAK/STAT signaling over-activation is able to compensate for ISC maintenance and proliferation defects caused by EGFR signaling disruption, and vice versa. Therefore, ISCs could be governed by a robust mechanism, signaling pathways could compensate with each other to safeguard ISC maintenance. The mechanisms of the molecular interactions among these pathways in ISC maintenance remains to be investigated. In mammals, ISCs in the small intestine are primarily controlled by Wnt signaling pathways, and there are other ISC specific markers not controlled by Wnt signaling. In addition, mammalian ISCs in vitro strictly depend on both EGFR and Wnt signals, indicating that EGFR and Wnt signaling may also cooperatively control mammalian ISC fate. It is suggested that combinatory signaling control of stem cell maintenance could be a general mechanism for ISCs throughout evolution (Xu, 2011).

The involvement of EGFR signaling in Drosophila ISC regulation may bring out important implications to understanding of intestinal diseases, in which multiple signaling events could be involved. For example, in addition to Wnt signaling mutation, gain-of-function K-Ras mutations are frequently associated with colorectal cancers in humans. Moreover, activation of Wnt signaling caused by the loss of adenomatous polyposis coli (APC) in humans initiates intestinal adenoma, but its progression to carcinoma may require additional mutations. Interestingly, albeit controversial, Ras signaling activation is suggested to be essential for nuclear β-catenin localization, and for promoting adenoma to carcinoma transition. In the Drosophila midgut, loss of APC1/2 genes also leads to intestinal hyperplasia because of ISC overproliferation. Given that EGFR signaling is generally activated in ISCs, it would be interesting to determine the requirements of EGFR signaling activation in APC-loss-induced intestinal hyperplasia in Drosophila, which might provide insights into disease mechanisms in mammals and humans (Xu, 2011).

Previous studies suggest that intestinal VM structures the microenvironment for ISCs by producing Wg and Upd maintenance signals. This study identified Vn, an EGFR ligand, as another important ISC maintenance signal produced from the muscular niche. Therefore, ISCs are maintained by multiple signals produced from the muscular niche. In addition, Spi and Krn, two additional EGFR ligands, were identified that function redundantly as possible autocrine signals to regulate ISCs. These observations are consistent with a previous observation that paracrine and autocrine EGFR signaling regulates the proliferation of AMPs during larval stages, suggesting that this mechanism is continuously utilized to regulate adult ISCs for their maintenance and proliferation. The only difference is that the proliferation of AMP cells is unaffected when without autocrine Spi and Krn, due to redundant Vn signal from the VM, whereas autocrine Spi/Krn and paracrine Vn signals are all essential in adult intestine for normal ISC maintenance and proliferation. It was found that Vn and secreted form of Spi have similar roles in promoting ISC maintenance and activation, but additional regulatory or functional relationships among these ligands require further investigation, as the necessity of multiple EGFR ligands is still not completely understood. It is known that secreted/activated Spi and Krn are diffusible signals, but clonal analysis data show that Spi and Krn can display autonomous phenotypes. This observation indicates that these two ligands could behave as very short range signals in the intestinal epithelium, or they could diffuse over long distance but the effective levels of EGFR activation could only be achieved in cells where the ligands are produced. Interestingly, palmitoylation of Spi is shown to be important for restricting Spi diffusion in order to increase its local concentration required for its biological function. Whether such modification occurs in intestine is unknown, but it is speculated that Vn, Spi and Krn, along with the possibly modified forms, may have different EGFR activation levels or kinetics, and only with them together effective activation threshold could be reached and sustained in ISCs to control ISC behavior. Therefore, a working model is proposed that ISCs may require both paracrine and autocrine mechanisms in order to achieve appropriate EGFR signaling activation for ISC maintenance and proliferation.

Mechanisms of JAK/STAT signaling activation is rather complex. In addition to Upd expression from the VM, its expression could also be detected in epithelial cells with great variability in different reports, possibly due to variable culture conditions. Upon injury or pathogenic bacterial infection, damaged ECs and pre-ECs are able to produce extra cytokine signals, including Upd, Upd2 and Upd3, to activate JAK/STAT pathway in ISCs to promote ISC division and tissue regeneration. Several very recent studies suggest that EGFR signaling also mediates intestinal regeneration under those stress conditions in addition to its requirement for normal ISC proliferation. Therefore, in addition to basal paracrine and autocrine signaling mechanisms that maintain intestinal homeostasis under normal conditions, feedback regulations could be employed or enhanced under stress conditions to accelerate ISC division and epithelial regeneration (Xu, 2011).

Evidence so far has indicated a central role of N signaling in controlling ISC self-renewal. N is necessary and sufficient for ISC differentiation. In addition, the downstream transcriptional repressor Hairless is also necessary and sufficient for ISC self-renewal by preventing transcription of N targeting genes in ISCs. Therefore, N inhibition could be a central mechanism for ISC fate maintenance in Drosophila. High Dl expression in ISCs may lead to N inhibition, though how Dl expression is maintained in ISCs at the transcriptional level is not clear yet. Hyperactivation of EGFR, Wg or JAK/STAT signaling is able to induce extra Dl+ cells, suggesting that these three pathways might cooperatively promote Dl expression in ISCs. It is also possible that these pathways regulate Dl expression indirectly. As Dl-N could have an intrinsically regulatory loop for maintaining Dl expression and suppressing N activation, these pathways could indirectly regulate Dl expression by targeting any component within the regulatory loop. Identifying their respective target genes by these signaling pathways in ISCs would be an important starting point to address this question (Xu, 2011).

Regulation of stem cell proliferation and cell fate specification by Wingless/Wnt signaling gradients enriched at adult intestinal compartment boundaries

Intestinal stem cell (ISC) self-renewal and proliferation are directed by Wnt/β-catenin signaling in mammals, whereas aberrant Wnt pathway activation in ISCs triggers the development of human colorectal carcinoma. This study utilized the Drosophila midgut, a powerful model for ISC regulation, to elucidate the mechanisms by which Wingless (Wg)/Wnt regulates intestinal homeostasis and development. It was shown that the Wg signaling pathway, activation of which peaks at each of the major compartment boundaries of the adult intestine, has essential functions. Wg pathway activation in the intestinal epithelium is required not only to specify cell fate near compartment boundaries during development, but also to control ISC proliferation within compartments during homeostasis. Further, in contrast with the previous focus on Wg pathway activation within ISCs, it was demonstrated that the primary mechanism by which Wg signaling regulates ISC proliferation during homeostasis is non-autonomous. Activation of the Wg pathway in absorptive enterocytes is required to suppress JAK-STAT signaling in neighboring ISCs, and thereby their proliferation. The study concludes that Wg signaling gradients have essential roles during homeostasis and development of the adult intestine, non-autonomously controlling stem cell proliferation inside compartments, and autonomously specifying cell fate near compartment boundaries (Tian, 2016).

Larval (part 1/2)

Wingless and segmentation

Very little information is available about gene expression during the larval period, a developmental interval critical to the formation of the adult. To what extent does gene expression during this period resemble that in the embryonic stages, and how does gene expression during the larval period contribute to segment polarity in the adult? In fact, all the genes expressed during embryonic segment polarity also play a similar role in the formation of the adult. Cells destined to form the cuticle of the adult abdomen are present as clusters of small, non-dividing diploid cells (the anterior dorsal, posterior dorsal and ventral histoblast nests) located at stereotyped postions in the larval epidermis. These cells, just as do their embryonic counterparts, express engrailed, hedgehog, wingless, patched, cubitus interruptus and sloppy paired in a stereotyped manner dependent on their positions within each segment. Each segment is subdivided into an anterior (A) and posterior (P) compartment, distinguished by activity of the selector gene engrailed (en) in P but not A compartment cells. The ventral epidermis of each abdominal segment forms a flexible cuticle, the pleura, with a small plate of sclerotised cuticle, the sternite, centered on the ventral midline. The pleura is covered with a uniform lawn of hairs, all pointed posteriorly, whereas the sternite contains a stereotyped pattern of bristles. Posterior compartments are to a large degree devoid of hairs and bristles, while the sternite cuticle of the A compartment consists of an anterior-to posterior progression of six types of cuticle distinguished by ornamentation and pigmentation. Just anterior to the posterior compartment, A6 is unpigmented, with hairs and none of the larger ornaments called bristles. A5 is darkly pigmented with hairs and bristles of large size. A4 and A3 are darkly and lightly pigmented respectively with moderately sized hairs and bristles. A2 is lightly pigmented with hairs, and A1, adjacent to the next more anteriorly located "posterior" compartment is unpigmented without hairs (Struhl, 1997a).

Hedgehog (Hh), a protein secreted by engrailed expressing P compartment cells, spreads into each A compartment across the anterior and the posterior boundaries to form opposing concentration gradients that organize cell pattern and polarity. Anteriorly and posteriorly situated cells within the A compartment respond in distinct ways to Hh: they express different combinations of genes and form different cell types. patched is expressed at both boundaries. patched is expressed in a graded fashion within each stripe, just anterior to each P compartment. ci peaks at high level in those cells abutting Hh- secreting cells of the P compartment and declines progressively in cells further away. wingless is also expressed in this domain and sloppy paired is expressed in the same region as wingless. decapentaplegic is expressed only in the ventral pleura in those A compartment cells neighboring P compartment cells within the same segment. dpp is not expressed immediately behind posterior compartments (Struhl, 1997).

Expression in Eye/Antennal disc

Wingless is a negative regulator of the morphogenetic furrow and affects tissue polarity in the developing Drosophila compound eye. A wave of pattern formation and cell-type determination sweeps across the presumptive eye epithelium in the morphogenetic furrow. This coordinates the cell division cycle, cell shape, and gene expression to produce evenly spaced neural cell clusters that will eventually form the adult ommatidia. As these clusters develop, they rotate inward to face the eye's equator and establish tissue polarity. Wingless is strongly expressed in the dorsal margin of the presumptive eye field, ahead of the morphogenetic furrow. Initiation of the morphogenetic furrow by Hedgehog is restricted to the posterior margin by the presence of Wingless, which represses hedgehog, at the lateral margins; removal of wingless allows lateral initiation. Inactivation of wg results in the induction of an ectopic furrow that proceeds ventrally from the dorsal margin (Treisman, 1995). This ectopic furrow is normal in most respects, however the clusters formed by it fail to rotate. A second consequence of this inactivation of wg is that the dorsal head is largely deleted. ptc loss-of-function mosaic clones induce circular ectopic morphogenetic furrows (Ma, 1995).

hedgehog and wingless both have roles in specifying adult head structures. Reduction of hedgehog activity results in flies completely lacking medial head structures, while loss of wingless results in deletion of lateral (orbital) and mediolateral (frons) head structures. Ectopic expression of hh results in the induction of ectopic ocelli at more lateral locations, while ectopic wg results in an invasion of mediolateral frons cuticle into the ocellar region. In orthodenticle mutants, specifically ocelliless regulatory mutations, wg expression fails to disappear from the medial region and instead persists across the entire primordium of the head vertex. At the same time, hh expression in lost. In a complementary fashion, hh also seems to have a positive role in otd expression; ectopic hh activates otd, suggesting that otd expression in the head vertex primordium may be activated by hh during normal eye-imaginal disc development. Thus otd is required for regional head development, and has a critical role in regulating wg and hh expression (Royet, 1996).

The eye-antennal imaginal discs of Drosophila melanogaster form the head capsule, the eyes and the antenna of the adult fly. Unlike the limb primordia, each eye-antennal disc gives rise to morphologically and functionally distinct structures. As a result, these discs provide an excellent model system for determining how the fates of primordia are specified during development. An investigation has been carried out of how the adjacent primordia of the compound eye and dorsal head vertex are specified. Subdivision of the eye-antennal disc is not based on compartmentalization: this is in contrast to the basis for subdivision in the wing and leg discs. Therefore, selector gene-mediated division of the disc into compartments, mediated by engrailed and invected, as in the wing disc for example, is not likely to be the basis for regionalization within the antennal primordium. Instead, in this region, the genes wingless and orthodenticle are expressed throughout the entire second instar eye-antennal disc, conferring a default fate of dorsal vertex cuticle. Mutations that decrease dpp expression in the eye primordia lead to the formation of severely reduced eyes. Similarly, the loss of otd or wg function in the vertex primordia causes the elimination of dorsal head structures (Royet, 1997).

Transplantation experiments show that the eye primordium occupies most of the posterior half of the eye-antennal disc (the so-called 'eye disc'). The head vertex forms from the dorsomedial region of the disc, while the antenna develops from the anterior half of the disc (the so-called 'antennal disc'). During the early third instar stage (70-80 hours after egg laying), dpp is expressed in a horseshoe-shaped domain along the ventral, posterior and dorsal periphery of the eye disc. Dorsal dpp expression does not extend as far anteriorly as ventral expression, but instead ends at the vertex primordium. At this stage, otd expression covers the vertex primordium and extends along the edge of the antennal disc. The posterior boundary of otd expression in the vertex anlage coincides, approximately, with the anterior boundary of the dpp domain. At the same stage of disc development, wg is expressed in two regions of the eye disc. One region corresponds to the future gena (the lateral part of the head capsule bounded above by the eye) and the other to the head vertex (Royet, 1997).

dpp expression prevents dorsal head development in the eye primordium. Flies homozygous for the dppd-blk allele that reduces dpp activity in the eye primordium greatly reduces the compound eye giving rise to an eye with only a few residual ommatidia. In these mutants the eyes are largely replaced by frons cuticle, which normally appears only on the dorsal areas of the head. This ectopic frons lies between the orbital cuticle and the remaining ommatidia, and to the anterior, between the shingle cuticle and the ommatidia. In other eye loss mutants, such as sine oculis or eyes absent, the eyes are completely lost but are not replaced by ectopic frons. This suggests that dorsal head cuticle does not result simply from loss of the eyes, but is caused instead by loss of dpp function. Clones of Mothers against dpp, coding for a protein involved in transmission of the Dpp signal, likewise transform ommatidia into frons (Royet, 1997).

Activation of decapentaplegic expression in the posterior eye disc eliminates wg and otd expression, thereby permitting eye differentiation. In dppd-blk mutants, the otd domain expands toward the anlagen of the shingle cuticle and the compound eyes, consistent with the location of ectopic frons cuticle on dppd-blk mutant heads. wg expression also expands in these mutant discs. Ectopic activation of the wingless pathway (the result of the generation of clones mutant for shaggy/zeste-white 3) in the eye primordium induces otd expression and vertex formation. Loss of shaggy function results in constitutively activated wg signaling and ectopic otd expression. This suggests that otd expression in the vertex primordium is normally activated or maintained by wingless. Early activation of dpp depends on hedgehog expression in the eye anlage prior to morphogenetic furrow formation. Loss of hh activity during the second instar larval stage eliminates dpp expression along the posterior and lateral margins of the eye disc and in the antennal primordium. This loss of dpp expression is associated with a dramatic expansion of the otd expression domain. wg expression also expands into the eye primordium (Royet, 1997).

Unlike the limb discs, which derive from single trunk segments, each eye-antennal disc arises from multiple embryonic head segments. Divisions between segment primordia within the disc could contribute to certain aspects of regional specification. It is proposed that wg and otd expression in the eye-antennal discs are inherited from the embryo, where the two genes are expressed in segments from which these discs are derived. The almost ubiquitous expression of these two genes serves to program the early disc for a vertex fate. Later, hh expression in the posterior region of the future eye disc induces dpp expression along the margins of the eye primordium. dpp represses wg, permitting the formation of the eye primordium (Royet, 1997).

The posteriorly expressed signaling molecules Hedgehog and Decapentaplegic drive photoreceptor differentiation in the Drosophila eye disc, while at the anterior lateral margins Wingless expression blocks ectopic differentiation. Mutations in axin prevent photoreceptor differentiation and leads to tissue overgrowth; both these effects are due to ectopic activation of the Wingless pathway. In addition, ectopic Wingless signaling causes posterior cells to take on an anterior identity, reorienting the direction of morphogenetic furrow progression in neighboring wild-type cells. Signaling by Dpp and Hh normally blocks the posterior expression of anterior markers such as Eyeless. Wingless signaling is not required to maintain anterior Eyeless expression and in combination with Dpp signaling can promote Ey downregulation, suggesting that additional molecules contribute to anterior identity. Along the dorsoventral axis of the eye disc, Wingless signaling is sufficient to promote dorsal expression of the Iroquois gene mirror, even in the absence of the upstream factor pannier. However, Wingless signaling does not lead to ventral mirror expression, implying the existence of ventral repressors (Lee, 2001).

Two characteristics distinguish anterior from posterior behavior in the eye disc: growth occurs in the anterior, with the exception of the second mitotic wave, and differentiation occurs in the posterior. Wg signaling regulates both of these properties. Wg signaling promotes the growth of eye disc cells. Loss of axin causes dramatic overgrowth and outgrowth of cells in the eye disc, and this phenotype is due only to excessive Wg pathway activity, since it can be blocked by a dominant negative form of dTCF/Pangolin. The strength of the phenotype may reflect higher levels of Wg signaling than are induced by loss of sgg; perhaps Axin contributes to retaining Arm in the cytoplasm, in addition to promoting its phosphorylation. Vertebrate Axin has been shown to associate with mitogen-activated protein kinase kinase kinase 1 and activate the c-jun N-terminal kinase (JNK) pathway. However, JNK signaling does not appear to be essential for the growth or differentiation of cells in the Drosophila eye disc, and it does not contribute to the axin mutant phenotype in the eye. The ability of Wg signaling to promote overgrowth in the eye disc is consistent with the reduction in the size of the eye disc caused by loss of Wg signaling (Lee, 2001).

Loss of axin function at the posterior margin results in outgrowths from the disc, over-riding the normal control of organ size. axin mutant clones also form smooth borders with surrounding cells, suggesting that their ability to adhere to wild-type cells is decreased. Growth control requires the formation of normal junctions between cells, so it is possible that the outgrowth results from this loss of adhesion. Because the posterior margin is the site of dpp expression prior to initiation, the outgrowth observed could also require Dpp signaling; in the leg disc, overlap between dpp and wg promotes the extension of a proximal-distal axis. However, punt;axin double mutant clones show a similar degree of overgrowth, suggesting that Dpp signaling does not contribute to this (Lee, 2001).

Early transplantation experiments and other studies have suggested that the region of the eye disc anterior to the morphogenetic furrow contains intrinsic positional information; the subsequent finding that Hh is essential for furrow movement has been taken to mean that all this information originates posterior to the furrow. The observations presented here challenge this view by showing that Wg signaling can generate a source of anterior positional information that appears to attract the morphogenetic furrow toward itself. Cells mutant for axin autonomously express ey and other markers for the region anterior to the furrow, including hth, tsh and mirr. Cells adjacent to an axin mutant clone show a reorientation of the pattern of Atonal expression and cells at the internal border of the clone express Hairless, which is likely to be activated by Dpp signaling from adjacent wild-type cells. This suggests that axin mutant cells produce a non-autonomous signal that maintains nearby cells in an Ato-expressing state; since wg-lacZ expression is activated in axin mutant clones, the signal could itself be Wg (Lee, 2001).

It is proposed that Wg, which is present throughout the first instar eye disc, initially establishes the entire eye field as anterior. The maintenance of Ey staining in anterior dsh clones implies that the anterior domain is stable without continuous Wg input; reception of Hh and Dpp is required for cells to undergo a transition from anterior to posterior. Ectopic activation of the Wg pathway in the posterior prevents this transition; the simplest explanation of this is that high levels of Wg signaling can block cells in the eye disc from responding to both Hh and Dpp. During normal development, growth may dilute Wg protein in the anterior to a level that can no longer block the action of Dpp and Hh. This system would allow a continuous transition from anterior to posterior, a necessity given the dynamic nature of the AP border. Correct AP patterning is crucially dependent on the initial expression patterns of wg, hh and dpp; however, the upstream regulators of these genes remain to be identified. hh expression in the posterior compartment of the leg and wing imaginal discs depends on en, but en is not required in eye development (Lee, 2001).

Although Wg signaling is sufficient to establish but not necessary to maintain anterior identity in the eye disc, Dpp signaling has complementary properties; it is sufficient to promote but not essential to maintain posterior identity. Ectopic Dpp can downregulate ey in the anterior, but loss of components of the Dpp pathway has a variable effect on ey expression and does not lead to hairless expression or induce reorientation of the furrow in neighboring cells. It is possible that this is due to redundancy with Hh signaling, which also has a weak and variable effect on ey expression. Clones that are mutant for cell-autonomous components of both the Hh and Dpp pathways do not differentiate, while loss of one or the other pathway only delays differentiation; however, even loss of both pathways does not reorient adjacent wild-type cells (Lee, 2001).

Downregulation of ey and photoreceptor differentiation appear to be independent events, since smo clones have a weaker effect on ey expression than Mad clones, despite their stronger effect on differentiation. Anterior Dpp can also downregulate ey over a much longer range than that over which it promotes photoreceptor differentiation, and loss of slimb downregulates ey without leading to ectopic differentiation (Lee, 2001).

The complementary effects of Wg and Dpp on AP polarity appear to be independent of one another. The effect of Dpp is not simply due to its repression of wg, and posterior Wg signaling can upregulate ey even when the Dpp pathway is also activated. However, activation of both pathways reveals the existence of another mechanism that distinguishes anterior from posterior, since the anterior of the disc is more sensitive to the effects of Dpp and the posterior is more sensitive to the effects of Wg. This leads to a striking repolarization of the eye disc when both pathways are activated, resulting in initiation of an ectopic morphogenetic furrow from the anterior margin as well as reduction or elimination of the normal posterior furrow. Since normal development requires anterior cells to be gradually converted to posterior by the action of Dpp and Hh, it is important for Dpp to overcome the effects of Wg in this region. This also underscores the importance of establishing the early expression patterns of wg and dpp (Lee, 2001).

It is not clear how differential sensitivity to the two pathways is controlled; it may require other localized factors such as Hth or Eyegone. Because the differential sensitivity is observed even when both pathways are activated at the intracellular level, it is not likely to be due to regulation of receptor levels as occurs in the wing disc. However, the intracellular dpp antagonist daughters against dpp (dad) is induced by Dpp signaling and could be present at lower levels in the anterior of the eye disc, making this region more sensitive to ectopic Dpp. No intracellular antagonist of Wg has yet been shown to be induced by the Wg pathway (Lee, 2001).

In addition to providing anterior information to the eye disc, Wg acts early in development to define its dorsal domain. Dorsal wg expression is controlled by pnr, and ectopic eye formation caused by loss of pnr. This can be blocked by restoring wg, suggesting that wg is a downstream effector of pnr. In addition, Wg signaling is necessary to maintain the expression of mirr and can induce ectopic mirr expression along the ventral margin. axin mutation has been used to clarify the relationships between pnr, wg and mirr. Small clones of cells mutant for pnr maintain mirr expression, showing that pnr does not have a direct effect on mirr, but acts through one or more non-autonomous factors. This is consistent with the expression of pnr in a smaller domain than mirr. Restoring Wg signaling to pnr mutant eye discs, by making pnr;axin double mutant clones in a Minute background, allows the expression of mirr; thus, no other factor downstream of pnr can be essential for mirr expression (Lee, 2001).

hh was expressed dorsally in early eye discs and activation of the Hh pathway in ventral ptc clones leads to ectopic mirr expression. The results are consistent with two possible roles for Hh. Dorsal hh expression could be independent of pnr and contribute to mirr activation in the absence of both pnr and axin. The dorsal domain of hh expression in the eye disc is indeed not stably established until the second larval instar, while wg and pnr are expressed dorsally from late embryonic stages. Alternatively, Hh could act downstream of pnr but upstream of wg to activate mirr. In support of this hypothesis, anterior ventral ptc clones activate mirr non-autonomously, and have also been shown to activate wg expression. Because ventral axin clones do not activate mirr expression, this mechanism would imply that Hh activates factors in addition to wg that allow ventral expression of mirr (Lee, 2001).

The restriction of mirr to the dorsal domain in axin clones must reflect either a requirement for Hh (in addition to Wg) for its activation, or the existence of ventral repressors. Ventral repression could explain the sharp expression boundary of mirr, which would otherwise be difficult to accomplish in a region of low and graded Wg activity. The most likely candidate for a ventral repressor is the Unpaired (Upd) ligand for the Janus kinase/signal transducer and activator of transcription (STAT) pathway. At third instar, upd is expressed at the optic stalk, in the center of the posterior margin, and also at the anterior ventral margin, but its removal from the optic stalk region is sufficient to derepress mirr ventrally. Consistent with the findings of this study, upd expression is not affected by ectopic Wg; it is not known whether it can be regulated by Hh, although the phenotypes of loss of upd and overexpression of hh are very similar (Lee, 2001).

Wg has three roles in early eye disc development: establishment of anterior identity, establishment of dorsal identity, and promotion of growth. Prior to furrow initiation, Pnr, expressed at the dorsal margin, activates wg expression in a broader domain; Wg then activates mirr and the other Iro-C genes throughout the dorsal compartment. Hh may contribute to the activation of these genes through Wg or act independently of Pnr. Upd, which is present at the optic stalk also contributes to the ventral repression of mirr. Mirr represses fng, forming a boundary of fng expression at the DV midline that leads to activation of N in this region. During furrow progression, Hh is expressed in the differentiating photoreceptors and Dpp in a stripe in the morphogenetic furrow. These two signals act to downregulate genes expressed in the anterior such as ey, and allow an anterior to posterior transition. Wg establishes the anterior state, probably at an earlier stage, while another factor (X) contributes to its maintenance. Other factors are necessary to modify the response to Wg in order to determine which cell fate should be specified; this is consistent with data suggesting that Wg signaling alters chromatin structure to allow access to transcription factors. A requirement for multiple signaling systems also ensures accuracy in cell fate determination (Lee, 2001).

In leg and antennal discs, the posterior compartment is maintained by engrailed, while the anterior compartment becomes asymmetric in the D/V axis, with decapentaplegic expression defining dorsal anterior leg, and wingless expression defining ventral anterior leg. Both dpp and wg are maintained in the anterior compartment by Hedgehog signaling. Unlike the wing disc, dpp is not expressed at the A/P compartment boundary. In addition, unlike wing discs, no D/V compartment has been demonstrated in legs or antennae. How are the dorsal anterior and ventral anterior territories defined and maintained? wg inhibits dpp expression and dpp inhibits wg expression in leg and eye/antennal discs. Loss of DPP signaling leads to ectopic wg expression. DPP signaling is transduced by its receptor Punt, and punt mutation results in an expansion of wg expression into the dorsal region of leg discs. The antennal portion of the eye-antennal disc is analogous to the leg disc, but inverted in the D/V and A/P axes. Thus wg, which is expressed in the ventral region of the leg disc, is expressed in the dorsal region of the wild-type antennal disc. In punt mutants, wg expression expands into the ventral domain of the antenna to form a continuous stripe along the A/P boundary from dorsal to ventral. In addition, wg expression expands from its normal location at the periphery of the eye anlage into the morphogenetic furrow. Likewise, loss of Wg signaling leads to ectopic activation of dpp transcription. This mutual repression provides a mechanism for maintaining separate regions of wg and dpp expression in a developing field. The term 'territory' is proposed to describe regions of cells that are under the domineering influence of a particular morphogen. Territories differ from compartments in that they are not defined by lineage but are dynamically maintained by continuous morphogen signaling. Thus it is thought that the anterior compartment of the leg disc is divided into dorsal and ventral territories by the mutual antagonism between WG and DPP signaling (Theisen, 1996).

The Decapentaplegic and Notch signaling pathways are thought to direct regional specification in the Drosophila eye-antennal epithelium by controlling the expression of selector genes for the eye (Eyeless/Pax6, Eyes absent) and/or antenna (Distal-less). The function of these signaling pathways in this process has been investigated. Organ primordia formation is indeed controlled at the level of Decapentaplegic expression but critical steps in regional specification occur earlier than previously proposed. Contrary to previous findings, Notch does not specify eye field identity by promoting Eyeless expression but it influences eye primordium formation through its control of proliferation. Analysis of Notch function reveals an important connection between proliferation, field size, and regional specification. It is proposed that field size modulates the interaction between the Decapentaplegic and Wingless pathways, thereby linking proliferation and patterning in eye primordium development (Kenyon, 2003).

This paper analyzes the role of Dpp and Notch in the regional specification of the eye-antennal disc. This study makes four observations: (1) domains of regional identity emerge in a complex pattern starting early in L2; (2) formation of eye and antenna primordia depend upon specific domains of dpp expression that emerge in early-L2 (eye) and mid-L2 (antenna); (3) neither Notch nor Dpp control the establishment of separate eye and antennal fields; (4) Notch can influence the establishment of an eye primordium through its control of proliferation in the eye field. Current models of regional specification have been evaluated based on these results and a new perspective on the emergence of regional identity in this tissue is presented (Kenyon, 2003).

It has been proposed that allocation of eye field and antennal field identity occurs in the latter half of L2 through the restriction of eye selectors, such as Ey, and antennal selectors, such as Dll, to distinct regions of the disc. However, two observations reported in this paper are not consistent with this interpretation: (1) Dll is not expressed ubiquitously at any time during disc development; (2) eye and antennal fields are clearly established by mid-L2 as evidenced by the restricted expression of Ey (eye field) and Cut (antennal field), and by distinct Dpp/Wg patterning centers within each field. These observations place the emergence of separate eye and antennal fields in the first half of L2 and not in the second half as previously proposed. Moreover, onset of Eya occurs in early-L2 and so is expressed by mid-L2. The beginning of eye primordium formation in early-L2, prior to the appearance of distinct fields, indicates that regional specification within this disc does not follow a two-step mechanism (i.e., establishment of separate fields followed by induction of organ primordia) but occurs in a more complex pattern. Further analysis of the transcription factors and signaling molecules active in the late-L1 and early-L2 disc is necessary to better understand how the establishment of eye field identity relates to eye primordium formation and the emergence of an antennal field (Kenyon, 2003).

The effect of field size on eye primordium formation cannot be simply mediated by Dpp but is likely due to the influence of a third signaling system, the Wingless pathway. Wg functions as a negative regulator of eye development and is known to antagonize Dpp signaling in L3 discs. This antagonistic interaction occurs at least in part at the posttranscriptional level and is likely established earlier in development. At the time of onset of Eya expression, early in L2, the sources of dpp and wg are localized to opposing regions of the eye-antennal disc -- dpp along the posterior margin and wg across the dorsal anterior region. Hence, the relative concentration of Dpp and Wg experienced by disc cells likely depends on their location within and the size of the morphogenetic field. Since Dpp induces Eya expression and Wg antagonizes Dpp signaling, field size becomes a critical variable in determining the response to Dpp/Wg signaling and thus influences eye primordium formation (Kenyon, 2003).

This model readily accounts for the changes in Eya expression observed in the various genetic backgrounds. In discs expressing Notch antagonists, dpp and wg are still expressed; however, inhibition of cell proliferation results in a smaller disc and a smaller morphogenetic field. This reduction in size changes the balance between Dpp and Wg signaling resulting in a lack of Eya induction. In this context, stimulation of cell proliferation by CycE increases field size, thus restoring relative levels of Dpp and Wg signaling compatible with Eya induction. A simple prediction of this model is that modification of Dpp/Wg signaling in favor of Dpp should restore Eya expression in small ey-Gal4 UAS-SerDN discs. This was tested by removing one wild-type copy of the wg gene and thus lowering Wg signaling in SerDN-expressing discs. In agreement with the model, Eya expression is significantly rescued in late-L2/early-L3 wg+/+ey-Gal4 UAS-SerDN discs regardless of disc/field size (Kenyon, 2003).

Different classes of photoreceptors (PRs) allow animals to perceive various types of visual information. In the Drosophila eye, the outer PRs of each ommatidium are involved in motion detection while the inner PRs mediate color vision. In addition, flies use a specialized class of inner PRs in the 'dorsal rim area' of the eye (DRA) to detect the e-vector of polarized light, allowing them to exploit skylight polarization for orientation. Homothorax plays a critical role for DRA development: hth is expressed specifically in maturating inner PRs of the DRA and maintained through adulthood. homothorax is both necessary and sufficient for inner PRs to adopt the polarization-sensitive DRA fate instead of the color-sensitive default state. Loss of hth results in the transformation of the DRA into color-sensitive ommatidia, and misexpression of hth forces color-sensitive inner PRs to acquire the typical features of polarization-sensitive DRA cells. Homothorax increases rhabdomere size and uncouples R7-R8 communication to allow both cells to express the same opsin rather than different ones as required for color vision. Homothorax expression is induced by the Iroquois complex and the Wingless (Wg) pathway. However, crucial Wg pathway components are not required, suggesting that additional signals are involved (Wernet, 2003).

Although activation of the Wg pathway (via overexpression of an activator form of Armadillo) strongly induces DRA throughout the IRO-C compartment, the DRA develops normally when Fz and DFz2, dsh, or TCF are inactivated. It is possible that low levels of wild-type protein persist long enough in the clones for DRA development to proceed, although this is unlikely considering the late onset of Hth expression. Therefore, redundant factors might exist, such as the Derailed receptor which has recently been shown to mediate Wnt5 function. Alternatively, another diffusible factor could act in parallel with the Wg/Fz pathway to induce the DRA, possibly acting downstream of Wg as a 'relay signal'. Indeed, cell nonautonomous inductive effects downstream of both wg and Arm have been reported to influence cell fate determination at the periphery of the fly retina, including the DRA (Wernet, 2003).

Wingless eliminates ommatidia from the edge of the developing eye through activation of apoptosis

The Drosophila compound eye is formed by selective recruitment of undifferentiated cells into clusters called ommatidia during late larval and early pupal development. Ommatidia at the edge of the eye often lack the full complement of photoreceptors and support cells, and undergo apoptosis during mid-pupation. This cell death is triggered by the secreted glycoprotein Wingless, which activates its own expression in peripheral ommatidia via a positive feedback loop. Wingless signaling elevates the expression of the pro-apoptotic factors head involution defective, grim and reaper, which are required for ommatidial elimination. It is estimated that approximately 6%-8% of the total photoreceptor pool in each eye is removed by this mechanism. In addition, the retinal apoptosis previously reported in apc1 mutants occurs at the same time as the peripheral ommatidial cell death and also depends on head involution defective, grim and reaper. The implications of these findings for eye development and function in Drosophila and other organisms is considered (Lin, 2004).

What purpose does the elimination of these perimeter ommatidia achieve? The answer probably lies in the connections between the six outer R cells of each ommatidium and their post-synaptic targets in the laminal layer of the optic ganglia. The lamina is organized into units called cartridges, which underlie each ommatidium and form synapses with the outer R cells. Because of the precise arrangement of the photoreceptors in the ommatidia and the curvature of the eye, a single line of sight is perceived by six different outer photoreceptors (R1-R6) residing in six different ommatidia. These R cells do not innervate the underlying cartridge; rather, each cell forms a synapse with a distinct adjacent cartridge. In this way, visual excitation in the curved surface of the retina is transformed into a smooth topographic map in the optic ganglia. Because there is not a one-to-one relationship between ommatidia and lamina cartridges, a problem arises at the edge of the eye, where there are not enough adjacent cartridges to form synapses with the perimeter ommatidia. The development of the optic ganglia is stimulated by the projection of axons from the overlying ommatidia during larval and early pupal development. The peripheral-most ommatidia are thought to induce underlying cartridges before they are eliminated. Thus, there are extra laminal targets at the eye's edge for the remaining perimeter outer R cells to innervate. Removal of ommatidia by wg-dependent PCD should minimize the number of incorrect connections that would compromise the fly's peripheral vision (Lin, 2004).

What is the signal triggering the Wg autoactivation circuit? The cells at the edge of the eye express Wg from 6 hours APF. However, Wg from these edge cells does not activate Wg in the adjacent ommatidia until 26-32 hours APF. Wg signaling alone is not sufficient to trigger Wg autoactivation, since elevated Wg expression is not observed in apc1 mutant eyes, even though the pathway is activated enough to induce R cell apoptosis. There must be some other signal(s) that triggers the wg self-activation in the perimeter ommatidia. The edge cells expressing Wg could also express another signal that would allow the Wg signal to activate Wg expression in the ommatidia at the appropriate time. This simply moves the problem back one step; i.e., what then triggers the expression/activation of this signal? An alternative is the presence of a signal that counteracts Wg signaling until 24-28 hours APF. One candidate is Ras signaling, which can block the activity of Wg in the eye. It has been previously suggested that elevated Ras signaling during larval and early pupal stages prevents overexpression of a stablized form of Arm from inducing PCD until mid-pupation. However, it was found that overexpression of an activated form of ras does not block the perimeter apoptosis. Therefore, it is thought unlikely that a decrease in the Ras pathway is the signal allowing Wg to initiate the apoptotic cascade (Lin, 2004).

Some eliminated ommatidia appear to have the full complement of cell types, so incompleteness does not appear to be the signal. However, the ommatidia destined to die are almost always smaller than normal. Small size combined with Wg from the edge cells could initiate Wg expression in the ommatidia. Another candidate for the trigger is the failure of synapse formation between the R cell neurons and their targets in the optic ganglia. Projection of the R cell axons into the ganglia is complete by early pupation, but the formation of synapses between the outer R cells and the lamina neurons does not occur until 24-38 hours APF. Perhaps the R cells from perimeter ommatidia cannot find enough post-synaptic targets because they are at the edge of the field. The absence of a retrograde signal from neurons in the optic ganglia could lead to the accumulation of Wg in these ommatidia (Lin, 2004).

A role for wingless in an early pupal cell death event that contributes to patterning the Drosophila eye

Programmed cell death (PCD) is utilized in a wide variety of tissues to refine structure in developing tissues and organs. However, little is understood about the mechanisms that, within a developing epithelium, combine signals to selectively remove some cells while sparing essential neighbors. One popular system for studying this question is the developing Drosophila pupal retina, where excess interommatidial support cells are removed to refine the patterned ommatidial array. Data is presented indicating that PCD occurs earlier within the pupal retina than previously demonstrated. As with later PCD, this death is dependent on Notch activity. Surprisingly, altering Drosophila Epidermal Growth Factor Receptor or Ras pathway activity has no effect on this death. Instead, a role for Wingless signaling is indicated in provoking this cell death. Together, these signals regulate an intermediate step in the selective removal of unneeded interommatidial cells that is necessary for a precise retinal pattern (Cordero, 2004).

In the course of examining hid mutant retinae, it was noticed that blocking cell death in the earliest pupal stages -- prior to known stages of cell death -- led to a clear increase in the number of interommatidial cells. With this in mind, pupal retinas was examined at earlier developmental stages from 14 to 24 h APF by using an antibody to the junction protein Armadillo; apoptotic cell death was also directly assessed with TUNEL staining. Prior to approximately 20 h APF, the retina is composed of a loosely patterned array of ommatidia consisting of photoreceptor neurons and cone cells; primary pigment cells (1°s) first emerge and enwrap the cone cells at 20 h APF, and secondary and tertiary pigment cells (2°/3°s) begin organizing at about this stage as well. Approximately one third of the interommatidial cells observed at 24 h APF (25°C) are selected to die by PCD during the following 10-12 h (Cordero, 2004).

Prior to 18 h APF, no significant amount of death was observed. At 18 h APF, a sharp band of death was observed in the anterior portion of the retina; some of this death is within the retina, and some is just outside the retinal field. Between 20 and 24 h APF, additional death is observed towards the middle of the retina in addition to the anterior death band. Levels of apoptosis are highest in anterior regions of the eye, but the center of the eye, for example, also contains significant levels of death. At 24 h APF, this early wave of death rapidly declines; the remaining interommatidial cells have reorganized end-to-end by this stage. At 26 h APF, the known, previously described burst of death commences. The increasing amount of TUNEL staining correlates with a decrease in the number of interommatidial cells. These results indicated that the pupal retina undergoes two separate surges of cell death that occur between 18-24 and 26-36 h APF; for convenience, these events are referred to as 'early-stage' and 'late-stage' cell death events in the pupal eye, respectively. During the early-stage death 1.8 cells are removed per ommatidia. The early-stage has not been described previously, and it was examined whether the pathways known to regulate late-stage death also regulate its predecessor (Cordero, 2004).

The baculovirus protein P35 interferes with apoptosis by binding to and inhibiting caspase activity; it is effective in inhibiting cell death including late-stage death in the Drosophila eye. Targeted over-expression of P35 with the eye-specific promoter GMR led to a near complete block of early-stage death: only a line of anterior cell death remained in GMR-p35 retinas. This result indicates that the early-stage cell death occurred by caspase-dependent apoptosis. In addition, it confirmed the assessment, based on TUNEL staining, that some of the anterior-most apoptotic cell death occurs in a region of future head cuticle just anterior to the retina (and is therefore outside of the expression domain of the GMR promoter (Cordero, 2004).

The head involution defective hid gene is a central regulator of cell death in Drosophila including late-stage cell death pathway in the retina. Hid induces PCD through activation of caspases. Retinas lacking functional hid activity looses all evidence of early-stage PCD. The number of cells within the GMR-p35 and hid-/- retinas at 20 and 21 h APF, respectively was in fact higher than the number of cells in a 18 h APF control retina. In these mutant genotypes the ommatidia are disorganized when compared with the control retinas due to the excess of cells. It was often found that hid-/- retinas are attached to what seems to be the antennal disc, suggesting that this early-stage death may include events required for separation of the eye-antennal discs. Together these results suggest that, similar to late-stage death, early-stage death is regulated by a caspase-mediated apoptosis pathway (Cordero, 2004).

The Egfr/Ras-1 pathway has been implicated in multiple stages of fly eye development including cell proliferation, survival and differentiation. Loss of function mutations in the Egfr leads to excessive cell death of the interommatidial cells. Activation of Egfr leads to activation of dRas1, which promotes cell survival by repressing the activity and expression of hid (Cordero, 2004).

Activated Egfr and dRas1V12 was expressed under the control of an inducible, heat shock promoter. As expected, late-stage cell death (26 h APF) is almost completely blocked by each transgene. Surprisingly, no alteration was seen in either the pattern of death or the cell number in 21 h APF retinas, suggesting that early-stage death is insensitive to the Egfr/dRas1 pathway. Consistent with these results, no effect on cell death was seen upon over-expression of the Egfr antagonist Argos. These findings are especially surprising because of the results indicating that hid is required for early-stage death: unlike larval or late-stage death, hid activity appears to be regulated by a Egfr-independent mechanism during early-stage cell death (Cordero, 2004).

Notch pathway signaling is also required to remove excess interommatidial cells during late-stage cell death. Using the temperature-sensitive allele Nts1, Notch function was reduced during early-stage cell death. A significant reduction in TUNEL positive cell was seen in Nts1 retinas when compared with controls; this correlates with the presence of excess cells in the Nts1 background. However, a role for Notch in directly regulating this cell death cannot be unambiguously assigned. In a normal 21 h APF retina, 1°s have emerged to enwrap the ommatidia and interommatidial cells have already re-organized. Reduction of Notch activity between 14 and 21 h APF leads to a block in 1° differentiation, a failure of interommatidial cells to re-organize, and improper ommatidia alignment. This is consistent with previous reports on the effects of reducing Notch in the developing retina (Cordero, 2004).

Reducing activity of the downstream wingless inhibitor has been shown to provoke cell death of photoreceptor neurons at late stages in the developing retina; its role during the stages of patterning and cell fate determination has not been assessed. The effects of inhibiting the wingless pathway on early-stage death in the pupal retina was tested by placing a temperature-sensitive allele in trans to a null. A small but significant decrease was observed in cell death in wgts/- retinas shifted to the non-permissive temperature beginning at the earliest steps in early-stage death. Similar results were observed in wg-/- clones. These data suggest that the Notch and wingless pathways provide a signal that is necessary to provoke early-stage cell death. Wingless localization primarily in the cone cells was identified by antibody staining, implicating these cells as the source of Wingless protein. Similar results were observed using an enhancer trap line. Interestingly, during late-stage cell death -- which requires different pathways such Egfr/dRas1 -- 1°s are required to regulate cell death. Early-stage cell death occurs through the period that the 1°s emerge, and one possibility is that the cone cells make use of a different signaling pathway to distinguish their influence on the interommatidial cells from the 1°s (Cordero, 2004).

These data indicate that the Notch and wingless pathways provide a signal that is required for cell death to occur during early-stage apoptosis in the Drosophila retina. Localization studies suggest that the cone cells provide Wingless to the surrounding interommatidial cells. In the wing, Notch and wingless regulate expression of each other at the DV boundary; loss of wingless activity leads to an increase in cell death in the wing, an activity opposite that of the loss of death observed in the retina. Both pathways are involved in early-stage death in the pupal retina, and further studies will be required to determine if mutual regulation occurs in the context of the retina as well. Surprisingly, no effects were found of Egfr/dRas-1 signaling during early-stage cell death. This observation leaves open the question as to which pathway opposes wingless and Notch activity during early-stage events; this opposition would ensure that a sufficient number of cells survive to undergo the second round of selection during late-stage death (Cordero, 2004).

Restricted teashirt expression confers eye-specific responsiveness to Dpp and Wg signals during eye specification in Drosophila

In Drosophila, the eye primordium is specified as a subdomain of the larval eye disc. The Zn-finger transcription factor teashirt (tsh) marks the region of the early eye disc where the eye primordium will form. Moreover, tsh misexpression directs eye primordium formation in disc regions normally destined to form head capsule, something the eye selector genes eyeless (ey) and twin of eyeless (toy) are unable to do on their own. Evidence suggests that tsh induces eye specification, at least in part, by allowing the activation of eye specification genes by the wingless (wg) and decapentaplegic (dpp) signaling pathways. Under these conditions, though, terminal eye differentiation proceeds only if tsh expression is transient (Bessa, 2005).

The specification of the eye primordium within the main epithelium (ME) of L2 eye discs correlates with tsh expression, suggesting that tsh might be involved in this specification. If this is the case, it would be expected that ectopic tsh expression will transform the overlaying squamous layer, the peripodial epithelium (PE) cells into an eye primordium, characterized by: (1) columnar morphology of the epithelial cells; (2) eye-specific gene expression, and (3) eye-specific response to key signaling pathways. Each of these points has been analyzed in turn by inducing the expression of tsh in marked clones of cells in the PE (Bessa, 2005).

Cells expressing tsh in the margin of the disc or in the PE overproliferate, adopt a columnar shape, with elongated nuclei, and are more densely packed than non-expressing cells. Some of these clones further show a sorting behavior, by which the tsh-expressing cells arrange themselves as hollow sacs with their apical sides pointing inwards, as monitored by expression of armadillo/ß-catenin, which localizes to adherens junctions. Such a sorting behavior is usually considered to be the consequence of the cells adopting a new identity (Bessa, 2005).

In order to test if tsh is sufficient to induce eye primordium identity in PE cells, the expression of the eye selector gene ey, as well as that of the early retinal genes eya and Dac, was examined in tsh-expressing clones. tsh-positive cells show increased Ey expression. In addition, PE tsh-expressing clones that lie close to the posterior margin activate eya and the eya target Dac, indicating that these cells adopt an eye primordium-like fate. PE clones overexpressing ey are not able to induce eya, neither are similar toy-expressing clones, in which ey expression is upregulated. In these PE clones, tsh expression is not induced. Therefore, it is concluded that neither ey upregulation nor the joint overexpression of toy and ey are able to re-specify the peripodial epithelium. In addition, overexpression of eya in PE clones do not turn Dac on either, which reinforces the idea that PE re-specification as eye primordium occurs only if tsh is expressed (Bessa, 2005).

Expression of tsh activates eya expression mostly in the center and posterior half of the PE, but not in the anterior half. Clones in this anterior region retain the expression of hth, which is normally expressed in all PE cells. Since dpp and wg are expressed in the domains of the posterior and anterior discs, respectively, it was reasoned that these differences in the response of tsh-expressing cells could be the result of these signaling pathways acting differently in anterior and posterior domains of the PE (Bessa, 2005).

To test this hypothesis, the response of normal PE cells to variations in both wg and dpp pathways was tested. Clones where the dpp pathway was hyperactivated through the expression of a constitutively active dpp-receptor, thick veins (tkvQD), or blocked by removing the signal transducer Mothers against dpp (Mad), showed no induction of eya expression or cell morphology changes. Neither did anterior clones expressing Axin, a negative regulator of the wg pathway or overexpressing wg. Nevertheless, when alterations in the dpp and wg pathways were performed in the presence of ectopic tsh, PE cells showed gene expression responses characteristic of the ME. Thus, whereas posterior tsh-expressing PE cells induce eya expression, tsh-expressing cells in which the dpp pathway has been blocked by removing Mad no longer express eya. Again, this is the behavior exhibited by tsh+ ME cells deprived of dpp signaling. Similarly, while anterior tsh-expressing PE cells retain hth expression, most clones expressing both tsh and Axin lose hth expression, as they do if Axin is expressed in the ME within the tsh domain. PE tsh+ tkv+ clones still fail to activate eya in anterior dorsal and anterior ventral regions, suggesting that even in these clones wg signaling can prevent PE re-specification. Clones of PE cells expressing tsh, tkvQD and Axin now activate eya anywhere in the disc, indicating that, in the presence of tsh, wg and dpp antagonize each other to regulate eya expression. It is noted, however, that the squamous to columnar cell shape change induced by tsh is independent of the activity of the wg and dpp pathways. These results suggest that tsh, when expressed in the PE, can reprogram this epithelial layer to respond to wg and dpp signals such that it develops in an eye primordium-specific manner (Bessa, 2005).

During the development of the eye disc, only cells of the ME will be specified as eye primordium. Although Wg and Dpp signals play essential roles during eye development, PE cells are relatively insensitive to these signaling pathways, as measured by cell survival, morphology, proliferation or gene expression changes. tsh starts being expressed in the ME around the time when the eye primordium is specified, and tsh has the potential to redirect eye disc PE cells towards eye development, an ability the eye selector genes toy and ey do not have on their own. These results indicate that the PE can be re-specified by tsh throughout most of the life of the larva. Thus, tsh-expressing clones induced during L1 and L2 induce eya and Dac expression. The transient expression of tsh during L2, or its induction by Gal4 drivers active during late-L2/L3, results in ectopic PE eyes (Bessa, 2005).

It is proposed that one way in which tsh might be involved in eye fate specification is by altering the response of eye disc cells to Dpp and Wg signals. The molecular mechanisms by which tsh might achieve this during eye development remain to be further investigated, but they might be similar to those already described during embryogenesis, where Tsh modulates wg and dpp pathways directly interacting with Armadillo, the wg signaling transducer, and with Brinker, a transcriptional repressor of the dpp pathway (Bessa, 2005).

In the eye disc, the cells specific response to wg and dpp enabled by tsh is superimposed onto the expression of eye-selector genes. Such combination of factors in turn would specify the eye primordium. The fact that Tsh and Ey have the potential to interact directly makes it possible for Ey to tether Tsh-containing transcriptional complexes to eye-specific targets genes (Bessa, 2005).

It is also observed that ato expression is induced in some of the tsh-overexpressing eye-disc cells. Therefore, tsh has the potential not only to sensitize eye disc cells to wg and dpp signals, but also to make them prone to neural differentiation. dpp and wg have been shown to regulate the spatial activation of ato to position several adult sensory organs, including the eye, within the corresponding imaginal discs. This mechanism for positioning ato would define a sensory organ prototype upon which selector genes, such as ey, would specify the final sensory type. Interestingly, the ectopic ato expression induced by tsh is not disc specific and, if tsh induction is transient, results in ectopic neurons. This ato induction might be mediated by tsh enabling cells to respond to dpp and wg (Bessa, 2005).

These results underlie the importance of the precise and dynamic spatiotemporal pattern of expression of tsh: although tsh expression must be confined to the ME layer of the eye disc, in order for eye development to proceed, tsh has to be first expressed in undifferentiated cells to be later turned off to allow retinal differentiation. The earlier paradox of tsh acting both as eye repressor and inductor, depending on the Gal4 promoters used, can now be explained as follows: Gal4 promoters that are not repressible by the gene expression changes induced upon tsh overexpression, such as ey-GAL4, will lead to sustained expression of tsh and, therefore, to a blockage of eye development. Other drivers that are turned off after tsh expression (i.e., MS1096, MD705) will mimic the situation found in the ME (that is, on/off), and in these cases, eye development will proceed. It is noted that in experiments where ey is ectopically expressed, eyes tend to develop in the proximal parts of appendages which derive from tsh-expressing domains in their respective imaginal discs. This correlation reinforces the idea of tsh as a potential eye-competence factor (Bessa, 2005).

At least three roles for tsh during eye development have been uncovered: promoting proliferation, acting as an eye repressor and acting as an eye inducer. The first two roles (proliferation and eye repression) are linked to the function of the transcription factor Hth. Thus, Tsh and Hth (together with Ey) maintain the eye disc cells in a proliferative, undifferentiated state, which is incompatible with eye differentiation. This state is kept as long as cells express hth, which is positively regulated by wg and repressed by dpp. Since tsh keeps hth on, sustaining tsh expression artificially in the disc blocks further eye differentiation. Once hth is repressed by Dpp signaling close to the MF, cells enter a preproneural state, that still maintains tsh expression, in which dpp activates the expression of retinal genes such as eya. The results suggest that tsh is required for the eye-specific interpretation of Wg and Dpp signals, and therefore for both the maintenance of proliferation and the specification of the retina. This model thus predicts that removal of the earliest tsh function (which corresponds to the most anterior regions in older discs) should result in eye loss due to either lack of proliferation or to the incorrect specification of the primordium; removal of later tsh function (which corresponds to more posterior regions of older discs) should cause a premature derepression of the eye differentiation program and excess of eye. In fact, both phenotypes have been described in tsh loss-of-function clones: eye loss and eye overgrowth. The current experiments, in which tsh function is reduced uniformly from early stages of eye development, agrees with an early role of tsh in eye specification and/or proliferation. This model of tsh function is further complicated by the fact that the dorsoventral genes also impinge on tsh function. Still, some tsh-clones show no phenotype. This might be explained by perdurance of the Tsh product, local differences in the requirement of tsh within the eye disc or the existence of compensatory functions (Bessa, 2005).

toy and ey lay atop the eye specification genetic network in Drosophila. However, neither Toy nor Ey is able to activate the expression of tsh in the PE, and tsh expression in maintained in ey mutant discs. The reverse is also true; tsh upregulates ey expression in the eye disc, but is unable to activate its expression de novo in any other disc. This indicates that tsh expression is regulated independently of the Pax6 genes in the eye disc. This situation is analogous to that of Optix, a Six3 homolog, which is expressed in the eye disc independently of ey with a pattern reminiscent of that of tsh. Nevertheless, Optix does not seem to regulate tsh; ectopic expression of Optix in the eye disc does not trigger tsh expression. Taking into account all these results, it is proposed that tsh functions in parallel to ey (and probably to toy) as an eye competence factor (Bessa, 2005).

Transformation of eye to antenna by misexpression of a single gene

In Drosophila, the eye and antenna originate from a single epithelium termed the eye-antennal imaginal disc. Illumination of the mechanisms that subdivide this epithelium into eye and antenna would enhance understanding of the mechanisms that restrict stem cell fate. This study shows that Dorsal interacting protein 3 (Dip3), a transcription factor required for eye development, alters fate determination when misexpressed in the early eye-antennal disc, and this observation has been taken advantage of to gain new insight into the mechanisms controlling the eye-antennal switch. Dip3 misexpression yields extra antennae by two distinct mechanisms: the splitting of the antennal field into multiple antennal domains (antennal duplication), and the transformation of the eye disc to an antennal fate. Antennal duplication requires Dip3-induced under proliferation of the eye disc and concurrent over proliferation of the antennal disc. While previous studies have shown that overgrowth of the antennal disc can lead to antennal duplication, these results show that overgrowth is not sufficient for antennal duplication, which may require additional signals perhaps from the eye disc. Eye-to-antennal transformation appears to result from the combination of antennal selector gene activation, eye determination gene repression, and cell cycle perturbation in the eye disc. Both antennal duplication and eye-to-antennal transformation are suppressed by the expression of genes that drive the cell cycle providing support for tight coupling of cell fate determination and cell cycle control. The finding that this transformation occurs only in the eye disc, and not in other imaginal discs, suggests a close developmental and therefore evolutionary relationship between eyes and antennae (Duong, 2008).

Dip3 is able to bind DNA in a sequence specific manner and activate transcription directly. Dip3 possesses an N-terminal MADF domain and a C-terminal BESS domain, an architecture that is conserved in at least 14 Drosophila proteins, including Adf-1 and Stonewall. The MADF domain directs sequence specific DNA binding to a site consisting of multiple trinucleotide repeats, while the BESS domain directs a variety of protein-protein interactions, including interactions with itself, with Dorsal, and with a TBP-associated factor (Bhaskar, 2002).

Antagonism between the N and EGFR signaling pathways influences developmental fate in the eye-antennal disc leading to a loss of eye tissue and the appearance of extra antennae. Although this phenotype was originally suspected to represent eye-to-antennal transformation, subsequent analysis suggests that it most likely represents antennal duplication. Specifically, the absence of the N signal leads to a failure in eye disc proliferation resulting in compensatory over-proliferation of the antennal disc and its subdivision into multiple antennae. Consistent with the idea that the extra antennae result from under-proliferation of the eye field, it was found that the phenotype was largely suppressed by over-expression of CycE to drive the cell cycle (Duong, 2008).

In this study, it was found that inhibition of eye disc growth leads to antennal duplication. But in addition, it was shown that the same treatment that leads to antennal duplication can also direct the transformation of eyes to antennae. These two phenotypes are anatomically distinct. This anatomical distinction is evident in adults: antennae resulting from antennal duplication are found anterior to the antennal foramen, while the antennae resulting from eye-to-antenna transformation are found posterior to the antennal foramen. It is also apparent in larval eye-antennal imaginal discs: antennal duplication discs exhibit multiple circular dac expression domains within a single sac of epithelium (the antennal disc), while eye-to-antennal transformation discs exhibit two or more circular dac expression domains spread over both the eye and antennal discs. The two types of discs display distinct molecular signatures as well: the antennal duplication discs exhibit duplicated Dll expression domains, while the eye discs undergoing transformation to antennae lack Dll expression (Duong, 2008).

Perhaps the most persuasive evidence that Dip3 can direct eye-to-antennal transformation is provided by the observation of eyes that are only partially transformed to antennae since is very difficult to reconcile these partial transformations with the idea of antennal duplication. In some cases, proximal antennal segments tipped with eye tissue are observed. In accord with this phenotype, some third instar larval eye discs display a central domain of Elav-positive differentiating photoreceptors surrounded by a circular dac domain (Duong, 2008).

These arguments support the idea that antennal duplication and eye-to-antennal transformation are mechanistically distinct phenomena, and the remainder of the discussion assumes this to be the case. However, the possibility that these two phenotypes are two manifestations of a single mechanism cannot be excluded. For example, the discs exhibiting duplicated Dll domains may represent complete transformations, while the discs lacking duplicated Dll domains, but containing Elav may represent partial transformations (Duong, 2008).

The data show that discs undergoing antennal duplication as a result of Dip3 expression are comprised of a severely diminished eye region and an enlarged antennal region. As shown by BrdU labeling experiments, these antennal duplication discs most likely result from suppression by Dip3 of cell proliferation in the eye field leading to overproliferation of the antennal disc. This conclusion is supported by the ability of factors that drive cell proliferation (e.g., Cyclin E) to alleviate the Dip3 misexpression defect (Duong, 2008).

Many experimental manipulations that reduce the size of the eye disc (e.g., surgical excision, induction of cell death, or suppression of cell proliferation) lead to enlargement and duplication of the antennal primordium. How might reduction of the eye field lead to antennal field over-growth? One possibility is that the eye field produces a growth inhibitory signal. Alternatively, the eye field and the antennal field may compete with each other for limited nutrients or growth factors. In support of this latter possibility, recent studies of the role of dMyc in wing development have demonstrated growth competition between groups of imaginal disc cells (Duong, 2008).

While the results imply that antennal disc overgrowth is required for antennal duplication, overgrowth is thought not to be sufficient for duplication. This conclusion derives from experiments in which an antennal disc specific driver is used to direct over-expression of CycE or Nact. This resulted in antennal overgrowth without concurrent reduction in the eye disc. In this case, antennal duplication was not observed. Thus, in addition to antennal overgrowth, antennal duplication also appears to require reduction or elimination of the eye disc. Regulatory signals from the eye disc may act to prevent antennal duplication (Duong, 2008).

The eye and antenna discs differ in several respects: (1) Specific expression of the organ-specification genes. The eye disc expresses the retinal determination gene network (RDGN) genes, including eyeless (ey), twin of eyeless (toy), eyes absent (eya), sine oculis (so), and dachshund (dac), while the antennal disc expresses Dll and hth. hth is also expressed in the eye disc but in a distinct pattern from that seen in the antennal disc. In the second instar eye disc, hth is expressed throughout the eye disc, and collaborates with ey and teashirt (tsh) to promote cell proliferation. The hth expression domain later retracts to only the anterior-most region of the eye disc. This pattern is different from the circular expression pattern observed in the antennal disc. (2) In the antennal disc, dpp is expressed in a dorsal anterior wedge and wg is expressed in a ventral anterior wedge. The intersection of Dpp and Wg signaling is required to specify the proximodistal axis in the leg and antenna. In the early eye disc, Wg and Dpp signaling may overlap. But as the disc grows in size, the wg and dpp expression domain are separated, so that there is probably no intersection between high levels of Wg and Dpp signaling. (3) Whereas the partial overlap of Dll and hth expression domains in the antennal disc is important for proximodistal axis specification, there is no Dll expression in the eye disc. Dll expression in the center of the antennal and leg discs is induced by the combination of high levels of Dpp and Wg signaling. Because there is no overlap of Dpp and Wg signaling in the eye disc, Dll is not induced (Duong, 2008).

Therefore, efficient transformation of the eye disc into an antennal disc requires at least three things: (1) repression of the eye fate pathway; (2) activation the antennal fate pathway; and (3) the intersection of Dpp and Wg signaling, mimicking the situation in the antenna and leg disc that induces proximodistal axis formation. Any one of these three conditions by itself is not sufficient. (1) Loss of the RDGN genes leads only to the loss of the eye. However, if apoptosis is blocked, or cell proliferation is induced, in the ey2 mutant (ey>p35 or ey>Nact in ey2), then Dll can be induced in the eye disc and extra antenna are formed. The induction of Dll is not ubiquitous in the eye disc, suggesting that the loss of ey does not autonomously lead to the expression of Dll and the transformation to the antennal fate. (2) Simply expressing the antennal determining genes Dll or hth in the eye disc does not change the eye fate into antennal fate. It was found that uniform expression of Dll in the eye disc (ey>Dll) resulted in mild eye reduction, whereas ey>hth completely abolished eye development. E132>Dll caused the formation of small antenna in the eye in about 46% of flies, whereas ptc>Dll and C68a>Dll induced extra antenna but not within the eye field. Therefore, although Dll and hth are important determinants for antennal identity, it is their specific spatial expression patterns that determine antennal development. (3) Creating the intersection of Wg and Dpp signaling does not change the eye into antenna. Such manipulation in the leg disc turned on vg and transdetermined the leg disc into wing disc. Therefore, the specific genes induced by Dpp and Wg signaling may depend on disc-specific factors. In the eye disc, turning on Wg signaling in the dpp expressing morphogenetic furrow only blocked furrow progression (Duong, 2008).

In this study, it was found that the ectopic expression of a single gene, Dip3, can cause eye-to-antenna transformation. Dip3 apparently satisfied all three requirements. (1) Overexpression of Dip3 repressed (non-cell-autonomously) ey and dac. The repression of ey may be due to the induction of ct. The ability of Dip3 to simultaneously repress multiple retinal determination genes is completely consistent with the many known cross-regulatory interactions between these genes. (2) ey>Dip3 turned on ct and hth. (3) By blocking cell proliferation, ey>dip3 reduced the eye field size and allowed the intersection of Dpp and Wg signaling. Furthermore, ey>Dip3 induced en, which probably created an ectopic A/P border and induced ectopic dpp/wg expression (Duong, 2008).

Interference with cell cycle progression appears to be a common link between the two phenotypes described in this study. In the case of antennal duplication, interference with eye disc growth leads to antennal disc overgrowth, which is a prerequisite for duplication. In the case of eye-to-antenna transformation, eye disc undergrowth allows the required intersection between Dpp and Wg signaling (Duong, 2008).

The observation that Dip3 misexpression can transform the eye field, but not other tissues, to an antennal fate suggests a close evolutionary relationship between the eye and the antenna. Previous studies have emphasized the homology between antennae and legs. The findings presented here that misexpression of a single transcription factor, namely Dip3, can transform eyes to antennae provides support for the notion that the eye and antenna may also, in some sense, be homologous to one another. Previous evidence in support of this idea comes from the observation that similar spatial arrangements of Wg and Dpp signaling along with a temporal cue provided by the ecdysone signal are required for the formation of the eye and the mechanosensory auditory organ. Small mechanosensory sensilla, such as Johnston's organ and the chordotonal organs (stretch receptors) are thought to represent the earliest evolving sense organs. Perhaps the eye resulted from a duplication and specialization of such a sensillum (Duong, 2008).

crinkled reveals a new role for Wingless signaling in Drosophila denticle formation

The specification of the body plan in vertebrates and invertebrates is controlled by a variety of cell signaling pathways, but how signaling output is translated into morphogenesis is an ongoing question. This study describes genetic interactions between the Wingless (Wg) signaling pathway and a nonmuscle myosin heavy chain, encoded by the crinkled (ck) locus in Drosophila. In a screen for mutations that modify wg loss-of-function phenotypes, multiple independent alleles of ck were isolated. These ck mutations dramatically alter the morphology of the hook-shaped denticles that decorate the ventral surface of the wg mutant larval cuticle. In an otherwise wild-type background, ck mutations do not significantly alter denticle morphology, suggesting a specific interaction with Wg-mediated aspects of epidermal patterning. This study shows that changing the level of Wg activity changes the structure of actin bundles during denticle formation in ck mutants. It was further found that regulation of the Wg target gene, shaven-baby (svb), and of its transcriptional targets, miniature (m) and forked (f), modulates this ck-dependent process. It is concluded that Ck acts in concert with Wg targets to orchestrate the proper shaping of denticles in the Drosophila embryonic epidermis (Bejsovec, 2012).

The ventral epidermis of Drosophila embryos is a well-established system for studying cell fate specification. At the end of embryogenesis, epidermal cells secrete a patterned array of cuticular structures that reflect the cell identities acquired in the epidermis at earlier stages of development. On the ventral surface of the larval abdomen, eight segmental belts of hook-shaped denticles alternate with expanses of flat, or naked, cuticle. Each belt contains roughly six rows of denticles, with each row displaying a characteristic size, shape and polarity. These distinct morphologies indicate unique positional values, at least some of which are imparted by signal transduction from the highly conserved Wg/Wnt growth factor pathway. During early embryogenesis, a cascade of transcription factors leads to activation of wg gene expression in segmental stripes that lie within the zone of cells that will secrete naked cuticle. Ectopic overexpression of wg across the segment, or hyperactivation of downstream components in the Wg signaling pathway, eliminates the denticle belts. Conversely, loss of wg activity causes all ventral epidermal cells to secrete denticles. The diversity of denticles is also reduced in wg null mutants, with most resembling the large denticles typical of the fifth row of the wild-type belt. Thus Wg signaling controls not only the segmental specification of naked cuticle expanses, but also generates the diversity of cell fates that give rise to the uniquely shaped denticles within the denticle belt (Bejsovec, 2012).

Denticles are formed by bundles of actin that accumulate apically and push out the apical membrane as they elongate. Incipient denticles first can be visualized as apical actin condensations in the ventral epidermal cells of stage 13 embryos, at roughly 10 hours after egg-laying (AEL). These actin condensations form preferentially along the posterior edge of the columnar epithelial cells, and over the next 2 hours become increasingly more organized and begin to elongate; during this elongation phase, microtubules become enriched at the base of the denticle and also within the core of the growing denticle. The mechanism by which the distinctive shapes of the denticles are specified is not well understood, but it requires Wg signaling between 4 and 6 hours AEL (Bejsovec, 2012).

This early phase of Wg activity stabilizes expression of engrailed (en) and its target, hedgehog (hh), in the adjacent row of cells. Wg and Hh signaling together control the expression of Serrate and rhomboid, which activate the Notch and EGF pathways, respectively, in defined rows within the segment; these gene activities are required to specify the diverse denticle types characteristic of a wild-type denticle belt (Bejsovec, 2012).

The organization of the actin-based denticle precursors and their transition to cuticular elements is directed by a set of structural proteins whose expression is controlled by the Wg-regulated transcription factor, Shaven-baby (Svb). Wg signaling represses svb, restricting its ventral expression to the domain of cells fated to secrete denticles. Ectopic svb expression in the naked region of the embryonic epidermis drives formation of apical actin extensions and subsequent production of ectopic denticles. A number of downstream targets of Svb have been identified; these include genes such as singed (sn) and forked, which encode known actin-remodeling proteins, and miniature, which encodes a membrane-anchored extracellular protein thought to mediate interaction between the cell membrane and the cuticle. However, the question remains as to how these structural proteins are deployed to form the distinct morphologies characteristic of each row of denticles. This study shows that the cytoplasmic myosin, Crinkled, interacts genetically with the Wg signaling pathway and plays a role in organizing the final shapes of the denticles during epidermal development (Bejsovec, 2012).

A genetic screen for modifiers of wg mutant phenotypes revealed an unexpected interaction between Wg signaling and the cytoplasmic myosinVIIA homolog, Ck, in shaping the denticles at late stages of embryonic development. Like other myosins, Ck/myosinVIIA has a typical actin-binding/ATPase head domain that mediates movement along actin filaments. However, the carboxy-terminus of Ck/myosinVIIA is unique in containing an SH3 domain and two FERM domains, which are shared by band 4.1, ezrin, radixin, moesin -- a family of proteins that link the actin cytoskeleton to membrane spanning proteins. These motifs are consistent with a role near the plasma membrane, possibly interacting with cell-surface receptors and/or adherens junctions. This raises the possibility that Ck may be involved in the association between the actin bundles of the incipient denticles and the apical membrane, where it could link the actin cytoskeleton to extracellular components of the cuticle through transmembrane proteins such as Miniature. Either loss or gain of function for the Wg target gene svb alters denticle morphology in the ck mutant background. Therefore, it is proposed that Ck myosin may help to distribute the products of some Svb target genes, such as Miniature, and thus facilitate the final morphology of the developing denticle. Genetic data suggest that wild-type Ck provides a buffering mechanism for the incorrect Svb target levels that accumulate in a wg null mutant (Bejsovec, 2012).

Wg signaling is most commonly associated with specifying the naked cuticle cell fate, but its other role in generating diverse denticle morphologies allowed requirements for Ck function in this process to be detected. The morphogenesis role requires lower levels of Wg signaling, as evidenced by weak mutations such as wgPE2 that can generate diversity but cannot specify naked cuticle cell fate. The finding that levels of svb and its target genes influence denticle morphology suggests that Wg signaling may generate denticle diversity in conjunction with Notch and EGF signaling by producing subtly graded differences in svb expression that are below the limits of current detection methods. Temperature shift experiments suggest that this is a continuing, independent role for Wg signaling, as it is detected after the point at which Wg input regulates the pattern of Serrate and rhomboid expression. It is proposed that late Wg signaling helps titrate the synthesis of svb target gene products to optimal levels required for shaping the denticles. The ck mutant provides a sensitized background that may allow further investigation of this possibility. The enhancement of denticle morphology defects along the dorsolateral edges of the denticle field also suggests input from dorsoventral patterning pathways, such as Dpp signaling (Bejsovec, 2012).

MyosinVIIA in humans is known to play a crucial role in hearing (reviewed by Hasson, 1999; Maniak, 2001; Petit, 2001; Dror, 2009). Stereocilia on the hair cells of the inner ear transduce the mechanical stimulation of sound waves into electrical impulses. Stereocilia are stabilized by bundles of actin filaments and microtubules, much like the denticles and bristles that decorate the fly epidermis. Mutations in the human myosinVIIA are associated with Usher syndrome, the most common hereditary deafness/blindness disorder, which results in disorganized stereocilia that cannot transduce sound. The precise role of myosinVIIA in organizing and maintaining these structures is as yet unknown. However, ck mutants in the fly also are deaf, and show morphological changes in the auditory sensory structures (Todi, 2005), suggesting that the fly is a powerful model system for exploring this aspect of myosinVIIA function. Indeed, the connection between Ck and Miniature may also be relevant to human hearing disorders. Mutations in α-tectorin, a human protein that shares functional domains with Miniature and organizes extracellular matrix in the cochlea, are associated with hereditary hearing loss (Bejsovec, 2012).

Cross regulation of intercellular gap junction communication and paracrine signaling pathways during organogenesis in Drosophila

The spatial and temporal coordination of patterning and morphogenesis is often achieved by paracrine morphogen signals or by the direct coupling of cells via gap junctions. How paracrine signals and gap junction communication cooperate to control the coordinated behavior of cells and tissues is mostly unknown. This study found that Hedgehog signaling is required for the expression of wingless and of Delta/Notch target genes in a single row of boundary cells in the foregut-associated proventriculus organ of the Drosophila embryo. These cells coordinate the movement and folding of proventricular cells to generate a multilayered organ. hedgehog and wingless regulate gap junction communication by transcriptionally activating the innexin2 gene, which encodes a member of the innexin family of gap junction proteins. In innexin2 mutants, gap junction-mediated cell-to-cell communication is strongly reduced and the proventricular cell layers fail to fold and invaginate, similarly as in hedgehog or wingless mutants. It was further found that innexin2 is required in a feedback loop for the transcriptional activation of the hedgehog and wingless morphogens and of Delta in the proventriculus primordium. It is proposed that the transcriptional cross regulation of paracrine and gap junction-mediated signaling is essential for organogenesis in Drosophila (Lechner, 2007).

In both vertebrates and invertebrates, the posterior foregut constitutes a center of organogenesis from which gut-associated organs such as the lung in vertebrates or the proventriculus in Drosophila develop. Proventriculus development involves the folding and invagination of epithelial cell layers to generate a multiply-folded organ. Two cell populations, the anterior and the posterior boundary cells, were shown previously to control cell movement and the folding of the proventriculus organ. In the posterior boundary cells, which organize the endoderm rim of the proventriculus, the JAK/STAT signaling cascade cooperates with Notch signaling to control the expression of the gene short stop encoding a cytoskeletal crosslinker protein of the spectraplakin superfamily. Thereby the Notch signaling pathway is connected to cytoskeletal organization in the posterior boundary cells, which have to provide a stiffness function to enable the invagination of the ectodermal foregut cells. The findings in this paper provide evidence that hedgehog is essential for the Notch signaling-dependent allocation of the anterior boundary cells. In amorphic hedgehog mutants, evagination and the formation of the constriction at the ectoderm/endoderm boundary are not affected, however, the inward movement of the anterior boundary cells is not initiated at the keyhole stage. The lack of cell movement of the ectodermal proventricular cells is consistent with the finding that hedgehog specifically controls Notch target gene activity in the anterior boundary cells. Genetic experiments further identify wingless as a target gene of hedgehog in the anterior boundary cells. wingless, in turn, controls the transcription of the innexin2 gene, which is expressed in the invaginating proventricular cells. When wingless is re-supplied in the genetic background of hedgehog mutants, innexin2 expression is rescued, providing further evidence that innexin2 is a target gene of wingless in the proventriculus primordium. Innexin2 encodes a member of the innexin family of gap junction proteins and is essential for the development of epithelial tissues. In the proventriculus, innexin2 mRNA is initially expressed in the early evagination stage in a broad domain covering both the ectodermal and endodermal precursor cells of the proventriculus primordium. When the ectodermal cells start to invaginate into the proventricular endoderm, innexin2 expression is upregulated in the ectodermal cell layer. Invagination of the ectodermal cells fails in hedgehog, wingless and kropf mutant proventriculi and dye tracer injection experiments demonstrate that hedgehog and kropf mutants show a strong reduction of gap junction communication. These data suggest that the direct coupling of cells via Innexin2-containing gap junctions, which are induced in response to hedgehog and wingless activities, is important for the coordinated movement of the ectodermal cell layer. It is known from extensive studies in mammals that the coupling of cells and tissues via gap junctions enables the diffusion of second messengers, such as Ca2+, inositol-trisphosphate (IP3) or cyclic nucleotides to allow the rapid coordination of cellular behavior during morphogenetic processes such as cell migration and growth control. Cell movement and folding involves a modulation of cell adhesion and of cytoskeletal architecture of the proventricular cells. A functional interaction of innexin2 with the cell adhesion regulator DE-cadherin, which is a core component of adherens junctions has been shown recently by co-immunoprecipitation, yeast two-hybrid studies, and genetic analysis. In mutants of DE-cadherin, Innexin2 is mislocalized and vice versa suggesting that the regulation of cell adhesion and gap junction-mediated communication may be linked. Similar evidence for a coordinated regulation of connexin activity and N-cadherin has been obtained in mammals during migration of neural crest cells (Lechner, 2007).

In kropf mutants or innexin2 knockdown animals, hedgehog, wingless and Delta transcription is strongly reduced as shown by in situ hybridization and by quantitative RT PCR experiments using mRNAs isolated from staged embryos. Furthermore, hedgehog, wingless and Delta are ectopically expressed and their mRNA is upregulated in embryos in which innexin2 is overexpressed. In summary, these experiments provide strong support that the gap junction protein Innexin2 plays an essential role enabling or promoting transcriptional activation of hedgehog, wingless and Delta. These data point towards an essential requirement of gap junction communication for the transcriptional activation of morphogen-encoding genes activating evolutionary conserved signaling cascades essential for patterning in animals. It is of note that gap junctions are established at very early stages of embryonic development, correlating with a maternal and zygotic expression of innexin2 and other innexin family members. kropf mutant animals, which are devoid of maternal and zygotic innexin2 expression are early embryonic lethal and develop no epithelia, consistent with a fundamental role of gap junctions in development, on top of which pattern formation of tissues and organs may occur. It has been shown previously that gap junctions are essential for C. elegans, Drosophila, and vertebrate embryogenesis from early stages onwards (Lechner, 2007 and references therein).

In the nematode C. elegans, a transient network formed by the innexin gap junction protein NSY-5 was recently shown to coordinate left-right asymmetry in the developing nervous system. Previous findings in chick and Xenopus laevis embryos have suggested an essential role of connexin43-mediated gap junction for the determination of the left-right asymmetry of the embryos. Treatment of cultured chick embryos with lindane, which results in a decreased gap junctional communication, frequently unbiased normal left-right asymmetry of Sonic hedgehog and Nodal gene expression, causing the normally left-sided program to be recapitulated. An important role of connexin43 (Cx43)-dependent gap junction communication for sonic hedgehog expression was also observed in limb patterning of the chick wing. Additionally, modulation of gap junctions in Xenopus embryos by pharmacological agents specifically induced heterotaxia involving mirror-image reversals of the heart, gut, and gall bladder. These data in combination with the current findings indicate that the transcriptional regulation of hedgehog and other morphogen-encoding genes by gap junction proteins may be evolutionary conserved between deuterostomes (vertebrates) and protostomes (Drosophila), although the Drosophila innexin gap junction genes share very little sequence homology with the connexin genes. The molecular mechanism underlying innexin2-mediated transcriptional regulation of hedgehog, wingless and Delta is not clear. It has been proposed that the nuclear localization of the carboxy-tail of connexin43 may exert effects on gene expression and growth in cardiomyocytes and HeLa cells. This would infer a cleavage of connexin43 to release the C-terminus, however, in vivo evidence for this event is still lacking. Sequence analysis reveals a nuclear receptor recognition motif within the C-terminus of Innexin2. It has been demonstrated that this recognition motif mediates the interaction of coactivators with nuclear receptors. However, there is no immunohistochemical evidence for a nuclear localization of Innexin2 or the Innexin2 C-terminus in Drosophila embryonic cells indicating that a direct involvement of Innexin2 in regulating transcription of target genes may not occur. The direct association of a transcription factor with gap junctions has been recently proposed for the mouse homolog of ZO-1-associated nucleic acid-binding protein (ZONAB). This transcription factor binds to ZO-1, which is associated with oligodendrocyte, astrocyte and retina gap junctions. It is possible that innexin2-dependent transcriptional regulation may involve a similar type of mechanism: a still unknown transcriptional regulator associated with the C-terminus of innexin2-containing gap junctions could be released upon modulation of gap junction composition thereby modulating the transcription of innexin2-dependent target genes (Lechner, 2007).

miR-965 controls cell proliferation and migration during tissue morphogenesis in the abdomen

Formation of the Drosophila adult abdomen involves a process of tissue replacement in which larval epidermal cells are replaced by adult cells. The progenitors of the adult epidermis are specified during embryogenesis and, unlike the imaginal discs that make up the thoracic and head segments, they remain quiescent during larval development. During pupal development, the abdominal histoblast cells proliferate and migrate to replace the larval epidermis. This study provides evidence that the microRNA, miR-965, acts via string and wingless to control histoblast proliferation and migration. Ecdysone signaling downregulates miR-965 at the onset of pupariation, linking activation of the histoblast nests to the hormonal control of metamorphosis. Replacement of the larval epidermis by adult epidermal progenitors involves regulation of both cell-intrinsic events and cell communication. By regulating both cell proliferation and cell migration, miR-965 contributes to the robustness of this morphogenetic system (Verma, 2015).

The findings of this study link regulation of the miR-965 microRNA to the onset of histoblast proliferation at the larval to pupal transition. Previous reports have provided evidence that Ecdysone signaling activates string expression to trigger the onset of histoblast proliferation at the beginning of pupal development (Ninov, 2009). The current findings provide evidence that Ecdysone signaling works though regulation of miR-965, which in turn regulates string. Interestingly, evidence was also found for negative feedback regulation of miR-965 on EcR. Mutual repression circuitry of this type can contribute a switch-like function: EcR activity lowers miR-965 activity, which allows greater EcR expression/activity by alleviating miR-965 mediated repression. In a circuit of this design, there will be a delay between reduced transcription of the miRNA primary transcript and the decay of the mature miRNA product. Hence sustained EcR activity is needed to throw the switch (Verma, 2015).

EcR shows positive transcriptional autoregulation and this is buffered by miR-14 in a mutual repression circuit (Varghese, 2007). Positive feedback allows for a sharp switch-like response, but also makes the system very sensitive to stochastic fluctuation in EcR activity. Coupling EcR positive auto-feedback to miRNA-mediated repression allows a robust switch function upon Ecdysone stimulation, while protecting the system from the effects of biological noise. This study provides evidence that miR-965 plays an analogous role in regulating EcR response and suggests that miR-965 confers robustness to the EcR response in the histoblasts (Verma, 2015).

Upregulation of string in the miR-965 mutant contributes to the defects in histoblast proliferation. How misregulation of string might contribute to the migration defects is less immediately obvious. Previous work has shown that cell cycle progression in the histoblast population is required to trigger programmed cell death in the surrounding larval epidermal cells (LECs). Evidence has been provided that cell growth and the expansion of the histoblast nests may be required to elicit LEC apoptosis. Although the mechanism by which expansion of the histoblasts triggers LEC death is not clear, elevated string expression in the miR-965 mutant is likely to be responsible for the cell cycle progression defects during this phase, hindering normal LEC removal and histoblast migration (Verma, 2015).

Persistence of the LECs might also be a consequence of the increased expression of Wg protein in the mutant histoblast nests. Wg acts in combination with EGFR and Dpp signals to control abdominal segment patterning. These signals are thought to control differential cell adhesion, which may be important for elimination of the LECs as well as for proper segmental fusion of the histoblast nests. Elevated expression of Wg protein may lead to an expanded range of action, perhaps resulting in ectopic Wg activity in the LECs (Verma, 2015).

Each adult abdominal segment has a well-defined anterior-posterior polarity. Wg is required from 15–20 hr APF for bristle formation and from 18–28 hr APF for tergite differentiation and pigmentation. Overexpression of wg has been shown to cause ectopic bristle formation, and shaggy mutant clones, which constitutively activate wg signaling, can cause polarity reversal in abdominal bristles, while EGFR, FGF, dpp and Notch signaling have no effect on the polarity of bristles in adult epidermis. Wg levels are normally higher in the posterior region of the anterior histoblast nests and lower more anteriorly. The current finding that Wg levels were elevated and that the distribution of Wg was broader than normal suggests ectopic Wg activity throughout the histoblast nest, including cells that normally experience low Wg levels. Ectopic spread of Wg could be responsible for the formation of ectopic bristles and for the occasional instances of polarity reversal observed in the anterior part of tergites in the miR-965 mutants (Verma, 2015).

Replacement of the larval epidermis during metamorphosis involves regulation of both cell-intrinsic events in the abdominal histoblasts and communication between histoblasts and the larval cells they will replace. miR-965 acts on at least two separate processes required during histoblast morphogenesis. A miRNA with multiple targets can add a layer of regulation, acting across different pathways to integrate their activities. In doing so, the miR-965 miRNA appears to contribute to the robustness of this complex morphogenetic system (Verma, 2015).

Components of Intraflagellar Transport complex A function independently of the cilium to regulate canonical Wnt signaling in Drosophila.

The development of multicellular organisms requires the precisely coordinated regulation of an evolutionarily conserved group of signaling pathways. Temporal and spatial control of these signaling cascades is achieved through networks of regulatory proteins, segregation of pathway components in specific subcellular compartments, or both. In vertebrates, dysregulation of primary cilia function has been strongly linked to developmental signaling defects, yet it remains unclear whether cilia sequester pathway components to regulate their activation or cilia-associated proteins directly modulate developmental signaling events. To elucidate this question, this study conducted an RNAi-based screen in Drosophila non-ciliated cells to test for cilium-independent loss-of-function phenotypes of ciliary proteins in developmental signaling pathways. The results show no effect on Hedgehog signaling. In contrast, the screen identified several cilia-associated proteins as functioning in canonical Wnt signaling. Further characterization of specific components of Intraflagellar Transport complex A uncovered a cilia-independent function in potentiating Wnt signals by promoting β-catenin/Armadillo activity (Balmer, 2015).

A systematic screen was perfomred to test for potential roles of ciliary proteins in non-ciliated epithelial cells and developmental signaling pathway contexts. This approach identified roles for several cilia-associated proteins in regulating N, Wg, and EGFR signaling, demonstrating cilia-independent functions in vivo in Drosophila. Ciliary proteins have no effect on Hh signaling in non-ciliated cells (Balmer, 2015).

Components of the Hh/Shh pathways are highly conserved between Drosophila and vertebrates, with the main difference being that Shh/Hh signaling takes place in the cilium in vertebrate cells. The lack of a requirement for conserved ciliary proteins in Hh signaling in Drosophila epithelial cells thus suggests that the cilium provides a structural compartment for transducing Hh signals, rather than that specific cilia-associated proteins are required within the pathway. Consistent with this, it was recently demonstrated that Hh-signaling components are localized within the cilium in ciliated Drosophila neuronal cells (Kuzhandaive, 2014). The IFT-B protein IFT25, which has no effect on cilia assembly but is required for Hh signaling in vertebrates, is the exception. However, IFT25 is not conserved in Caenorhabditis elegans or Drosophila. The importance of IFT25 suggests that moving Shh-signaling components along the axoneme (in and out of the cilium) is also critical for Shh signaling in vertebrates, which is again consistent with the notion that Shh signaling needs to take place inside the cilium when cilia are present (Balmer, 2015).

Evolutionary aspects of the Hh/Shh pathway components, studied in planarians, revealed that Hh signaling might have originally been organized by the cilium, with cilia serving as a signaling compartment. The evolutionary loss of IFTs in planaria does not affect Hh signaling, consistent with the notion that the cilium serves as a Hh/Shh signaling hub but that specific components of ciliogenesis are not required for Hh signaling per se (Balmer, 2015).

This screen for developmental signaling requirements of ciliary proteins in non-ciliated cells identified several such factors as being required for canonical Wg/Wnt signaling. Previous studies have proposed conflicting effects of the loss of cilia on canonical Wnt signaling in vertebrates, ranging from reduction or loss of signaling to overactivation of the Wnt pathway. These different, or even opposing, outcomes were possibly caused by pathway crosstalks, context-specific interactions, or both. For example, Shh signaling and Wnt signaling often mutually affect each other, and when cilium biogenesis is impaired, Shh signaling is reduced or abolished. Thus, the potential positive effect of impaired ciliary function on Wnt signaling could be secondary to the loss of Shh signaling, leading to an overactivation of Wnt signaling in ciliary mutants. In particular, mutations in a dynein subunit of the retrograde transport complex (Dync2h1) have been shown to display cell-type-specific effects on cilium integrity and canonical Wnt signaling. The dync2h1 mouse embryonic fibroblasts (MEFs) and regions of dync2h1 mutant embryos that exhibit loss of cilia, and thus loss of Shh signaling, induce overactivation of Wnt signaling. However, in dync2h1 mutant embryos or small interfering RNA in MEFs that disrupt retrograde transport but leave the cilium intact (and thus do not disrupt Shh signaling) display reduced canonical signaling levels (Balmer, 2015).

This study demonstrates that a subset of IFT-A components, as well as other ciliary proteins, act positively in canonical Wnt/Wg signaling in a cilium-independent manner. This is consistent with the preceding data, in particular the effects of the dync2h1 mutants that leave cilia intact. Importantly, these data provide insight into the role of cilia-associated proteins outside of the ciliary structure. This mechanistic study in Drosophila has focused and the IFT-A proteins, and it was demonstrated that a subset of these is required for stabilization, localization, or both of β-cat/Arm prior to its activity in the nucleus. Taken with the previous observations, this non-ciliary function is likely conserved in vertebrates. Due to the omnipresence of primary cilia in vertebrate cells and the associated difficulty of uncoupling ciliary and non-ciliary functions of IFT-A proteins, Drosophila provides a unique and ideal model system to dissect their non-ciliary function. These data also suggest that IFT-A functions in a different configuration in ciliary versus non-ciliary contexts, because IFT144 (although efficiently knocked down) did not affect any detectable aspects of developmental signaling pathways tested. IFT144 is a core structural component of IFT-A in cilia, thus suggesting a different composition of IFT-A outside of the ciliary compartment. However, IFT43 and IFT121, which are described as peripheral within IFT-A in the cilium, show effects (although the IFT121 effects are generally weaker, both in KDs and in mutants, as compared to other IFT-A components), suggesting that some aspects of IFT-A are nonetheless preserved between the cilia and the cytoplasmic locations (Balmer, 2015).

Can these observations be related to disease aspects of the spectrum of ciliopathies? Mutations in human IFT-A components cause a specific subcategory of ciliopathies, called skeletal ciliopathy, which are characterized by limb morphogenesis defects, as well as extra-skeletal abnormalities, including retinal or renal defects. IFT-A mutations often lead to Shh/Hh pathway disruption, which in turn induces skeletal morphogenesis problems similar to the ones observed in skeletal ciliopathies. Importantly, the canonical Wnt-signaling pathway has been proposed to act downstream of Hh and bone morphogenetic protein signaling in bone morphogenesis, and it is therefore possible that defects observed in these syndromes are due to impaired Hh and Wnt signaling. Retinal and renal defects can also be induced by defective Wnt signaling levels. This work supports this concept and adds insight into the function of ciliary proteins with regards to Wnt signaling (Balmer, 2015).

It remains unclear whether the cytoplasmic IFT-A proteins (in their possibly altered configuration) associate with microtubular structures and whether such an association is required for function in Wnt/Wg signaling. Interestingly, a role for kinesin II in Wg signaling and the transport of *beta:-cat/Arm was recently reported (Vuong, 2014), consistent with a link to microtubules. One hypothesis is that the IFT-A proteins associate with microtubules and play a similar role to Costal-2 (Cos-2)/Kif7 in Hh signaling. In the absence of Hh signals, Cos-2 binds to the Hh/Shh effector Ci/Gli and tethers it to microtubules, together with Fused, Suppressor of Fused, and several kinases. Hh activation reverses this binding and frees Ci from the complex. Cos-2 is a negative regulator of Hh signaling, and the current data suggest that IFT-A proteins act antagonistically to the destruction complex by promoting β-cat/Arm stabilization. Sequestering Arm away from the destruction complex to prevent its phosphorylation and allow activation of the pathway makes this an attractive hypothesis for future study (Balmer, 2015).

Wingless mediated apoptosis: How cone cells direct the death of peripheral ommatidia in the developing Drosophila eye

Morphogen gradients play pervasive roles in development, and understanding how they are established and decoded is a major goal of contemporary developmental biology. This study examined how a Wingless (Wg) morphogen gradient patterns the peripheral specialization of the fly eye. The outermost specialization is the pigment rim; a thick band of pigment cells that circumscribes the eye and optically insulates the sides of the retina. It results from the coalescence of pigment cells that survive the death of the outermost row of developing ommatidia. This study investigated here how the Wg target genes expressed in the moribund ommatidia direct the intercellular signaling, the morphogenetic movements, and ultimately the ommatidial death. A salient feature of this process is the secondary expression of the Wg morphogen elicited in the ommatidia by the primary Wg signal. Neither the primary nor secondary sources of Wg alone are able to promote ommatidial death, but together they suffice to drive the apoptosis. This represents an unusual gradient read-out process in which a morphogen induces its own expression in its target cells to generate a concentration spike required to push the local cellular responses to the next threshold response (Kumar, 2015).

This paper used the Drosophila eye as a model system with which to study how morphogen gradients can be converted into sharply constrained tissue patterns. The action of the Wg morphogen gradient was examined and it was asked how the highest threshold response, the death of the peripheral ommatidia, is orchestrated. Three observations argue that the secondary Wg expressed by the cone cells combines with the primary Wg from the head capsule to generate a sufficient concentration to kill the ommatidia. First, when the Wg pathway is activated in all cone cells (pros->arm* were arm* is an N-terminally truncated form of Armadillo, a constitutive, cell autonomous activator of the Wg transduction pathway) there is an extended zone of apoptosis in the region where the primary Wg source is known to be high. Second, when the secondary Wg (that secreted by the cone cells) is removed the extended band of ommatidial death is lost. Third, when a level of Wg equivalent to that normally found in the peripheral regions is supplied to pros-arm* eyes all ommatidia now die. Thus, this represents a novel gradient read-out mechanism in which the primary morphogen (Wg derived from the head capsule) elicits a secondary morphogen expression (Wg expressed by the cone cells) in the target cells. Thereafter, the two sources unite to generate the high local morphogen concentration needed to direct the appropriate cell behaviors at that position (Kumar, 2015).

If there is a permissive zone in the periphery (∼3 ommatidial rows) in which the ommatidia will die if cone cell Wg expression occurs, then this raises the question of how the cone cells responses are normally tightly restricted to the peripheral-most row of ommatidia to ensure that only these ommatidia die. The following describes 1he mechanisms likely responsible for this restriction (Kumar, 2015).

(1) The high threshold of the ommatidial response: It is surmised that the cone cells have a high threshold response to the morphogen, and the initial responses to the primary Wg source (diffusing from the head capsule) is restricted to the outermost ommatidia. However, it can be envisioned that the secondary Wg secreted by the outer cone cells could diffuse and elicit the same output in the next ommatidial row, and an extreme view could see a relay mechanism in which even more internal rows of ommatidia could express Wg in their cone cells (Kumar, 2015).

(2) The role played by Notum:
The expression of Notum is similar to Snail family transcription factors in that it is expressed in the cone cells and 2°/3° PCs of the outermost ommatidia, and since Notum functions to inhibit the free diffusion of Wg, it likely acts to prevent Wg diffusion into more interior ommatidia. Indeed previous studies have shown that in notum mutant clones the zone of death expanded out into more interior rows. Thus Notum (and other mechanisms for preventing Wg diffusion) is seen as playing a critical role in restricting the ommatidial death to the outermost row of ommatidia (Kumar, 2015).

(3) Combining the high threshold response with the restriction of Wg diffusion: Consider the primary Wg diffusing from the head capsule. It enters the outer row of ommatidia and is of sufficient concentration to elicit the appropriate responses (the various expressions in the cone cells and 2°/3° PCs) but not at a level high enough to kill the ommatidia. The cone cells of the outermost row now begin to secrete the secondary Wg, but the concomitant expression of Notum by the cone cells and 2°/3° PCs of these ommatidia provide a barrier to the movement of both the primary and secondary sources of Wg. This restriction of Wg movement not only protects the more internal ommatidia, but ensures that the high levels of morphogen are constrained in the outermost ommatidia to provide the requisite signal for apoptosis (Kumar, 2015).

In addition to uncovering the synergy between the Wg derived from the head capsule and the cone cells, a number of phenomena relating to the behavior of the various cell types have been detected (Kumar, 2015).

(1) The early cone cell death: Following the collapse of the cone cells, the ommatidial apoptosis program begins with the death of cone cells themselves, followed ∼two hours later by the other ommatidial cells. This precocious cone cell death may represent a lower apoptosis threshold for these cells, but it is noted that they are sources of Wg secretion and likely experience autocrine and paracrine (between cone cells of the same ommatidium) Wg signaling as they collapse, and as such are more likely to reach the critical Wg activation level before the other cells (Kumar, 2015).

(2) The cone cell immunity to death: In pros-arm* eyes, in which all cone cell nuclei fall to the photoreceptor layer and express Wg, there is a wide swath of extended death at the periphery in which all cells of the ommatidia die (including the cone cells). But upon prevention of the cone cell nuclear fall by the expression of esg RNAi, the cone cells survive while the photoreceptors in the extended peripheral zone still die. In these ommatidia, levels of Wg needed to drive apoptosis are achieved, but the cone cells appear invulnerable to it. Whether this invulnerability results from the absence of Snail family transcription factors needed to prime the cone cells for the death signal, or whether by remaining in the apical location they somehow avoid the full level of Wg exposure remains unclear (Kumar, 2015).

(3) The fall of the cone cell nuclei: The maintenance of cone cell cell-bodies in the appropriate apical location is seemingly critical for the ommatidial stability and integrity, as their fall leads to the disruption of corneal lens units and delamination of photoreceptors. This fall appears to be directed by their expression of Snail family transcription factors. In pros-arm* eyes, the expression of esg.RNAi prevents the fall, and correspondingly the ectopic expression of esg in otherwise wild type cone cells engenders their nuclear fall (albeit prematurely). It was asked whether the fall of the cone cell nuclei resulted from a wholesale collapse of the apical junctions of the cone cells, but D/E-cadherin staining showed a normal apical junction pattern many hours after the nuclei had migrated basally. Thus it does not appear that the cone cells nuclei move basally because the cells lose their apical attachments, rather it is inferred that expression of the Snail family transcription factors reprograms some other behaviors of the cone cells. Such a behavior could be a switch in cell-type affinity. If cone cells normally maintain an apical location by adhesive differences with the photoreceptors, and if these adhesive differences are switched, then cone cell plasma membranes will then preferentially move to the photoreceptor layer. Since the nucleus defines the site of maximum cell body profile with corresponding maximum membrane area, then the fall of the nuclei may simply result from the cone cells acquiring an adhesive affinity with the photoreceptors. Other mechanisms can also be envisaged, in which, for example, motor machinery of the cell is used to reposition the cone cell nuclei in the more basal location (Kumar, 2015).

An appropriate Gal4 driver line is not available to activate gene expression selectively in the 1° PCs, and the mechanism of their death remains unresolved. In GMR.wg eyes, their death was observed coincident with the photoreceptors (following the apoptosis of the cone cells) and it is surmised that it is the high level of Wg derived from head capsule and the cone cells that directs their death. However, there are a number of indications from that offer clues to a more nuanced understanding of their behavior. Initially the nuclei of the 1° PCs flank the clustered photoreceptor nuclei in their more apical region, but when the cone cell nuclei fall, those of the 1° PCs are shunted more basally. This movement deeper into the photoreceptor layer may play a role in their death. A similar argument can be made from the analysis of * eyes in which 1° PCs are lost, but when Snail family transcription factors are removed from this background, the cone cell nuclei do not fall, and the 1° PCs do not die. Hence the 1° PCs behave in a similar manner as the cone cells; if their position is maintained they do not die even though ambient Wg concentrations are sufficient for their death. This may indicate a general principle; that cells need to be in the correct topological position to experience the death signal (Kumar, 2015).

Furthermore, in * eyes, the cone cell nuclear fall is accompanied by the loss of the 1° PCs even though the cone cells themselves do not die. The removal of Wg expression from the pros-arm* cone cells rescues the 1° PCs indicating that their loss is normally triggered by the cone cell Wg expression, and it is suspected that the low-level apoptosis seen in the main body of pros-arm* eyes may represent the death of the 1° PCs. If this is the case, then this suggests that the 1° PCs have a lower threshold Wg response for their apoptosis than the cone cells and photoreceptors (Kumar, 2015).

The death of the photoreceptors appears to simply require the additive of effects of the two Wg sources to trigger their death. But another feature has emerged from these studies – the idea that chronic exposure to sub-lethal levels of Wg triggers photoreceptor degeneration. Consider pros-arm*/esgRNAi eyes; here the photoreceptor death occurs only at the widened zone of peripheral apoptosis, but in the main body of the adult eyes ommatidia show degenerate rhabdomere-like tissue in the apical retinas. The presence of rhabdomere-like tissue suggests the differentiation and subsequent degeneration of the photoreceptors leaving them alive but in a runtish condition. Since this phenomenon is Wg dependent (it is absent when wgRNAi is additionally included) it is inferred that the persistent Wg expression from the cone cells chronically signals to the photoreceptors. Indeed, when GMR.wg/GMR.P35 eyes (in which the apoptosis mechanism is suppressed and the photoreceptors are therefore subject to chronic Wg exposure), were examined a similar degenerate phenotype occurred. This observation suggests another function for the removal of the outer-most row of ommatidia: if they were not removed, chronic exposure to high levels of Wg emanating from the head capsule would lead them to deteriorate into a runtish condition (Kumar, 2015).

A striking feature of the peripheral patterning mechanism is the timing aspect. The peripheral ommatidia are exposed to head capsule-derived Wg from the time of their birth. And yet they only respond to this Wg signal at defined times. The first occurs shortly after pupation when ac/da transcription is repressed and hth expression is induced. This corresponds with the surge in ecdysone expression that occurs in the animals at this time. The second response is the death of ommatidia at 42 h APF and this mechanism is closely tied with the large peak of ecdysone expression that occurs in the second day of pupation. Thus, it is speculated that Wg provides the spatial signal for peripheral patterning, but that the hormone system of the fly provides the temporal cue that determines when the spatial information can be utilized (Kumar, 2015).

It is concluded that the periphery of the fly eye is an excellent model system with which to study how morphogen gradients are decoded into discrete tissue types, and this study has delved into the mechanism that precisely restricts the spatial positioning of one of those tissue types. An intricate mechanism has been uncovered in which initial threshold responses lead to the local boosting of the morphogen signal while at the same time upregulating mechanisms to prevent the spread of the morphogen. Evidence is also provided to support the idea that appropriate spatial, temporal and topological context is required for the peripheral ommatidia to undergo developmental apoptosis (Kumar, 2015).

Larval (part 2/2)

Expression in wing and leg discs

Continue: Wingless Developmental biology part 2/2

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

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