spitz

DEVELOPMENTAL BIOLOGY

Embryonic

Temporal and spatial expression patterns of SPItranscripts indicate that spi is expressed throughout the embryo with enrichment in the procephalic region [Images], ventral midline, mesodermal layers and possibly peripheral nervous system (Rutledge, 1992).

When secreted Spitz protein is expressed in embryos, an alteration in cell fates is observed in the ventral ectoderm, such that lateral cells acquire the ventral-most fates. Graded activation of the EGF-R pathway therefore may normally give rise to a repertoire of discrete cell fates in the ventral ectoderm (Schweitzer, 1995).

The midline glia of the Drosophila embryonic nerve cord undergo a reduction in cell number after facilitating commissural tract morphogenesis. The numbers of midline glia entering apoptosis at this stage can be increased by a loss or reduction of function in genes of the spitz group or the Drosophila EGF receptor pathway. Argos, a secreted molecule with an atypical EGF motif, is postulated to function as a Egf-r antagonist. argos function reflects or is involved in the process that restricts midline glia numbers developmentally. In this study, the role of argos is assessed in the determination of midline glia cell numbers. Fewer midline glia enter apoptosis in embryos lacking argos function. Ectopic expression of argos is sufficient to remove all Egf-r-expressing midline glia from the nerve cord, even those that already express argos. Egf-r expression is not terminated in the midline glia after spitz group signaling triggers changes in gene expression. Paradoxically, although all midline glia express Egf-r, argos expression is restricted to the midline glia that do not enter apoptosis. It is therefore likely that an attenuation of Egf-r signaling by Argos is integrated with the augmentation of Egf-r signaling by Spitz throughout the period of reduction of midline glia numbers, and argos-expressing midline glia depend upon continued Spitz activation of the EGF-R at levels higher than adjacent non-argos expressing midline glia to overcome possible autocrine inhibition by released Argos. It is suggested that signaling by Spitz (but not Argos) is restricted to adhesive junctions. In this manner, midline glia not forming signaling junctions remain sensitive to juxtacrine Argos signaling, while an autocrine Argos signal is excluded by the adhesive junction (Stemerdink, 1997).

The transcription factors encoding genes tailless (tll), atonal (ato), sine oculis (so), eyeless (ey) and eyes absent (eya), and Efgr signaling play a role in establishing the Drosophila embryonic visual system. The embryonic visual system consists of the optic lobe primordium, which, during later larval life, develops into the prominent optic lobe neuropiles, and the larval photoreceptor (Bolwig's organ). Both structures derive from a neurectodermal placode in the embryonic head. Expression of tll is normally confined to the optic lobe primordium, whereas ato appears in a subset of Bolwig's organ cells that are called Bolwig's organ founders. Phenotypic analysis of tll loss- and gain-of-function mutant embryos using specific markers for Bolwig's organ and the optic lobe, reveals that tll functions to drive cells to an optic lobe fate, as opposed to a Bolwig's organ fate. Similar experiments indicate that ato has the opposite effect, namely driving cells to a Bolwig's organ fate. Since tll and ato do not regulate one another, a model is proposed wherein tll expression restricts the ability of cells to respond to signaling arising from ato-expressing Bolwig's organ pioneers. The data further suggest that the Bolwig's organ founder cells produce Spitz (the Drosophila TGFalpha homolog) signal, which is passed to the neighboring secondary Bolwig's organ cells where it activates the Epidermal growth factor receptor signaling cascade and maintains the fate of these secondary cells. The regulators of tll expression in the embryonic visual system remain elusive, no evidence for regulation by the 'early eye genes' so, eya and ey, or by Egfr signaling is found (Daniel, 1999).

The Drosophila visual system comprises the adult compound eye, the larval eye (Bolwig's organ) and the optic lobe (a part of the brain). All of these components are recognizable as separate primordia during late stages of embryonic development. These components originate from a small, contiguous region in the dorsal head ectoderm. During the extended germband stage, the individual components of the visual system can be distinguished morphologically as well as by spatially localized expression of the homeobox gene so and the adhesion molecule Fas II. Initially centered as an unpaired, oval domain straddling the dorsal midline, the anlage of the visual system subsequently elongates in the transverse axis and narrows in the anteroposterior axis. By late gastrulation (stage 8), the anlage occupies two bilaterally symmetric stripes that are anterior and adjacent to the cephalic furrow. The domain of so expression at this stage contains two regions with a high expression level [ol_ex (the external fold of the optic lobe) and ol_in]. Only these two regions will ultimately give rise to the optic lobe and Bolwig's organ; the so-positive cells dorsal and posterior to these domains will either form part of the dorsal posterior head epidermis (dph) or undergo apoptotic cell death. During the extended germband stage, the anlage of the visual system expands further ventrally until, around stage 10, it reaches the equator (50% in the dorsoventral axis) of the embryo. Shortly thereafter, ol_in, the portion of the anlage that will give rise to most of the optic lobe and Bolwig's organ, reorganizes into a placode of high cylindrical epithelial cells that differ in shape from the surrounding more squamous cells of the head ectoderm. During stage 12, this placode starts to invaginate, forming a V-shaped structure with an anterior lip (ol_al) and a posterior lip (ol_pl). Bolwig's organ, which consists of a small cluster of sensory neurons, derives from the basal part of the posterior lip and can be recognized during stage 12 as a distinct, dome-shaped protrusion. During stage 13, invagination of the optic lobe separates it from the head ectoderm; only the cells of Bolwig's organ remain in the ectoderm. The ectodermal region ol_ex is also internalized and forms an external 'cover' of the optic lobe; many cells of this population undergo apoptosis (Daniel, 1999).

Epidermal growth factor receptor is activated in midline regions of the head neurectoderm, in particular in the anlage of the visual system. Moreover, increased and/or ectopic activation of Egfr results in a 'cyclops' phenotype very similar to what has been described for ectopic tll expression. Egfr signaling has been shown to be required in both chordotonal organs and compound eye for the inductive signaling triggered by ato expression. Two questions raised by these observations have been investigated: (1) is Egfr signaling required for tll expression in the optic lobe and (2) is Egfr signaling involved in the recruitment of the secondary (non-atonal-expressing) Bolwig's organ cells? The answer to both these questions is no. tll expression is unaltered when levels of Egfr signaling are manipulated, suggesting that Egfr signaling is not required for tll expression. To investigate the second question, a test was performed for the presence of Egfr-relevant mRNAs or proteins: Rhomboid mRNA, which would be expected to be present only in the signaling cells, and phosphorylated MAPK, Pointed and Argos mRNAs, which would be expected to be expressed in all cells receiving an Egfr-mediated signal. In stage 12 embryos, rho is expressed only in the small group of Bolwig's organfounder cells (the same cells expressing ato). In contrast, activated (phosphorylated) MAPK is present in a larger cluster of cells including the entire Bolwig's organ and part of the adjacent optic lobe. Consistent with this, pnt and aos, both known to be switched on in cells receiving the Spi signal, are expressed at the same stage throughout the entire Bolwig's organ primordium. These gene expression and MAPK activation patterns are consistent with the idea that the Spi signal is activated by rho in the Bolwig's organ founders and passed to the neighboring secondary Bolwig's organ cells where it activates the Egfr signaling cascade. Supporting this view, only 3-4 photoreceptor neurons are found in the Bolwig's organ of embryos lacking rho or spi; furthermore, the size of the posterior lip of the optic lobe is reduced in such embryos. The fact that absence of secondary Bolwig's organ cells in rho or spi mutant embryos can be rescued by blocking cell death in the background of a deficiency that takes out the reaper complex of genes indicates that the Spi signal is not necessary for the specification (recruitment) of secondary Bolwig's organ cells, but rather, for their maintenance (Daniel, 1999).

Small Wing PLCγ is required for ER retention of cleaved Spitz during eye development in Drosophila

The Drosophila EGF receptor ligand Spitz is cleaved by Rhomboid to generate an active secreted molecule. Surprisingly, when a cleaved variant of Spitz (cSpi) was expressed, it accumulated in the ER, both in embryos and in cell culture. A cell-based RNAi screen for loss-of-function phenotypes that alleviate ER accumulation of cSpi identified several genes, including the small wing (sl) gene encoding a PLCsγ. sl mutants compromised ER accumulation of cSpi in embryos, yet they exhibit EGFR hyperactivation phenotypes predominantly in the eye. Spi processing in the eye is carried out primarily by Rhomboid-3/Roughoid, which cleaves Spi in the ER, en route to the Golgi. The sl mutant phenotype is consistent with decreased cSpi retention in the R8 cells. Retention of cSpi in the ER provides a novel mechanism for restricting active ligand levels and hence the range of EGFR activation in the developing eye (Schlesinger, 2004).

The accumulation of cSpi in the ER appears to reflect a novel mechanism for ER retention. In contrast to ER retention of the full-length Spi precursor, cSpi remains in the ER when the retrograde trafficking machinery from the Golgi to the ER is compromised following incubation with dsRNA for COPI. Utilization of a novel ER retention and export machinery has been identified for the SREBP protein-regulating cholesterol synthesis (Schlesinger, 2004).

To identify the mechanism responsible for cSpi retention and assess its biological significance, a screen was conducted for dsRNAs that would compromise this property. The analysis focussed on the sl gene, in view of previous observations demonstrating that it is a negative regulator of EGFR signaling in the eye. Sl is broadly expressed. Compromising the levels of Sl in embryos, either by dsRNA injection or in a sl mutant background, led to efficient release of cSpi. Thus, Sl is also required in vivo for the retention of cSpi. The actual retention mechanism remains unknown. Sl is a cytoplasmic protein, while cSpi is a secreted protein that is retained within the ER lumen. Additional proteins must participate to form a physical link. While sl encodes a PLCγ, it is believed that its enzymatic activity is not necessary for the retention process. sl interacts genetically with EGFR signaling only in the eye. sl mutants that were defective in the catalytic domain did not give rise to an eye phenotype. In addition to the catalytic domain, Sl also contains several motifs that may mediate protein-protein interactions, including SH2, SH3, and PH domains. It is thus possible that in addition to its enzymatic role, Sl serves as a scaffold protein in other contexts. In mammalian cells, PLCγ has been shown to function in the cells receiving the signal, downstream to receptor-tyrosine kinases (Rhee, 2001). The implicated role of Sl/PLCγ in the cells producing the signal points to a novel function of this protein (Schlesinger, 2004).

While Sl is broadly expressed, the EGFR hyperactivation phenotype of sl null flies is manifested only in the eye. This phenotype entails recruitment of extra R7 photoreceptor cells and misrotation of ommatidia. The restricted effect led to an examination of the possibility that cSpi is normally generated in the ER only in the eye. The cleavage of EGFR ligands depends upon a distinct family of serine proteases that carry out intramembrane proteolysis. Rhomboid-1 is the primary player, and hence mutations in this gene give rise to embryonic phenotypes that are similar to spi or Star (Schlesinger, 2004).

Two additional members of the family, Rhomboid-2/Brho and Rhomboid-3/Roughoid (Ru), are expressed in the oocyte and the eye, respectively. Recently, expression of Ru was also detected in the embryonic VUM neurons, where it plays a role in guidance of tracheal migration in the CNS. Homozygous null mutations for ru demonstrated that it is essential for normal eye development, but its role is partially redundant with Rhomboid-1, since some photoreceptor cells are recruited in ru mutants. The question is whether there are properties of Ru that are distinct from those of Rhomboid-1 and may account for the generation of cSpi in the ER during eye development (Schlesinger, 2004).

As far as substrate specificity is concerned, the Rhomboid 1-3 proteins are all capable of cleaving the membrane precursors of the EGFR ligands Spi, Keren, and Gurken. In addition, all three proteins are enriched in the Golgi when expressed in mammalian cells. However, functional assays in cell culture, including both Drosophila and mammalian cells, suggest that in contrast to Rhomboid-1, Ru may be capable of cleaving the Spi precursor already in the ER. However, cSpi is secreted only upon coexpression of Star. While Ru is located primarily in the Golgi, this cleavage may take place en route to the Golgi. The failure to secrete cSpi in the absence of Star likely represents the property of ER retention that was uncovered in this work (Schlesinger, 2004).

ru and sl give rise to opposite phenotypes in the eye. It is assumed that they act in a sequential manner, i.e., Ru generates cSpi in the ER and Sl mediates the retention of this ligand, to avoid excessive secretion. The genetic interaction experiments between ru and sl can be interpreted within this context. Indeed, the eye phenotype of hypomorphic mutations in ru could be efficiently rescued by mutations in sl. While the level of cSpi in the ER was compromised, more efficient secretion was facilitated in the sl background, thus compensating for the initial defect. Surprisingly, even null mutations in ru were partially rescued by sl mutants. In a ru null background, Rhomboid-1 is the only other known Rhomboid family protease that is functional in the eye. It is suggested that residual levels of cSpi may also be generated in the ER by Rhomboid-1. An efficient release of these low levels in the absence of Sl may lead to the partial rescue that was observed in ru null mutants (Schlesinger, 2004).

Finally, the requirement for Sl specifically in the cells processing the ligand was demonstrated by the capacity to rescue sl null flies by expressing Sl in the R8 cells. Normally, expression of Rhomboid-1 and Ru in the differentiating photoreceptor cells is induced by EGFR activation, thus making these cells a source for subsequent rounds of photoreceptor cell recruitment. Incomplete rescue by expression of Sl in R8 cells may be explained by the failure to restore ER retention of cSpi in the other photoreceptor cells expressing Ru (Schlesinger, 2004).

In conclusion, it has been demonstrated that the cleaved form of Spi is efficiently retained in the ER through a novel mechanism. This retention is significant only in the developing eye, where the Rhomboid-3/Ru protein may normally generate the cleaved ligand in the ER. Thus, in spite of efficient cleavage of mSpi in the ER, only the molecules that will overcome retention by association with Star will be secreted to activate EGFR in the neighboring cells. small wing, encoding a PLCγ, provides a link to the retention mechanism, and sl mutants exhibit EGFR hyperactivation phenotypes mainly in the eye. The eye is a tissue where the restricted range of EGFR activation is particularly crucial. The number of undifferentiated precursor cells is limited. EGFR activation is responsible for sequential inductions of the different cell types comprising the mature ommatidia. It is thus imperative to restrict the number of cells that are induced at every cycle. Negative feedback loops that are transcriptionally induced by EGFR activation in the cells receiving the signal were previously shown to be central to this restriction. This study demonstrates that fine tuning the level of ligand that is released by the cells providing the signal represents another cardinal tier of regulation (Schlesinger, 2004).

EGF receptor signaling regulates pulses of cell delamination from the Drosophila ectoderm: A single chordotonal organ precursor and its progeny provide the source of secreted Spitz relevant for oenocyte induction.

Many different intercellular signaling pathways are known but, for most, it is unclear whether they can generate oscillating cell behaviors. Time-lapse analysis of Drosophila embryogenesis has been used to show that oenocytes delaminate from the ectoderm in discrete bursts of three. This pulsatile process has a 1 hour period, occurs without cell division, and requires a localized EGF receptor (EGFR) response. High-threshold EGFR targets are sequentially activated in rings of three cells, prefiguring the temporal pattern of delamination. Surprisingly, widespread misexpression of the relevant activating ligand, Spitz, is compatible with robust delamination pulses. A single chordotonal organ precursor (called C1) and its progeny provide the source of secreted Spi relevant for oenocyte induction. Although Spitz ligand becomes limiting after only two pulses, artificially prolonging its secretion generates up to six additional cycles, revealing a rhythmic underlying mechanism. These findings illustrate how intercellular signaling and cell movements can generate multiple cycles of a cell behavior, despite individual cells experiencing only one cycle of receptor activation (Brodu, 2004).

The induction of larval oenocytes in Drosophila has been used as a simple model system for investigating the developmental regulation of EGFR signaling. Oenocytes are induced from the dorsal ectoderm of abdominal segments by a fixed and highly restricted source of Spi. This triggers a local EGFR response within a ring of overlying dorsal ectodermal cells, termed a whorl, leading to the upregulation of numerous oenocyte-specification genes and subsequent cell delamination. The simple cell geometry of the oenocyte whorl, together with time-lapse microscopy, was used to explore the timing of Spi secretion, EGFR-target activation, early cell induction, and later cell delamination. These studies reveal that oenocytes delaminate in bursts of three and identify the cell-counting mechanism as an EGFR-dependent pulse generator converting the time window of Spi secretion into final oenocyte number. This represents the first example of a rhythmic cell behavior other than the cell cycle to be reported in the Drosophila embryo (Brodu, 2004).
Rather than delaminating from the ectoderm in a continuous stream, oenocyte precursors segregate in discrete well-separated bursts of three cells. Genetic backgrounds affecting the pattern of cell segregation but not early fate specification were used to show how these pulses are regulated by EGFR signaling. The signaling parameters regulating the time of onset, time of cessation, and in particular, the cyclical nature of cell delamination of oenocytes are discussed (Brodu, 2004).

Spi secretion begins during stage 10, triggering weak activated Rolled/ERK but not the first morphological readout for oenocyte induction, the sickle-shape change, until 1 hr later. This early inhibition of EGFR induction occurs upstream of Pointed P1 and requires Delta-dependent Notch signaling. Although the supply of Spi ligand is not rate limiting for initiating induction, it does specify the final number of delamination pulses. In turn, this depends upon the duration of Rhomboid-1 expression by the C1 lineage, which is regulated by the Hox protein Abdominal-A. In this regard, it is interesting that oenocyte number is higher than six in many other winged insects. For example, in the parasitic wasp Phaenoserphus viator, oenocyte clusters of 'about 20 cells' have been reported, tempting speculation that this species may undergo seven rather than two delamination pulses (Brodu, 2004).

The sequence of events during wild-type oenocyte induction and delamination was identified using time-lapse movies. EGFR signaling initially induces all six precursors within a whorl to adopt a sickle-shape change within 10 min. There then follow two complete cycles of pulsatile delamination. Each 1 hr cycle comprises a 45 min pause, during which time no precursors leave the ectoderm, followed by a 15 min delamination phase, where three cells segregate rapidly, at 7.5 min intervals. Each cycle is reset when the inner-ring triplet delaminates and migrates away from the whorl site, allowing the remaining outer-ring cells to move into the inner position before they too delaminate (Brodu, 2004).

The mechanism involved in pulse generation was revealed, at least in part, by testing the roles of several different EGFR-signaling parameters. Surprisingly, although Spi ligand is essential for oenocyte induction and delamination, it plays only a permissive role in pulse generation. Thus, overexpression of Rhomboid-1 or secreted Spi in a widespread or prolonged manner does not suppress pulses of delamination nor alter their initial frequency, but it does produce up to six additional cycles. This leads to three main conclusions: (1) although only two pulses normally occur, the underlying mechanism is cyclical and has the potential to generate many more; (2) neither the frequency nor the number of cells per cycle are altered by increasing Spi-ligand levels; (3) pulses do not need Spi secretion to be pulsatile or even restricted to the Spitz normal source, C1. In addition, C1 does not provide any other essential rhythmic cue, because when it is eliminated, resupplying widespread Rhomboid-1 can rescue periodic delamination (Brodu, 2004).

In contrast to constitutive Spi secretion, widespread activation of the EGFR or its downstream effector, Ras1, disrupts delamination pulses. Loss of rhythmicity is also observed when the EGFR pathway is deregulated by removing the Yan or Argos inhibitors. Together, these functional data demonstrate that the spatiotemporal pattern and/or the levels of EGFR activation and downstream signal transduction are critical for pulse generation. For Ras1 overactivation or argos inactivation, it was also shown that some oenocytes fail to switch on a late differentiation marker at the appropriate time. Thus, one function of pulses may be to ensure cell-to-cell consistency in the duration or level of the oenocyte EGFR response, in turn promoting homogeneous cell differentiation (Brodu, 2004).

Using a panel of markers for double- and single-ring stages, it was possible to place gene expression 'snapshots' in temporal order with the cell movements recorded in movies. Three generic EGFR targets (activated Rolled/ERK, Yan, and argos) and three oenocyte-specific EGFR targets (Sal, svp^lacZ, and svp^lacZΔ18) were analyzed. In wild-type embryos, the high-threshold EGFR outputs of argos and svp^lacZ expression, detectable Rolled activation, and strong Yan downregulation are all inner ring specific, whereas lower-threshold outputs such as Sal upregulation and svp^lacZΔ18 expression are present in both precursor rings. Delamination itself also appears to be a high-threshold EGFR response and is thus confined to the inner ring (Brodu, 2004).

argos is a particularly interesting high-threshold target, as its expression is normally confined to the inner ring but its activity is required in the outer ring to tone down the EGFR response, as measured by Rolled activation. This remote inhibitor role is consistent with several previous studies, and real-time analysis shows that it promotes oenocyte pulses by preventing premature outer-ring delamination. During wild-type embryogenesis, such negative feedback would be transiently downregulated each time the inner-ring source of Argos is physically removed via delamination, thus facilitating upregulation of the EGFR response in the next triplet. In addition, Argos may play a more subtle autocrine role in ring-1, since loss of function not only eliminates a second 45 min pause phase completely but also partially reduces the first pause to 25 min (Brodu, 2004).

Together, the real-time cell tracking, the expression analysis of the EGFR response, and the oenocyte counts in EGFR pathway altered backgrounds are consistent with the notion that pulses require at least some components of the high-threshold EGFR response to be more strongly expressed by inner- than outer-ring cells. It thus follows that one critical molecular transition underlying pulse generation occurs after each round of delamination, when cells of the outer triplet move centrally and upregulate a subset of EGFR-target genes (Brodu, 2004).

At least two distinct mechanisms ensure that strong expression of high-threshold EGFR targets is restricted to the dynamic population of inner-ring cells. The first of these arises from inner-ring cells being closer to C1 and therefore exposed to higher levels of secreted Spi. Hence, when Spi ligand is widely overexpressed, high-threshold EGFR readouts such as detectable activated Rolled/ERK and argos expand ectopically into the outer ring (Brodu, 2004).

A second mechanism that is not dependent on localized Spi-ligand secretion also enhances the inner-ring EGFR response. This was initially revealed in four different genetic backgrounds where Spi secretion is delocalized yet pulses remain. In UAS-rho1 embryos, real-time and EGFR-target analyses showed that, despite Spi secretion throughout the En stripe, oenocyte delamination and the full repertoire of inner-ring markers, including strong svp^lacZ expression and Yan downregulation, remain confined to the inner ring. It was not possible, however, to detect such a clear and consistent inner-versus-outer difference in levels with activated Rolled/ERK and argos expression, either reflecting technical limitations or indicating that some high-threshold EGFR targets remain more tightly restricted than others. Nevertheless, these studies provide evidence that, when exposed to the same Spi ligand concentration, inner-ring precursors express some components of the oenocyte EGFR response more strongly than their neighbors. One molecular explanation for this bias is revealed by the reduced sensitivity of inner-ring cells to the delamination-blocking effects of argos overexpression. Thus, the argos sensitivity difference may account for why pulses remain in UAS-rho1 embryos. In wild-type embryos, both this mechanism and graded Spi ligand would be expected to contribute to promoting robust pulses. The basis for differential argos sensitivity is not yet understood but it likely initiated independently of EGFR signaling. In addition, the parameters regulating whorl geometry and thus setting the size of delamination quanta to three cells remain unclear. In this regard, it is intriguing that among all the EGFR pathway components tested, only activated Ras1 produced oenocyte counts suggestive of an altered quantal size, in this case two cells (Brodu, 2004).

The EGFR-dependent pulse generator drives rhythmic clearance of cells from their induction site, one solution to the problem of how to induce a large number of cells using a point source of short-range signal. Coupling intercellular signaling to cell movement in this way also allows the generation of multiple output cycles, even though individual cells experience only one intracellular cycle of EGFR activation. This contrasts with the vertebrate segmentation clock, where cells undergo multiple intracellular oscillations of gene expression, in this case involving Notch signaling. One aspect that is shared with many oscillating systems, including the segmentation clock, is the essential contribution of negative feedback, which in the oenocyte context is mediated by Argos. The relative simplicity of the oenocyte oscillator may prove particularly amenable for constructing and testing future mathematical models of intercellular signaling rhythms. Similar real-time analyses of other inductive processes, especially those of a reiterative nature, should clarify whether pulsatile cell behaviors are commonly associated with EGFR and other intercellular signaling pathways (Brodu, 2004).

Larval and Pupal

Reiterative use of the EGF Receptor triggers differentiation of all cell types in the Drosophila eye

Use of a dominant-negative form of the EGF-R in the eye reveals that EGF-R is required for differentiation of all photoreceptor cell types (R1-R8), including R7 which is also subject to the Sevenless signal. DN-EGF-R is truncated in the 13 amino acids C-terminal to the transmembrane domain. Receptor tyrosine kinases dimerize and transphosphorylate each other upon activation. The removal of the intracellular domain produces a dominant-negative function because receptor molecules without the intracellular tyrosine kinase domain can dimerize with wild-type receptors, but the dimer is unable to signal. Expression of DN-EGF-R behind the morphogenetic furrow causes complete loss of the adult retina. As well as eight photoreceptors, each ommatidium comprises four cone cells and eight pigment cells (see The Drosophila Adult Ommatidium: Illustration and explanation with Quicktime animation). Expression of DN-EGF-R in the presumptive cone or pigment cells leads to them not differentiating. Overexpression of secreted Spitz, the ligand of EGF-R causes overrecruitment of all cell types in the ommatidium. Spitz has extracellular protease cleavage sites that allow a fragment with an EGF repeat to be released. Overexpression of membrane-bound full-length Spitz has no effect on eye development. In all cases the source of the extra photoreceptors is the same: transformation into photoreceptors of the "mystery cells" (early members of the cluster, later destined to leave and apparently rejoin the surrounding undetermined cells) (Freeman, 1996).

Just as with EGF-R, overexpression of activated Sevenless recruits extra cells into the ommatidium. Sevenless is also able to recruit additional cone and pigment cells when expressed in the pupal retina. Sevenless can also replace EGF-R function in the wing. Finally, overexpression of secreted Spitz can replace the need for Sevenless. It is concluded that there is no significant difference in the intracellular effects of activation of these two RTKs, even in the R7 cell, where both receptors are required (Freeman, 1996).

A model is proposed for eye development based on these and other observations. First, Spitz activation of DER can trigger all the cell types in the ommatidium, the choice of fate being dependent on when the activation occurs. Argos is an extracellular inhibitor of DER activation (Schweitzer, 1995). Third, the expression of Argos is dependent of EGF-R activation, establishing a negative feedback loop (Golembo, 1996). Fourth, Argos can diffuse further than Spitz. Fifth, the successive waves of induction of each cell type (photoreceptors, cone cells, primary pigment cells, and second/tertiary pigment cells) occur in concentric rings around the ommatidium: each cluster resembles a bullseye. In this model, Spitz is initially produced by the three central cells R8, R2 and R5 and that this recruits the immediately neighboring cells and photoreceptors. In R7, the later activation of Sevenless by its ligtand, Boss, is also required. As cells differentiate, they express Argos, which diffuses outwards, preventing more distal cells from responding to Spitz; Argos is unable to block cells that have already started to differentiate or cells that are exposed to high level of Spitz. Later, more cells start to produce Spitz, overcoming Argos inhibition in the nearest cells. This allows the next concentric ring of cells around the potoreceptors to be recruited, but now as a different cell type, cone cells. Again, Argos prevents more remote cells from responding by diffusing beyond the cone cells (now themselves producing it). Later still, the Spitz source expands again, now recruiting the pigment cells (Freeman, 1996).

A retinal axon fascicle uses spitz, an EGF receptor ligand, to construct a synaptic cartridge in the brain of Drosophila

Photoreceptor axons arriving in the Drosophila brain organize their postsynaptic target field into a precise array of five neuron "cartridge" ensembles. Hedgehog, an initial inductive signal transported along retinal axons from the developing eye, induces postsynaptic precursor cells to express the Drosophila homolog of the epidermal growth factor receptor (Egfr). HH alone is not sufficient for this cartridge assembly process, which depends on the presence of retinal axons. The Egfr ligand Spitz, a signal for ommatidial assembly in the compound eye, is transported to retinal axon termini in the brain where it acts as a local cue for the recruitment of five cells into a cartridge ensemble. Hedgehog and Spitz thus bring about the concerted assembly of ommatidial and synaptic cartridge units, imposing the "neurocrystalline" order of the compound eye on the postsynaptic target field (Huang, 1998).

In the mutant sine oculis, where only a few ommatidia may form in the eye disc, lamina development is restricted to the immediate vicinity of the small number of axons that grow into the lamina target field. The reduced number of arrays of retinal axons in these animals induces a proportionately reduced field of Dachshund-positive cells. A subset of these cells expresses the neuronal marker Elav. The onset of Dac expression is under the control of Hedgehog and neuronal differentiation (as indicated by Elav expression) involves a distinct signal. Is the putative signal for neuronal differentiation restricted to the immediate vicinity of a retinal axon fascicle? When axon fascicles enter a large field of postmitotic lamina precursor cells (LPCs) induced by hh+ somatic clones, Elav-positive cells are found only in the immediate vicinity of retinal axon fascicles. This local inductive effect is also observed for the expression of the gene argos (aos). aos is a direct transcriptional target of Egf receptor activation and encodes a secreted EGF-like product that can act as a negative regulator of Egfr signaling. In the lamina, Aos displays a punctate distribution surrounding the Elav-positive cartridge cells. As in the case of Elav, Hh is not sufficient to induce aos expression in the absence of retinal axons. Retinal axons thus appear to harbor a locally acting neuronal differentiation signal that is distinct from Hh. The local induction of Aos suggests that this signal may act via the Egf receptor (Huang, 1998).

The Drosophila homolog of the Egf receptor is strongly and specifically expressed by LPCs within the lamina target field. The onset of Egf receptor expression coincides with the terminal division of LPCs at the lamina furrow and the appearance of early markers such as Dac. Egf receptor immunoreactivity is found at higher levels among the older LPCs at the posterior of the lamina. The expression of the EGF receptor in the lamina depends on retinal innervation and is not detected in mutant animals, such as eyes absent (eya), that lack photoreceptor cells. Hh is sufficient for the onset of Egfr expression, as determined by ectopically expressing hh⁺ in the brain of an "eyeless" animal. R cell differentiation is blocked by the eya¹ mutation or by maintaining hh^ts2 animals at the nonpermissive temperature from a time point in early larval development. Ectopic hh⁺ expression induces the expression of the EGF receptor in its normal anterior-to-posterior gradient. This and additional experiments show that Hh is both necessary and sufficient for the onset of Egf receptor expression in the lamina (Huang, 1998).

The notion that Egfr activity might play a role in cartridge neuron differentiation is suggested by the observation that elav expression in prospective L1-L5 neurons coincides with the expression of aos, a transcriptional target of Egfr activation in many tissues. To determine whether Egf receptor activity is required for cartridge neuron differentiation, a dominant-negative form of the Egf receptor (DN-Egfr) was used to block Egfr signal reception. In the developing eye, strong ectopic expression of DN-Egfr prevents the formation of ommatidial cell clusters. This effect of DN-Egfr is suppressed by a wild-type Egfr transgene, consistent with the notion that the truncated receptor acts by interfering with Egfr signal reception. Widespred induction of DN-Egfr results in a normal array of photoreceptor axons innervating the lamina target field and induces an apparently normal field of DAC-positive LPCs. However, Elav-positive cells are absent from the lamina in these animals. Large DN-Egfr-expressing somatic clones also lack Elav-positive cells when they include the lamina target field. These observations indicate that Egfr signal reception is required for cartridge neuron differentiation but not for the early steps of lamina development that are under Hh control (Huang, 1998).

In the developing eye, the Egfr ligand Spitz is required for the differentiation of all ommatidial cell types, with the exception of the founding R8 cell. Spi is expressed initially by the R8 cell and later by additional cells as they join the ommatidial unit. Spi antigen is found on retinal axons as they project into the lamina. Within the developing lamina, Spi is found on retinal axon fascicles immediately adjacent to Elav-positive cartridge neurons. Spi is thus present at the right time and place to be an Egfr-activating ligand required for cartridge neuron differentiation. Additional experiments show that spi is required for cartridge formation (Huang, 1998).

Spi is synthesized as a transmembrane molecule. An artificially truncated form of Spi (secreted Spi, or sSpi), containing most of the extracellular portion of the molecule, has been shown to activate the Egf receptor both in vivo and in cell culture. In the developing eye, ubiquitous expression of sSpi induces the differentiation of ommatidial precursors without their assembly into ommatidial cell clusters. sSpi expression likewise can trigger ectopic neuronal differentiation within the lamina. To determine whether these ectopic neurons included a bona fide L-neuron cell type, the specimens were stained with an antibody against the brain specific homeobox (Bsh) protein. Bsh expression is an early marker of L5 differentiation and coincides with the onset of elav expression in a single medial layer in the posterior two-thirds of the lamina. With the expression of sSpi, ectopic Bsh-positive neurons are found throughout the three medial cell layers of the lamina. These include cells at the anterior of the lamina, where Bsh-positive cells are not seen in the wild type. Thus, LPCs that are normally destined for elimination by apoptosis or that undergo neuronal differentiation precociously can assume a proper L-neuron identity. In sum, these data indicate that spi⁺ activity is sufficient for the onset of cartridge neuron differentiation in the lamina (Huang, 1998).

The role of an individual ommatidial fascicle as the "founder" of a cartridge ensemble, together with the precision of axon pathfinding in this system, serve to impose the "neurocrystalline" order of the compound eye on the developing postsynaptic field. This mechanism yields a precise numerical match of ommatidial and cartridge units. The component axons of an ommatidial fascicle might additionally make important individual contributions to the specification of the number and type of postsynaptic cells in a cartridge. For example, individual R axons may make important individual contributions to the spatial and temporal pattern of Spi expression. A dynamic interplay between the extracellular levels of SPI and its negative regulator, Aos, might provide the tight localization of spi⁺ activity necessary for this axon-cell signaling. Following cartridge neuron differentiation, a remarkable feat of "axon-shuffling" occurs as the six R1-R6 axons of an ommatidial fascicle separate and migrate laterally to form their synaptic connections in a set of six neighboring cartridges. In the adult lamina, a synaptic cartridge thus receives its complement of R1-R6 axons from six ommatidial units whose axons did not contribute to its induction. The assembly of this precise circuitry nonetheless relies on the order imposed on the lamina during its initial inductive phase. A test of this notion may provide a significant insight into the establishment of precise synaptic circuitry in this and other systems (Huang, 1998).

The Drosophila TGFalpha homolog Spitz acts in photoreceptor recruitment in the developing retina

spitz function is required in developing ommatidia for the first step in cell recruitment: the recruitment of R2/R5 photoreceptor precursors. In spitz mutant clones, the initial single neuron stage ommatidia appear normal, as does their spacing. However, no further progression occurs, and the clusters appear to be arrested at the one-neuron stage. A marker that is expressed in the founding R8 photoreceptor cell, BOSS, is unaffected by spitz mutation, and neither are two genes that act upstream of R8 cell specification, Atonal and Scabrous. Spitz pro-protein is expressed in the retinal neurons as they begin to differentiate. Once R2 and R5 begin to differentiate they also secrete Spitz protein, which increases the local Spitz concentration,and may help with the recruitment of R3/R4. As R3 and R4 differentiate they add yet more Spitz to the local domain of the developing cluster, and this may be an inductive signal for the development of the remaining photoreceptors (Tio, 1997).

Antagonism between EGFR and Wingless signalling in the larval cuticle of Drosophila

Signaling by the epidermal growth factor receptor (EGFR) plays a critical role in the segmental patterning of the ventral larval cuticle in Drosophila: by expressing either a dominant-negative EGFR molecule or Spitz, an activating ligand of EGFR, it is shown that EGFR signaling specifies the anterior denticles in each segment of the larval abdomen. rhomboid, spitz and argos are expressed in denticle rows 2 and 3, just posterior to denticle row 1 in the engrailed expression "posterior" domain of larval ectoderm. These denticles derive from a segmental zone of embryonic cells in which EGFR signaling activity is maximal (Szuts, 1997).

Both Egfr and spitz are expressed fairly uniformly throughout the embryonic epidermis. However, Spi appears to be incapable of activating Egfr unless it is processed into a secreted form; there is genetic evidence that the membrane-spanning products of the rhomboid and Star genes may be mediating this process. In young embryos (before the germ band is fully extended) rhomboid is expressed in distinct segmental stripes. These stripes remain visible until stage 16 by which time they fade away. They become circumferential, and one cell wide in the dorsal half of the embryo. In the ventral half, they are also one cell wide in the thoracic segments, a bit wider in the first abdominal segment, and at least two cells wide in all other segments. These stripes correspond roughly to the cells giving rise to denticle rows 2 and 3. argos is observed in, and adjacent to, cells expressing rhomboid, and indeed is expressed in circumferential stripes after completion of germ-band retraction. The segmental stripes of argos are fairly similar to the rhomboid stripes but a bit wider. Argos is an inhibitor of Egfr function, so Argos reduction is expected to result in overactivation of Egfr signaling. argos loss of function mutants result in an entire additional row of denticles anterior to row 1 (Szuts, 1997).

High EGFR signalling activity depend on bithorax complex gene function. In mutants lacking abdominal-A and Ultrabithorax, rhomboid expression is very weak. In these mutants, there is very little expression of Argos. These homeotic genes account for the main difference in shape between abdominal and thoracic denticle belts (Szuts, 1997).

senseless repression of rough is required for r8 photoreceptor differentiation in the developing Drosophila eye

An outstanding model to study how neurons differentiate from among a field of equipotent undifferentiated cells is the process of R8 photoreceptor differentiation during Drosophila eye development. Senseless is a Zn finger transcription factor that is expressed and required in the sensory organ precursors (SOPs) for proper proneural gene expression. In senseless mutant tissue, R8 differentiation fails and the presumptive R8 cell adopts the R2/R5 fate. senseless repression of rough (ro) in R8 is an essential mechanism of R8 cell fate determination and misexpression of senseless in non-R8 photoreceptors results in repression of rough and induction of the R8 fate. Surprisingly, there is no loss of ommatidial clusters in senseless mutant tissue and all outer photoreceptor subtypes can be recruited, suggesting that other photoreceptors can substitute for R8 to initiate recruitment and that R8-specific signaling is not required for outer photoreceptor subtype assignment (Frankfort, 2001).

The analysis of sens mutant ommatidia reveals that the process of R8 differentiation fails very early in development. Despite this, recruitment and differentiation of outer photoreceptors occurs. These findings are unique and paradoxical because R8 is thought to initiate the recruitment of all other photoreceptors, although ato-independent photoreceptor differentiation has been observed. Moreover, loss-of-function mutations in all other genes known to be cell-autonomously required in R8 for normal eye development lead to the complete failure of photoreceptor recruitment. How can photoreceptor recruitment, a process known to require R8, occur without an R8 cell? At present, R8 is believed to have two distinct functions in the process of photoreceptor recruitment. First, R8 is thought to recruit photoreceptors by providing the initial source of Spitz (Spi), a positive ligand for the EGFR during eye development. Spi activates the EGFR which induces Ras signaling and differentiation of all photoreceptors (except R8), cone cells, and pigment cells. Second, R8 recruits the R7 photoreceptor via direct Boss/Sevenless interactions. The second function of R8 is clearly abrogated in sens mutant clones. This is expected because R7 is induced by physical contact with the Boss ligand, which is normally expressed solely on R8, and Boss is never expressed within sens mutant clones. However, the first function of R8, while somewhat compromised, is not eliminated (Frankfort, 2001).

Current models predict that after Spi is first secreted from R8, it binds to the EGFR and induces Ras signaling in two neighboring cells, the emerging R2 and R5 photoreceptors. Subsequently, Spi is also secreted from R2 and R5 (and later R3 and R4) and the increased Spi concentration leads to recruitment of all later photoreceptors. In one model, it is specifically the timing of induction of the EGFR pathway that determines photoreceptor subtype. However, an equally plausible model for the recruitment of R2 and R5 (and perhaps later photoreceptors) is one that is similar to R8-mediated induction of R7, where both activation of the EGFR pathway by Spi and ligand/receptor interactions (Boss/Sevenless) are required together for induction of the R7 fate. All outer photoreceptor subtypes can be recruited in sens mutant ommatidia. Because sens mutant ommatidia lack a differentiated R8 cell, these observations rule out all models for subtype specification that involve any R8-specific signaling, either in the form of spatial cues or ligand/receptor interactions. Other models that rely on timing, signaling from other photoreceptors, retinal prepatterning, combinatorial signaling, the actions of surrounding undifferentiated cells, or instructive signaling from the presumptive R8 prior to overt R8 differentiation remain possible, likely in combination with one another (Frankfort, 2001).

In sens mutant ommatidia, the selection of the presumptive R8 cell is not affected, but the presumptive R8 differentiates as an R2/R5 cell at approximately the same time as R8 would normally differentiate. Thus, it is likely that while the identity of the cell initially producing Spi is different in the absence of sens function, the timing of initiation of Spi secretion remains more or the less the same. Because all photoreceptors are recruited, it therefore appears that an R2 or R5 photoreceptor can largely fulfill the previously presumed function of R8 in outer photoreceptor recruitment. Thus, it has been specifically demonstrated that R8 is dispensable for photoreceptor recruitment and it is likely that Spi produced from an alternate source (in this case R2/R5) at roughly the same time is entirely sufficient to initiate the process of recruitment. However, as fewer photoreceptors are recruited in sens mutant tissue, it is clear that activation of recruitment from this alternative source is suboptimal. Indeed, decreased levels of dpERK expression in sens mutant ommatidia reflect a reduction in Ras signaling, which is perhaps due to decreased secretion of Spi. Nevertheless, it is now certain that activation of the recruiting pathway mediated by Spi occurs independently of R8 differentiation (Frankfort, 2001).

Spitz/EGFr signalling via the Ras/MAPK pathway mediates the induction of bract cells in Drosophila legs

The specification of bract cells in Drosophila legs has been analyzed. Mechanosensory bristles induce bract fate in neighboring epidermal cells, and the RAS/MAPK pathway mediates this induction. Spitz and EGF receptor have been identified as the ligand and the receptor of this signaling; by ubiquitous expression of constitutively activated forms of components of the pathway it has been found that the acquisition of bract fate is temporally and spatially restricted. The role of the poxn gene in the inhibition of bract induction in chemosensory bristles has also been studied (del Álamo, 2002).

Drosophila legs are covered by a constant and leg-specific pattern of different types of external sensory organs, mainly mechanosensory (MB) or chemosensory (ChB) bristles. Bristles on the legs can be classified by the presence of bracts. Bracts are small epidermal structures that appear associated to MB in specific places on adult femur, tibia and the tarsal segments of all legs. Bracts appear on the proximal side of the bristles, and share the same polarity. Bracts are also present in the proximal costa of the wing, showing the same morphology as in the leg (del Álamo, 2002).

Are bristle and bract related by lineage? Sensory organ precursors (SOPs) undergo a specific pattern of cell divisions that give rise to four cells: two epidermal cells, the shaft and socket, and two neural cells, a neuron and a sheath cell. Previous clonal analyses of leg disc have suggested a lack of lineage relationship between bristles and bracts. These results were confirmed by labelling clones of cells induced in early third instar larvae; the bract cell does not belong to the SOP lineage (del Álamo, 2002).

How is bract fate specified? The results indicate that the acquisition of bract fate is controlled at three levels. One level of control takes place in the receptor cell, where the competence to acquire bract fate is spatially and temporally controlled. Ubiquitous expression of activated Raf provided in short pulses of time indicated that the competence to acquire bract fate is spatially restricted to specific regions of imaginal discs. There is also a temporal restriction to early pupal development, with peak competence between 8-12 hours APF. These results are consistent with there being a temporally and spatially restricted expression of a tissue-specific regulator that gives the receptor cell the competence to activate bract fate (del Álamo, 2002).

This view is similar to that observed in the specification of bristles by the AS-C genes. Although the expression of AS-C genes is spatially and temporally restricted to cell clusters, ubiquitous ac and sc expression provided at different developmental times, in a genetic background that lacks the endogenous genes, results in a pattern of bristles of the correct type, located in the wild-type positions. Therefore, both pattern and type of bristles are defined by the developmental context in which AS-C genes are expressed (del Álamo, 2002).

The Delta/Notch pathway represses bract fate specification. Overexpressing both activated Raf (Raf*) and constitutive active Notch (Nintra), results in a strong reduction in the number of ectopic bracts produced by Raf*. This result suggests that N signaling acts downstream of Raf in the inhibition of the RAS/MAPK pathway (del Álamo, 2002).

Another level of control occurs in the bristle cell that sends the inductive signal. Spi protein requires the functions of rho1 and S genes to be processed into a soluble, activated form. S and spi are ubiquitously expressed, and rho1 is expressed in SOP cells. The phenotype of rho1 mutant cells in clones indicates that rho1 is required for the induction of bract fate. Nevertheless, rho1 is also expressed in bract-less ChBs, and ubiquitous overexpression of S and rho1 results in a mild phenotype of extra bracts in wild-type positions. Together these results suggest that another component, whose expression must be restricted to the SOP of MBs, is required for bract induction (del Álamo, 2002).

The expression of the poxn gene is both necessary and sufficient for the specification of bract-less ChBs. Since ChBs are specified before epidermal cells are competent for bract induction this provides an explanation for the bract-less phenotype of ChBs. Nevertheless, Poxn overexpression suppresses bracts, and the result of the combined overexpression of Poxn and activated Raf or sSpi indicates that Poxn acts in the SOP cells to repress Spi signaling. Poxn and Rho1 are co-expressed in the SOP. Therefore, these results do not allow the molecular mechanisms by which poxn expression represses Spi signaling to be deduced. Nevertheless, as the result of Star and Rho overexpression indicates that they are not sufficient to induce bracts, and that at least one other component present in the SOP cell is required, it can be tentatively suggested that poxn may act upstream of this other gene (del Álamo, 2002).

Bracts always appear on the proximal-most side of the bristle, which raises the question of whether the position of the bract is defined by a polarized signal. Several lines of evidence suggest that the SOP cell is polarized. In all the experiments that result in extra bracts, the SOP cells appear clustered on the proximal-most side. It seems likely that the polarization of the SOP cell leads to a polarization of Spi signaling, which results in a constant position of the bract, although other possibilities cannot be rejected (del Álamo, 2002).

Concerning the number of bracts per bristle, the results indicate that the argos (aos) gene plays a role in mediating a lateral inhibition mechanism. Loss of aos leads to clusters of bracts, while overexpression removes all bracts. It is reasonable to think, therefore, that as a result of the activation of the RAS/MAPK pathway in the presumptive bract cell, aos expression is being activated in this cell to inhibit the pathway in neighboring epidermal cells (del Álamo, 2002).

Bristles induce bracts via the EGFR pathway on Drosophila legs

A long-standing mystery in Drosophila has been how certain bristles induce adjacent cells to make bracts (a type of thick hair) on their proximal side. The apparent answer, based on loss- and gain-of-function studies, is that these bristles emit a signal that neighbors then transduce via the epidermal growth factor receptor pathway. Suppressing this pathway removes bracts, while hyperactivating it evokes bracts indiscriminately on distal leg segments. Misexpression of the diffusible ligand Spitz (but not its membrane-bound precursor) elicits extra bracts at normal sites. What remains unclear is how a secreted signal can have effects in one specific direction (Held, 2002).

The tibia (Ti) and basitarsus (Ba) of the second leg were the main subjects for analysis. These segments have ten (Ti) or eight (Ba) longitudinal rows of MS bristles, all of which normally possess bracts. They also have a few chemosensory (CS) bristles that lack bracts (~8 on Ti and ~5 on Ba). Mechanosensory (MS) bristles can be distinguished from CS bristles by their shapes (straight vs. curved and thick vs. thin) even when their bracts are artificially suppressed (Held, 2002).

In wild-type males the second legs have an average of 139 MS bristles on the Ti and 74 MS bristles on the Ba. Thus, a wild-type fly is '100%Ti and 100%Ba', while a fly lacking bracts would be '0%Ti and 0%Ba'(Held, 2002).

The EGFR pathway is involved in the development of ommatidia of the fly eye via dosage-sensitive interactions between loss-of-function (LOF) alleles of Star and Ras1. If the EGFR pathway were instrumental in bract development, then those same alleles might be expected to also manifest dosage effects on the frequencies of bracts. Indeed, they do. Star⁵⁶⁷¹/+ heterozygotes have a missing-bract phenotype (31%Ti and 89%Ba), which is aggravated slightly in deficiency heterozygotes such as Df(2L)ast4/+ (17%Ti and 78%Ba). In contrast, Ras1^e1B/+ heterozygotes look nearly wild-type (96%Ti and 100%Ba). The double heterozygote shows synergistic effects: Star⁵⁶⁷¹/+; Ras1^e1B/+ flies have fewer bracts than either heterozygote alone (2%Ti and 60%Ba). In each of the above genotypes, the Ti was more strongly affected than the Ba. This disparity was seen in other contexts as well. Another trend in differential sensitivity was found among the basitarsal bristle rows: dorsal bristles tend to lose bracts more readily than ventral ones (Held, 2002).

If all epidermal cells are competent to make bracts in response to EGFR stimulation, then it should be possible to fool them into 'thinking' that they have 'heard' a signal (when in fact they have not) by activating the pathway downstream of the receptor. For this purpose, a constitutively active Ras1 transgene was used under the control of a heat shock promoter. When hs-Ras1*^M11.2 males are heat-shocked at any time from 5 to 27 h AP, their legs acquire extra bracts. On the Ti, these excess bracts are patchily distributed. On the Ba, the bracts are also patchy (mainly found near bristles) for shocks between 11 and 27 h AP, but earlier shocks (5-10 h AP) typically yield a confluent lawn of unpigmented bracts (Held, 2002).

Since Star acts upstream of the EGF receptor, the missing-bract defect of Star⁵⁶⁷¹/+; Ras1^e1B/+ heterozygotes should be rescueable by hyperactivating Ras1. When the hs-Ras1*^M11.2 transgene is introduced and the resulting Star⁵⁶⁷¹/hs-Ras1*^M11.2; Ras1^e1B/+ pupae are heat-shocked during the extra-bract sensitive period (24 h AP), a partial rescue is indeed observed. The shocked flies have significantly more bracts (30%Ti and 59%Ba) than their unshocked control siblings (0%Ti and 40%Ba) (Held, 2002).

The recent availability of a temperature-sensitive LOF allele for the Egfr gene (Egfr^ts1a) makes it possible to define the sensitive period when the Egfr protein is needed for signal transduction. In the upshift series, Egfr^ts1a/Egfr^CO mutants (Egfr^CO is a null allele) were raised at the permissive temperature of 18°C and then shifted to the restrictive temperature of 29°C at different times for the duration of development. In the downshift series, flies of the same genotype were raised at 18°C (to bypass earlier lethal periods), transferred to 29°C at pupariation (before the sensitive period for bract induction begins) and shifted back to 18°C at different times. Flies raised continuously at 18°C have a wild-type pattern of bracts (99%Ti and 100%Ba), while those kept at 29°C during the pupal period lack all bracts (0%Ti and 0%Ba). For the Ti, the 50% midpoint for bract removal is 17 h AP for upshifts and 28 h AP for downshifts. The sensitive period for the Ti would thus be defined as 17-28 h AP. For the Ba, this period begins 4 h earlier (13-28 h AP) (Held, 2002).

Basitarsal bristle rows are heterogeneous in their timecourses. Relative to the bracts of the ventral rows, the bracts of the dorsal rows acquire immunity to upshifts (Egfr inactivation) later but lose their ability to be rescued by downshifts (restoration of Egfr function) earlier (Held, 2002).

To confirm the role of the EGFR pathway, attempts were made to activate or repress the pathway by the Gal4-UAS method ('driver>slave' ). Two types of Gal4 drivers were used: scabrous-Gal4 (sca-Gal4) is expressed in bristle SOPs and in the proneural clusters (PNCs) whence they arise, whereas Distal-less-Gal4 (Dll-Gal4) is expressed throughout the tarsus and distal Ti. In the first series of experiments, the UAS slaves encoded ligands: UAS-mSpi, UAS-sSpi, and UAS-argos. Spitz (Spi) is a ligand that activates Egfr in various tissues. It is synthesized as a membrane-bound precursor (mSpi) that must be cleaved and released by the action of Star and Rhomboid in order to activate Egfr. In contrast, the UAS-sSpi transgene was engineered to encode only the extracellular part, thus allowing sSpi to be secreted directly without cleavage. Argos is a diffusible inhibitor of Egfr that also does not require cleavage for its secretion. Misexpressing mSpi (via sca>mSpi or Dll>mSpi) has no detectable effect on bracts, while misexpressing sSpi causes some extra bracts at normal sites. For sca>sSpi the number of bristles with extra bracts averaged 1.5 per Ti, 7 per Ba, and 25 per leg overall, while for Dll>sSpi there were 1.4 per Ti, 12 per Ba, and 29 per leg overall. Within each sample, the frequencies varied from 4-36 or 1-63 multiply bracted bristles per leg, respectively. Most of the affected bristles have two adjacent bracts of normal size, while a few (one or two bristles per leg, respectively) have three adjacent bracts in a proximal arc. In contrast, misexpressing Argos reduces the number of bracts to 27%Ti and 85%Ba for sca>argos and 26%Ti and 30%Ba for Dll>argos. Both types of flies had fewer bracts in dorsal vs. ventral rows of the Ba. For sca>argos, 27% (row 4) and 44% (row 5) of the dorsal bristles had bracts vs. 85%-100% for the remaining rows, and on Dll>argos basitarsi the frequencies were 5% (row 4 and row 5) vs. 22%-44% elsewhere. Dll>argos legs also lacked claws and apodemes (Held, 2002).

In the second series of experiments, the UAS slaves encoded various versions of Egfr itself: UAS-Egfr (wild-type product), UAS-Egfr*^top4.2 (constitutively activated form), and UAS-Egfr^DN (dominant-negative form). Misexpressing the normal Egfr was expected to cause extra bracts, but in fact it eliminated bracts (0%Ti and 0%Ba for sca>Egfr and 10%Ti and 0%Ba for Dll>Egfr. Also surprising were the findings that (1) misexpressing the activated receptor had no detectable effect on bracts with either driver and (2) misexpressing the DN form via Dll-Gal4 likewise had no effect. These negative results cannot be ascribed to impotence of the transgenes, since other defects were obvious. To wit, tarsal segments 2-4 were shortened and fused in Dll>Egfr^DN (and Dll>Egfr) legs, and the entire tarsus was reduced to a bump on the end of a swollen Ti in Dll>Egfr*^top4.2 legs. No sca>Egfr^DN flies survived to the adult stage (Held, 2002).

Evidently, the EGFR pathway is necessary and sufficient for bract induction. Its necessity is shown by the ability of pathway suppression to remove bracts, and its sufficiency is shown by the ability of pathway hyperactivity to cause extra bracts or to restore bracts to defective mutants. Suppression was enforced by (1) heterozygosity for the LOF Star⁵⁶⁷¹ allele; (2) haploidy for Star in a deficiency heterozygote; (3) dosage interactions between Star^LOF and Ras1^LOF; (4) exposure of Egfr^ts1a mutants to restrictive temperature, and (5) misexpression of the Egfr inhibitor Argos via sca>argos and Dll>argos. Hyperactivation was achieved by (1) exposure of hs-Ras1*^M11.2 pupae to heat shocks and (2) misexpression of Spi via sca>sSpi and Dll>sSpi.

The inability of sca>mSpi and Dll>mSpi to affect bracts may be due to the fact that Star is essential to convert mSpi into its active form, but Star is present in stoichiometrically limiting amounts -- as is obvious from the sensitivity of bracts to Star dosage. The failure of activated (Egfr*) or dominant-negative (Egfr^DN) Egfr to affect bracts is baffling, given the drastic effects of these same agents on tarsal morphology -- a useful 'internal control' for their potency (Held, 2002).

Also perplexing is that overexpressing the wild-type Egfr causes missing bracts, rather than extra bracts. However, it is important to realize that both Gal4 drivers cause expression of the UAS transgenes not only in the cells surrounding the bristle SOP, but also in the SOP itself, where excess Egfr may interfere with production or secretion of the ligand needed for bract induction (Held, 2002).

Based on the extra-bract phenotypes of sca>sSpi and Dll>sSpi, the inductive ligand in wild-type flies could be sSpi itself. If so, then it is hard to understand why their effects are so mild compared with those of hs-Ras1*^M11.2. The weakness could be due to (1) low output of sSpi relative to the burst of Ras1 from the heat-shock promoter or (2) the presence of inhibitors like Argos, which would not affect Ras1 because Ras1 acts downstream of Egfr (Held, 2002).

Two spatial constraints on signaling were found. One is the greater tendency for the Ti (vs. Ba) to lose bracts when EGFR signaling dwindles. The other is seen on the Ba itself: dorsal rows lose bracts more readily than ventral rows. Both trends exist in (1) Star^LOF heterozygotes, (2) Star deficiency heterozygotes, (3) Star^LOF Ras1^LOF double heterozygotes, and (4) sca>argos flies. Dll>argos flies display the latter trend but not the former, presumably because Dll is expressed more strongly distally. Indeed, expression of Dll during larval life could be the key factor that enables a cell to make bracts in response to later EGFR input from a neighboring bristle cell. Its mode of action might resemble how the homeobox gene Ultrabithorax regulates hair development on different legs (Held, 2002).

Interestingly, both of these trends can also be seen in the data from the temperature shifts. The fact that the Egfr TSP begins 4 h later for the Ti may reflect a higher threshold for signaling there. That is, tibial bract cells may need more EGFR stimulation (longer duration) than basitarsal bract cells before they can differentiate on their own. The fact that dorsal rows lag behind ventral rows can be explained similarly. The inferred difference in thresholds could also explain why dorsal bristle rows of wild-type basitarsi are sensitive to bract loss from heat shocks, while ventral bristle rows are virtually immune (i.e. retain their bracts regardless) (Held, 2002).

Why should cells in different places need different levels of EGFR input to become bracts? The reason for the proximal-distal axis (Ti vs. Ba) is unclear, but the dorsal-ventral discrepancy might stem from differential cross talk between EGFR and the other pathways that govern dorsal (Decapentaplegic) vs. ventral (Wingless) patterning (Held, 2002).

Bract induction is the epitome of a private 'chat' between two cells, though more than one cell in the SOP clone may emit the signal. Its only rival is the famous tête-à-tête between the R8 photoreceptor precursor and a neighboring cell, whereby the latter is recruited to become an R7 photoreceptor. How are other neighbors prevented from 'hearing' the signal and thereby forming a ring of elements (bracts or R7s) around the 'speaker' cell, as is known to occur, for example, in the genesis of chordotonal organs and oenocytes? For R7 induction, various transcription factors limit the ability of other neighbors to respond to the R8 signal. For bract induction the answer is less clear. If a globally acting signal (e.g. emanating from segment boundaries) were enforcing the direction of bract induction, then bristles should always induce bracts on their proximal side, regardless of the orientation of the bristle itself (e.g., lichens only grow on the shady side of trees). However, this is not the case. Misoriented bristles typically make bracts on the side of their socket opposite to the direction in which their shaft points. The simplest way for a bristle cell to send its signal directionally (and to ensure that only one neighbor gets it) would be for it to present a membrane-bound ligand on part of its surface. However, overexpressing mSpitz (via sca>mSpi or Dll>mSpi) fails to evoke extra bracts, so this strategy seems unlikely (Held, 2002).

Oddly, expressing secreted Spitz (via sca>sSpi or Dll>sSpi) elicits extra bracts only on the proximal (usual) side of bristle sockets, rather than in a ring as might have been expected a priori. Evidently, it is not the signal that is localized on one side of the emitting cell but rather the receptor that must be localized on one side of the receiving cell -- or, indeed, perhaps on all epidermal cells. There are precedents for such polarization in the wing and eye, where Frizzled receptors localize on one specific face of each cell (Held, 2002).

Spitz acts redundantly with another Egfr ligand to control rotation in the Drosophila eye

Ommatidial rotation in the Drosophila eye provides a striking example of the precision with which tissue patterning can be achieved. Ommatidia in the adult eye are aligned at right angles to the equator, with dorsal and ventral ommatidia pointing in opposite directions. This pattern is established during disc development, when clusters rotate through 90°, a process dependent on planar cell polarity and rotation-specific factors such as Nemo and Scabrous. Epidermal growth factor receptor (Egfr) signalling is required for rotation, further adding to the manifold actions of this pathway in eye development. Egfr is distinct from other rotation factors in that the initial process is unaffected, but orientation in the adult is greatly disrupted when signalling is abnormal. It is proposed that Egfr signalling acts in the third instar imaginal disc to 'lock' ommatidia in their final position, and that in its absence, ommatidial orientation becomes disrupted during the remodelling of the larval disc into an adult eye. This lock may be achieved by a change in the adhesive properties of the cells: cadherin-based adhesion is important for ommatidia to remain in their appropriate positions. In addition, there is an error-correction mechanism operating during pupal stages to reposition inappropriately oriented ommatidia. These results suggest that initial patterning events are not sufficient to achieve the precise architecture of the fly eye, and highlight a novel requirement for error-correction, and for an Egfr-dependent protection function to prevent morphological disruption during tissue remodelling (Brown, 2003).

The Egfr ligand Keren was misexpressed in developing photoreceptors and cone cells under the control of sev-Gal4. Surprisingly, this caused a disruption in the orientation of ommatidia relative to WT, a phenotype not previously associated with excess Egfr signalling. In the WT adult eye, all ommatidia are oriented at 90° relative to the equator. By contrast, when Keren is misexpressed, many ommatidia are abnormally oriented, with some ommatidia having rotated more than 90° and some less than 90°. In general, excess Egfr signalling leads to over-recruitment of cells in the eye, but photoreceptor recruitment is not affected when Keren is expressed at these levels. However, analysis of the pupal retina shows that Keren misexpression causes over-recruitment of cone cells, consistent with it acting through the Egfr. Previous work has shown that recruitment of cone cells is more sensitive than photoreceptors to Egfr overactivation; these results support this view, and also suggest that rotation is more sensitive than photoreceptor recruitment to perturbation of Egfr signalling (Brown, 2003).

Further examination of the adult phenotype indicates that it is rotation specifically that is disrupted on overexpressing Keren; the chirality (i.e. the correct specification of R3 and R4) of the ommatidia remains unaffected. This distinguishes the UAS-Keren phenotype from disruption of PCP components, which can cause both rotational and chiral defects (Brown, 2003).

Is Egfr activity normally required for correct rotation? Several conditions were examined that decrease Egfr signalling, including a haploinsufficient Star allele (which has slight rotational defects), rho3/roughoid mutants, and expression of dominant-negative Egfr under the control of heatshock HS-Gal4. In all these cases, rotational defects are clearly seen in correctly specified ommatidia. In order to quantify and compare the rotational defects further, the rotation angles of approximately 600 ommatidia each were measured in WT, UAS-keren and ru¹ eyes. Strikingly, defects caused by too little or too much Egfr activity are very similar -- ommatidia are over- or under-rotated, although in both cases there is a bias towards rotation angles of greater than 90°. The similarity of the rotational defects caused by increasing and decreasing pathway activity is reminiscent of some PCP mutations (Brown, 2003).

The rotational phenotypes caused by perturbation of Egfr signalling are very similar to the published phenotype of the roulette mutation, one of the few mutations reported to specifically disrupt rotation and not chirality. Interestingly, roulette turns out to be allelic to argos. The roulette mutation is now referred to as argos^rlt (Brown, 2003).

There are four ligands that activate the Drosophila Egfr: Spitz, Gurken and Keren (which resemble mammalian TGFalpha), and Vein, a neuregulin-like molecule. Spitz is thought to mediate most of the Egfr functions in eye development, although spitz clones do not phenocopy Egfr clones in all respects. Specifically, spitz clones do not show defects in cell survival or ommatidial spacing, which are seen in Egfr loss-of-function clones. spitz hypomorphic eyes were examined to determine whether these show rotational defects. Under-recruited ommatidia are very common in the spi^scp1 hypomorph, indicating that Egfr activity is substantially impaired – to beneath the threshold for photoreceptor recruitment. Despite this, very few misrotated ommatidia are seen. In comparison, ru¹ eyes show only minor recruitment defects, indicating a less dramatic reduction of Egfr activity than spi^scp1. ru¹ eyes, however, show severe rotational defects. These data suggest that Spitz is not essential for normal rotation. They do not, however, rule out the possibility that Spitz acts redundantly with another ligand. To test this, a genetic interaction between Star and a spitz hypomorph was tested. As expected, heterozygosity for spitz enhances the recruitment defects in the S/+ eye. A significant enhancement of rotational defects is observed, implying that Spitz does function in ommatidial orientation. Together, these results suggest that Spitz acts redundantly with another Egfr ligand to control rotation. The fact that loss of Rho3/ru, a protease that activates Egfr ligands, results in rotational defects, whereas spitz mutants do not, implies the involvement of another cleaved ligand. Gurken is restricted to the germline. By elimination, it is therefore tentatively concluded that Keren also acts in the Egfr-dependent regulation of ommatidial rotation. Note, however, that keren expression is too low to detect by in situ hybridisation in any tissue so it is not possible to tell whether keren is transcribed appropriately. Confirmation of this hypothesis awaits the identification of a keren mutant (Brown, 2003).

EGFR signaling regulates the proliferation of Drosophila adult midgut progenitors

In holometabolous insects, which undergo development through metamorphosis involving muliple stages, the adult appendages and internal organs form anew from larval progenitor cells during metamorphosis. The adult Drosophila midgut, including intestinal stem cells (ISCs), develops from adult midgut progenitor cells (AMPs) that proliferate during larval development in two phases. Dividing AMPs first disperse, but later proliferate within distinct islands, forming large cell clusters that eventually fuse during metamorphosis to make the adult midgut epithelium. Signaling through the EGFR/RAS/MAPK pathway is necessary and limiting for AMP proliferation. Midgut visceral muscle produces a weak EGFR ligand, Vein, which is required for early AMP proliferation. Two stronger EGFR ligands, Spitz and Keren, are expressed by the AMPs themselves and provide an additional, autocrine mitogenic stimulus to the AMPs during late larval stages (Jiang, 2009).

The insect midgut, like the vertebrate intestine, is an endoderm-derived organ. Both the larval and adult Drosophila midguts are composed of a single layer of epithelial cells with two layers of visceral muscle (VM) wrapped outside. Inside the gut lumen, a peritrophic membrane separates the food from the intestinal epithelium. During both mammalian and insect embryonic development, Forkhead and GATA transcription factors play evolutionary conserved roles in the specification and subsequent morphogenesis of the digestive tract. Similarly, multiple signaling pathways, including the EGF, Wingless (Wnt), Dpp (TGFβ), Notch and Hedgehog pathways, are involved in the embryonic development of the Drosophila midgut and mammalian intestine. In both systems, cross-talk between mesodermal cells and endoderm-derived epithelial cells in the gut primordium plays important roles during embryonic gut development (Jiang, 2009).

Starting from embryonic development stage 11, the Drosophila midgut epithelium consists of two distinct cell populations: differentiating midgut epithelial cells (larval enterocytes, ECs) and undifferentiated adult midgut progenitors (AMPs, also referred to as midgut histoblast islets or midgut imaginal islets). In Drosophila embryos, AMPs can be marked by expression of asense or by one of several lacZ- or Gal4-expressing enhancer-trap insertions. AMPs first appear as spindle-shaped cells localized to the apical surface of the midgut epithelium, but later migrate to the basal surface of the epithelium where they remain throughout larval development. Notch signaling has been shown to be involved in the development of Drosophila AMPs. In Notch mutant embryos, the number of AMPs in the midgut rudiment is strongly increased at the expense of differentiated larval ECs (Hartenstein, 1992). During larval development, the ECs grow in both size and ploidy by undergoing several endocycles, reaching 64C (DNA content) by the wandering L3 stage. The AMPs remain diploid throughout larval development and appear as scattered islets of cells (hence the term 'midgut histoblast islets') in late-stage larval midguts. During pupal development, the ECs histolyze and a new adult midgut epithelium forms from the AMPs. Similar midgut progenitor cells have also been found in other insect species (Jiang, 2009).

The adult Drosophila midgut has been shown to undergo dynamic self-renewal, a process similar to that found in the mammalian intestine/colon. Fly and mammalian gut homeostasis are both powered by intestinal stem cells (ISCs), and Notch signaling plays similar roles in regulating their differentiation into mature gut cells. Thus, the Drosophila midgut may serve as a model to study gut homeostasis and the development of cancers, such as colorectal carcinoma, that are directly associated with this dynamic process in humans (Jiang, 2009).

This study describes the development of the AMPs in Drosophila larvae and pupae. Drosophila AMPs are shown to divide extensively throughout larval development, and their proliferation can be separated into two distinct phases. During early larval stages, the AMPs divide and disperse to form islets throughout the midgut, but during late larval development the dividing AMPs are contained within these islets. Furthermore, this study revealed that Drosophila EGFR signaling is both necessary and sufficient to induce the proliferation of AMPs during larval development (Jiang, 2009).

Drosophila AMPs were previously thought to be relatively quiescent during larval development, dividing just once or twice, and not initiating rapid proliferation until the onset of metamorphosis. This is the case for several other larval progenitor/imaginal cell types, such as the abdominal histoblasts and cells in the salivary gland, foregut and hindgut imaginal rings. Various studies have suggested that AMP proliferation might precede the onset of metamorphosis. However, these studies did not report the extensive proliferation of the AMPs that is describe in this study, and failed to recognize the early larval proliferative phase when the AMPs divide and disperse. The extensive proliferation of the AMPs is similar to that of the larval imaginal disc cells, which also proliferate throughout larval development, dividing about ten times (Jiang, 2009).

Lineage analysis revealed that the proliferation of the Drosophila AMPs occurs in two distinct phases. In early larvae, the AMPs divide and disperse throughout the midgut to form individual islets. During later larval development, the AMPs continue to divide but do so within these islets, forming large cell clusters. It is speculated that in the early larva, secretion of Vn from the midgut visceral muscle (VM) cells results in low-level activation of EGFR signaling in the AMPs, which is sufficient for their proliferation and might also promote their dispersal. No proliferation defects were detected in AMPs defective in shot function, suggesting that the mechanism of EGFR activation used by tendon cells during muscle/tendon development is probably not the same as in the larval midgut. Specifically, it is unlikely that the Short stop-mediated concentration of Vn on AMPs activates EGFR signaling in the AMPs during early larval development. Consistent with this, dpERK staining was only observed in AMP clusters and not in the isolated AMPs present at early larval stages (Jiang, 2009).

The mechanisms that regulate the transition between these two proliferation phases remain unclear. Fewer AMP clusters are observed when sSpi, sKrn, lambdaTOP (activated Egfr) or Ras^V12 were induced in the AMPs starting from early larval stages, suggesting that EGFR signaling, in addition to its crucial role as an AMP mitogen, might also play a role in AMP cluster formation. In the late larval midgut (96-120 hours AED), high-level EGFR activation, resulting from expression of spi and Krn in the AMPs themselves, might not only promote AMP proliferation, but might also suppress AMP dispersal and thus promote formation of the AMP clusters. How the timing and location of Spi- or Krn-mediated EGFR activation are regulated during larval development is also unclear. It is noted, however, that the pro-ligand form of Krn acted similarly to sKrn, and no functions were uncovered for the Rho-like gene products that regulate Spi and Krn function by proteolytic cleavage in other tissues. This suggests that the localized expression of these ligands in the AMP clusters might be the critical parameter that controls their effects. Consistent with this, Rho-independent cleavage and function of Krn have been documented (Jiang, 2009).

In the developing Drosophila wing, EGFR/RAS/MAPK signaling promotes the expression and controls the localization of the cell adhesion molecule Shotgun (Shg, Drosophila DE-cadherin). Ras^V12-expressing clones generated in the wing imaginal disc are round, much like the AMP clusters described in this study, owing to increased adhesive junctions. In developing Drosophila trachea, EGFR activity upregulates shg expression to maintain epithelial integrity in the elongating tracheal tubes. In the eye, EGFR activity leads to increased levels of Shg and adhesion between photoreceptors. Given these precedents, it seems reasonable to suggest that high-level EGFR activity in the AMP islets upregulates Shg and promotes the homotypic adhesion of the AMPs. Alternatively, changes in the differentiated cells of the midgut epithelium might promote AMP clustering. In either case, the dispersal of early AMPs and subsequent formation of late AMP clusters facilitate the formation of the adult midgut epithelium during metamorphosis (Jiang, 2009).

This study confirms previous reports that Drosophila AMPs replace larval midgut epithelial cells to form the adult midgut epithelium during metamorphosis. Furthermore, this study shows that the majority of AMPs lose esgGal4-driven GFP expression as they differentiate to form the new adult midgut epithelium. These cells lacked Prospero, which marks enteroendocrine cells in both the larval and adult midgut. They to through several rounds of endoreplication during late pupal development, and thus probably all differentiated into adult enterocytes (ECs). During early metamorphosis, some cells in the new midgut epithelium remain small and diploid and maintained strong esgGal4 expression. For several reasons, it is believed that these esg-positive cells are the future adult intestinal stem cells (ISCs). First, esgGal4 expression marks AMPs, including adult ISCs and enteroblasts. Second, mitoses in the adult midgut are observed only in ISCs, and mitoses were observed only in the esg-positive cells during metamorphosis. Third, esg-positive cells migrate to the basal side of the midgut epithelium, the location of adult ISCs. Fourth, AMP clones generated during early larval development contained just a few esg-positive cells when the new adult midgut first formed (24 hours APF), but when such clones were scored in newly eclosed adults, they contain large numbers of ECs, as well as cells positive for the enteroendocrine marker Prospero and the ISC marker Delta. This suggests that a small fraction of AMPs differentiate into adult ISCs. However, esg-positive cells in the new pupal midgut lack Delta expression until eclosion, suggesting that they are probably not mature adult ISCs (Jiang, 2009).

How a small fraction of AMPs are selected to become adult ISCs in the newly formed pupal midgut epithelium is not known. One possibility is that the adult ISCs are determined during larval development, long before the formation of the adult midgut. Another is that they are specified during early metamorphosis. This second hypothesis is preferred for several reasons. First, in lineage analysis, it was found that all AMP clones induced during early larval stages formed multiple clusters. This suggests that there are no quiescent AMPs in the larval midgut. Second, when AMP clones are induced at mid-third instar, the mosaic clusters always contains multiple GFP-positive cells, suggesting that all AMPs in the mid-third instar midgut remain equally proliferative. Third, during larval development, differentiation of the AMPs was never observed, as judged by their ploidy (diploid) and lack of expression of the enteroendocrine marker Prospero (not shown). Fourth, all AMPs appeared to express esgGal4 throughout larval development. Given the crucial role that Notch signaling plays in regulating AMPs during embryonic midgut development (Hartenstein, 1992) and ISCs in adult midgut homeostasis, it is expected that Notch might also function to specify adult ISCs during metamorphosis (Jiang, 2009).

EGFR signaling is both required and sufficient to promote AMP proliferation. Hyperactivation of EGFR signaling, such as by expression of activated Ras (Ras^V12), promoted massive AMP overproliferation and generated hyperplastic midguts that were clearly dysfunctional. In contrast, inhibiting EGFR/RAS/MAPK signaling dramatically reduced AMP proliferation. Furthermore, the ability of EGFR signaling to induce ectopic AMP proliferation is almost unique. With the exception of larval hemocytes, activated EGFR signaling does not promote cell proliferation in the imaginal discs, salivary gland imaginal rings, abdominal histoblasts, foregut and hindgut imaginal rings. This suggests that the regulation of AMP proliferation is different from that in other imaginal cells (Jiang, 2009).

Despite the obvious differences between adult ISCs and their larval progenitors, the AMPs, there are also similarities. First, when the new adult midgut epithelium forms, larval AMPs give rise to the new adult midgut including the adult ISCs. Many genes, such as esg, that are specifically expressed in the larval AMPs are also expressed in the adult ISCs. Second, the structure of the midgut epithelium with basal AMPs or ISCs is similar in larval and adult stages. Third, vn expression in larval VM persists in the adult midgut, suggesting that Vn from the adult VM might also regulate the ISCs (Jiang, 2009).

In two Drosophila stem cell models, the testis and ovary, stem cells reside in special niches comprising other supporting cell types. These niches maintain the stem cells and provide them with proliferative cues. For example, in the testis, germ stem cells attach to the niche that comprises cap cells. The cap cells release Jak/Stat and BMP ligands [Upd (Os) and Gbb/Dpp], which maintain the stem cells and induce their proliferation. Whether Drosophila ISCs utilize supporting cells that constitute a niche remains unclear. This study shows that multiple EGFR ligands are involved in the regulation of Drosophila AMP proliferation. During early larval development, the midgut VM expresses the EGFR ligand vn, which is required for AMP proliferation. Thus, the early AMPs might be considered to require a niche comprising non-epithelial VM. Later in larval development, however, the AMPs express two other EGFR ligands, spi and Krn, which are capable of autonomously promoting their proliferation and may render vn dispensable. It was found, however, that depleting spi and Krn in the AMPs did not affect AMP proliferation, suggesting that vn or another trigger of EGFR/RAS/MAPK activity might complement spi and Krn in late-stage larvae (Jiang, 2009).

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).

Regional control of Drosophila gut stem cell proliferation: EGF establishes GSSC proliferative set point & controls emergence from quiescence

Adult stem cells vary widely in their rates of proliferation. Some stem cells are constitutively active, while others divide only in response to injury. The mechanism controlling this differential proliferative set point is not well understood. The anterior-posterior (A/P) axis of the adult Drosophila midgut has a segmental organization, displaying physiological compartmentalization and region-specific epithelia. These distinct midgut regions are maintained by defined stem cell populations with unique division schedules, providing an excellent experimental model with which to investigate this question. This study has focused on the quiescent gastric stem cells (GSSCs) of the acidic copper cell region (CCR), which exhibit the greatest period of latency between divisions of all characterized gut stem cells, to define the molecular basis of differential stem cell activity. Molecular genetic analysis demonstrates that the mitogenic EGF signaling pathway is a limiting factor controlling GSSC proliferation. Under baseline conditions, when GSSCs are largely quiescent, the lowest levels of EGF ligands in the midgut are found in the CCR. However, acute epithelial injury by enteric pathogens leads to an increase in EGF ligand expression, specifically Spitz and Vein, in the CCR and rapid expansion of the GSSC lineage. Thus, the unique proliferative set points for gut stem cells residing in physiologically distinct compartments are governed by regional control of niche signals along the A/P axis (Strand, 2013).

The CCR epithelium is the exclusive site of large acid-secreting copper cells responsible for generating a low pH compartment in the midgut. Gastric stem cells in the CCR are normally quiescent but are robustly stimulated to replenish the unique differentiated cells of the gastric epithelium in response to injury by enteric pathogens or heat stress. This study resolvse outstanding issues related to the GSSC lineage, demonstrating the presence of tripotent GSSC lineages in the CCR. In addition, wa central role is demonstrated for the conserved EGF signaling pathway in controlling the emergence of gastric stem cells from quiescence. Taken together, two key differences between GSSCs and intestinal stem cells (ISCs) are now evident: the unique region specific cell lineages that they support (copper, interstitial, enteroendocrine vs. enterocyte and enteroendocrine) and their activity levels (quiescent vs. active). Thus, maintenance of physiologically and functionally distinct compartments of the adult midgut depends upon the activity of distinct stem cell lineages (Strand, 2013).

What is the nature of the unique molecular program that governs the observed differences in GSSC and ISC proliferative behavior? This study indicates that regional differences in gut stem cell proliferation are controlled by regional differences in EGF ligand availability. First, reporters of EGF pathway activity are normally very low in the CCR under baseline conditions, when GSSCs are quiescent. However, damage to the gastric epithelium by enteric infection increases local EGF ligand expression and Erk phosphorylation. This EGF activation directly correlates with an observed increase in proliferating GSSCs. Second, ectopic activation of the EGF pathway is sufficient to cell-autonomously promote GSSC proliferation in the absence of environmental challenge. Finally, functional EGF signaling is required for GSSC proliferation following enteric infection and for GSSC lineage expansion. Importantly, these studies of GSSCs in the CCR are similar to previous studies demonstrating that EGF signals are an essential part of the core niche program controlling the ISC lineage. Thus, regional control of EGF ligands, and perhaps other regulators of EGF pathway activity, are essential in generating gastrointestinal stem cell niches with distinct proliferative set points (Strand, 2013).

In this light, it is worth noting that over-expression of epidermal growth factors and their receptors are associated with human gastric cancer, the second leading cause of cancer-related deaths worldwide. In addition, Ménétrier’s disease is a hyperproliferative disorder of the stomach caused by over-expression of the EGF ligand TGF-α. Over production of TGF-α and increased EGF signaling is associated with an expansion of surface mucous cells and a reduction in parietal and chief cells. Gastric stem cells are the proposed cell-of-origin in Ménétrier’s disease, but this has not been directly tested due to a lack of gastric stem cell specific markers in the murine system. Advances in understanding how EGF ligand availability controls activity of the acid-secreting gastric stem cell lineage in Drosophila raises the possibility that hyperplastic conditions associated with the human stomach might arise when ectopic EGF ligands draw resident stem cells out of their quiescent state (Strand, 2013).

EGF signaling appears to be only one aspect of the region specific program controlling gastric stem cells in the adult copper cell region. Previous studies have shown that a Delta-lacZ enhancer trap line was not present in GSSCs under baseline conditions. In the course of this study, it was observed that Pseudomonas entomophila challenge also leads to an increase in Delta ligand expression in dividing cells, suggesting a role for Delta/Notch signaling in the GSSC lineage. In addition, elegant studies of GSSCs under baseline conditions have recently shown that the secreted BMP/Dpp signaling pathway is both necessary and sufficient to specify copper cells in the adult midgut and acts via the labial transcription factor. Interestingly, while the highest levels of Dpp pathway reporters are detected in the CCR, manipulation of the BMP/Dpp pathway did not affect GSSC proliferation. Thus, the GSSC lineage is influenced by secreted niche factors, which independently control both GSSC proliferation and cell fate specification (Strand, 2013).

In conclusion, understanding GI regionality and homeostatic diversity along the A/P axis is important for several reasons. It is now possible to gain insight into how the modification of a core GI niche program, which adapts each stem cell to its compartment specific physiology, leads to difference in lineage output. Second, disruption of regional identity along the GI tract is associated with a class of precancerous conditions called metaplasias, in which one region of the GI tract takes on the attributes of another. Finally, both the establishment and maintenance of tumorigenic lineages exhibit marked preferences along the A/P axis of the gut. The striking similarities between vertebrate and invertebrate GI biology, suggest that delving deeper into the mechanisms underlying Drosophila midgut regionalization will continue to provide important insights into these fundamental biological problems (Strand, 2013).

Epidermal growth factor signalling controls Myosin II planar polarity to orchestrate convergent extension movements during Drosophila tubulogenesis

Most epithelial tubes arise as small buds and elongate by regulated morphogenetic processes including oriented cell division, cell rearrangements, and changes in cell shape. Through live analysis of Drosophila renal tubule morphogenesis this study shows that tissue elongation results from polarised cell intercalations around the tubule circumference, producing convergent-extension tissue movements. Using genetic techniques, it was demonstrated that the vector of cell movement is regulated by localised Epidermal growth factor (EGF) signalling from the distally placed tip cell lineage, which sets up a distal-to-proximal gradient of pathway activation to planar polarise cells, without the involvement for PCP gene activity. Time-lapse imaging at subcellular resolution shows that the acquisition of planar polarity leads to asymmetric pulsatile Myosin II accumulation in the basal, proximal cortex of tubule cells, resulting in repeated, transient shortening of their circumferential length. This repeated bias in the polarity of cell contraction allows cells to move relative to each other, leading to a reduction in cell number around the lumen and an increase in tubule length. Physiological analysis demonstrates that animals whose tubules fail to elongate exhibit abnormal excretory function, defective osmoregulation, and lethality (Saxena, 2014: PubMed).

Specificity of Drosophila cytonemes for distinct signaling pathways

Cytonemes are types of filopodia in the Drosophila wing imaginal disc that are proposed to serve as conduits in which morphogen signaling proteins move between producing and target cells. The specificity was investigated of cytonemes that are made by target cells. Cells in wing discs made cytonemes that responded specifically to Decapentaplegic (Dpp) and cells in eye discs made cytonemes that responded specifically to Spitz (the Drosophila epidermal growth factor protein). Tracheal cells had at least two types: one made in response to Branchless (a Drosophila fibroblast growth factor protein, Bnl), to which they segregate the Bnl receptor, and another to which they segregate the Dpp receptor. It is concluded that cells can make several types of cytonemes, each of which responds specifically to a signaling pathway by means of the selective presence of a particular signaling protein receptor that has been localized to that cytoneme (Roy, 2011).

Cells in developing tissues are influenced by multiple signals that they process and integrate to control cell fate, proliferation, and patterning. An example is in the Drosophila wing imaginal disc, where cells depend on several signaling systems that are intrinsic to the disc. Dpp, Wingless (Wg), Hedgehog (Hh), and epidermal growth factor (EGF) are produced and released by different sets of disc cells, and receipt of these signaling proteins programs their neighbors to develop and grow. The mechanisms by which morphogen signaling proteins influence target cells must ensure both specificity and accuracy, and one possibility is that these proteins transfer at points of direct contact. Imaginal discs are flattened sacs that have a monolayer of columnar cells on one side and squamous peripodial cells on the other. Many cells in wing discs make filopodial extensions that lie along the surfaces of the monolayers, oriented toward morphogen-producing cells. These extensions have been termed cytonemes to denote their appearance as cytoplasmic threads and to distinguish them as specialized structures that polarize toward morphogen-producing regions (Roy, 2011).

In wing discs dissected from third instar larvae, cytonemes can be seen as filaments extending from randomly generated somatic clones engineered to express a fluorescent protein such as soluble, cytoplasmic green fluorescent protein (GFP) or a membrane-bound form such as mCD8:GFP (the extracellular and transmembrane domains of the mouse lymphocyte protein CD8 fused to GFP). To image disc cytonemes, unfixed discs were placed peripodial side down on a coverslip, covered with a 1-mm-square glass, and mounted over a depression slide with the disc hanging from the coverslip. Because fluorescence levels in cytonemes were low relative to background, recorded images were processed to increase intensity and were subjected to de-convolution. Expression of CD8:GFP in wing disc clones revealed cytonemes emanating from both the apical and basal surfaces of columnar cells, as well as from peripodial cells (whose apical and basal surfaces could not be distinguished). Most cytonemes were perpendicular to the anterior/posterior (A/P) axis of the disc and oriented toward the cells that produce Dpp at the A/P compartment border; others were oriented toward the cells that produce Wingless at the dorsal/ventral (D/V) compartment border. Disc-associated myoblasts also had filopodia (Roy, 2011).

In the eye disc, cells in the columnar layer organize into ommatidial clusters as a wave of differentiation [the morphogenetic furrow (MF)] passes from posterior to anterior. A second axis, centered at the equator, is orthogonal to the MF and defines a line of mirror-image symmetry where dorsal and ventral ommatidia are juxtaposed. The columnar cells divide during the third instar period but stop or divide only once after the MF passes. CD8:GFP expression was induced in somatic clones and the columnar cells were examined. Whereas clones of six to eight cells were present on both sides of the MF, only cells anterior to the MF had visible cytonemes. Cytonemes emanating from these clones oriented either toward the axis defined by the MF or toward the axis defined by the equator. Single clones with cytonemes oriented both toward the MF and toward the equator were not observed, and there was no apparent correlation between clone position and cytoneme orientation or cytoneme length. Cells in the peripodial layer of the eye disc also had cytonemes (Roy, 2011).

The EGF pathway is a key signaling system for eye development, and cells in the MF express the EGF protein Spitz (Spi). Because one of the two types of anterior cell cytonemes extended toward the MF and to explore the distribution of membrane-bound receptor proteins, clones were induced that expressed an epidermal growth factor receptor:GFP (EGFR:GFP) fusion protein. Anterior cells expressing EGFR:GFP had cytonemes that oriented toward the MF, and most of these cytonemes had fluorescent puncta; no cytonemes that were marked by EGFR:GFP oriented toward the equator. Other than their 'furrow-only' orientation, the cytonemes marked by EGFR:GFP were similar to those marked by CD8:GFP. In contrast, co-expression of CD8:GFP with (nonfluorescent) EGFR marked both furrow-directed and equator-directed cytonemes. Thus, expression of EGFR:GFP does not eliminate the equator-directed cytonemes, suggesting that the specific localization of EGFR:GFP to furrow-directed cytonemes is not a consequence of ectopic (over)expression of this fusion protein (Roy, 2011).

Evidence that the furrow-directed cytonemes depend on Spi/EGF signaling was obtained by expressing a dominant negative form of EGFR. Although EGFR is required for cell proliferation in the disc, small clones expressing EGFR^DN were recovered that co-expressed EGFR^DN and CD8:GFP; in these clones, only cytonemes that appeared to be randomly oriented were present, indicating that the long, furrow-directed cytonemes may require EGFR signal transduction in the cytoneme-producing cells (Roy, 2011).

Wing disc-associated tracheal cells also make cytonemes. The transverse connective (TC) is a tracheal tube that nestles against the basal surface of the wing disc columnar epithelium and that sprouts a new branch [the air sac primordium (ASP)] during the third instar period in response to Branchless (Bnl) expressed by the wing disc. Tracheal tubes are composed of a monolayer of polarized cells whose apical surfaces line a lumen. Expression of CD8:GFP throughout the trachea (btl-Gal4 UAS-CD8:GFP) made it possible to detect GFP fluorescence in several types of cytonemes emanating from the basal surfaces of the TC and ASP. Cytonemes at the tip of the ASP (length range, 12 to 50 μm; average length of 23 μm) contained the Breathless (Btl); the Drosophila fibrobast growth factor receptor (FGFR) and appeared to contact disc cells that express Bnl. Short cytonemes (length range, 2 to 15 μm; average length of 8.5 μm) extended from the TC cells in the vicinity of the ASP (Roy, 2011).

Tests were carried out to se whether Dpp, Spi, Bnl, and Hh affected wing disc, eye disc, and tracheal cytonemes differentially. Ubiquitous expression of Spi, Bnl, or Hh (induced by heat shock) did not alter the A/P-oriented apical cytonemes in the wing disc, and, in the eye disc, the long cytonemes of the columnar layer were unaltered after ubiquitous expression of Dpp, Bnl, or Hh. In contrast, long oriented cytonemes were absent in wing discs after ubiquitous expression of Dpp, and only short cytonemes that appeared to be randomly oriented were observed. Similarly, 0.5 to 3 hours after cSpi, a constitutively active form of EGF, was expressed ectopically by heat shock induction, clones expressing CD8:GFP in the eye disc had many short cytonemes that lacked apparent directional bias; in contrast to controls, no long cytonemes oriented toward the MF were observed. Cytonemes with normal orientation and length (including MF-directed cytonemes) were present in eye discs that were examined later, 8 hours after a pulse of cSpi expression. To monitor EGFR-containing cytonemes for sensitivity and responsiveness to Spi, cSpi was expressed by heat shock induction, and cells in clones expressing EGFR:GFP were examined. After a pulse of cSpi expression, the extensions oriented outward without apparent directional bias, and the EGFR:GFP puncta were present in all cytonemes (Roy, 2011).

To examine responses of the ASP tip cytonemes, Hh, Spi, Dpp, and Bnl were overexpressed by heat shock and GFP-marked cytonemes at the ASP tip were examined. No differences in number of cytonemes were detected until about 3 hours after heat shock. Four to 5 hours after heat shock, expression of Bnl increased the number of tip cytonemes by ~2.6 times, and although most of the cytonemes were <30 μm, the cytonemes >30 μm also increased (~3.2 times). Most of the long cytonemes in these preparations were oriented in directions other than toward the cells that normally express Bnl. The number of long cytonemes >30 μm did not change after overexpression of Hh, Spi, and Dpp (0.6 to 0.8 times); the number of short cytonemes increased after Dpp overexpression (~1.7 times) but not after overexpression of Hh or Spi (Roy, 2011).

Thus, the responses of apical wing disc cytonemes to overexpressed Dpp, of eye disc cytonemes to ubiquitous Spi, and of ASP tip cytonemes to exogenous Bnl (Drosophila FGF) are similar. These results suggest that the cytonemes detected in the wing discs and eye discs may have orientations and lengths that are dependent specifically on the respective sources of Dpp and Spi, whereas the ASP may extend cytonemes in response to more than one signaling protein. These results are, however, complicated by the heat shock mode of induction because both the cells that expressed GFP (and extended marked cytonemes) as well as the surrounding cells expressed the signaling proteins. To overcome this problem, a method was developed to induce two types of somatic clones in the same tissue, one that expressed GFP and another that expressed Dpp (Roy, 2011).

The GAL4 system was used to label cytonemes with CD8:GFP. Clones of GAL4-expressing cells were generated with heat shock-induced flippase (FLP recombinase). The second type of clone expressed a Dpp:Cherry fusion and was generated with a variant Cre-progesterone receptor recombinase that could be activated with a regime of heat shock and RU486. By adjusting the timing and strength of induction, wing discs were produced with small, independent, and relatively infrequent clones. In discs with clones that expressed ectopic Dpp as well as clones that expressed CD8:GFP, apical cytonemes tagged with GFP were detected that oriented toward nearby Dpp:Cherry-expressing cells and not toward either the A/P or D/V signaling centers. Such 'abnormally directed' cytonemes were never observed in control discs. The abnormally oriented cytonemes suggest that apical cytonemes in the wing blade respond directly to sources of Dpp and that their orientation reflects extant sources of signaling protein (Roy, 2011).

To characterize the relationship between tracheal ASP tip cytonemes and FGF signaling from the wing disc, the distribution of Btl (FGFR) was examined in ASP cells and in ASP cytonemes. In preparations from larvae with tracheal expression of both CD8:GFP and Btl:Cherry (btl-GAL4 UAS-CD8:GFP;UAS-Btl:Cherry), cytonemes were marked by CD8:GFP, some of which had fluorescent Btl:Cherry puncta. Each ASP had only a few long (>30 μm) cytonemes, most of which contained Btl:Cherry puncta. Few of the more numerous short cytonemes (<30 μm) contained Btl:Cherry puncta. To characterize Btl:Cherry after overexpression of Bnl, focus was placed on preparations obtained 1 to 2 hours post-induction (genotype btl-GAL4 UAS-CD8:GFP/HS-Bnl;UAS-Btl:Cherry/Gal80^ts), because during this time interval the ASP morphology was close to normal but cytonemes had changed. ASPs were ignored after longer postinduction intervals because of major malformations to ASP morphology after 3 to 4 hours. Long cytonemes with Btl:Cherry puncta were present 1 hour after a pulse of Bnl expression; but 2 hours after the pulse, most ASPs had no long cytonemes, and the number of short puncta-containing cytonemes increased at the tip and along the shaft of the ASPs. After control heat shock or heat shock-induced expression of Dpp, the distribution of Btl:Cherry puncta in the ASP tip cytonemes was similar to normal controls: Long cytonemes had Btl:Cherry puncta, but most short cytonemes did not (Roy, 2011).

Because the number of small cytonemes at the ASP tip may have increased after ectopic Dpp expression, whether the thickveins (tkv) gene, which encodes a subunit of the Dpp receptor, is expressed in the ASP was investigated. Expression of the tkv reporter, tkv-lacZ (P{lacW}tkv¹⁶⁷¹³), was detected in the ASP. When Tkv:GFP and Btl:Cherry were expressed together, Tkv:GFP and Btl:Cherry segregated to separate tip cytonemes at the ASP tip. Whereas Tkv-containing cytonemes were short (<30 μm), most of the Btl-containing cytonemes were longer (three of four of the Btl:Cherry-containing cytonemes were longer than 30 μm), and they lay in focal planes closer to the disc. These properties were consistent in all preparations examined in which both green Tkv and red Btl cytonemes were intact. Imaging these marked ASPs revealed that overexpressed Tkv:GFP and Btl:Cherry were present not only in the plasma membranes (as expected) but also in separate puncta in the cell bodies. This shows that Tkv and Btl receptors also segregated to separate locations in the ASP cell bodies (Roy, 2011).

These findings suggest that the ASP has long cytonemes that are specific to Bnl and specifically harbor Btl-containing puncta and that the ASP also has cytonemes that are specific to Dpp and specifically harbor Tkv. Similarly in the eye disc, the presence of EGFR:GFP in furrow-oriented cytonemes and not in equator-oriented cytonemes suggests that cytonemes in the eye disc also selectively localize receptors. And as was previously shown, apical cytonemes in the wing disc selectively localize Tkv. The apparent ligand specificities and contrasting makeup of these cytonemes suggest a diversity of functionally distinct subtypes: Cells appear to make cytonemes that respond specifically to the Dpp, EGF, or Bnl signaling proteins. The basal filopodia implicated in Delta-Notch signaling in the wing disc may represent yet another type (Roy, 2011).

The mechanism that endows cytonemes with specificity for a particular signaling protein cannot be based solely on tissue-specific expression of a receptor. Spi, Dpp, and Hh are active in eye discs, but only changes in Spi signaling affected the furrow-directed cytonemes. And in the wing disc, both the Hh and EGF signal transduction pathways are active in cells at the A/P compartment border, but the apical cytonemes only responded to overexpressed Dpp. The findings that tracheal cells in the ASP respond to both Dpp and Bnl and that the Tkv and Btl receptors are present in different cytonemes that the ASP cells extend suggest that specificity may be a consequence of the constitution of the cytoneme, not on which receptors the cells make. The mechanism that localizes receptors to different cytonemes is not known, but because the marked receptors that were expressed also segregated to different intracellular puncta, the processes that concentrate these receptors in separate locations may not be exclusive to cytonemes. There is a precedent for segregation of proteins to different cellular extensions, neurons segregate proteins to dendrites or axons, so extending projections with specific and distinct attributes may be a general property of cells (Roy, 2011).

Spitz and the immune response

Oxidative stress in the haematopoietic niche regulates the cellular immune response in Drosophila

Oxidative stress induced by high levels of reactive oxygen species (ROS) is associated with the development of different pathological conditions, including cancers and autoimmune diseases. This study analysed whether oxidatively challenged tissue can have systemic effects on the development of cellular immune responses using Drosophila as a model system. Indeed, the haematopoietic niche that normally maintains blood progenitors can sense oxidative stress and regulate the cellular immune response. Pathogen infection induces ROS in the niche cells, resulting in the secretion of an epidermal growth factor-like cytokine signal that leads to the differentiation of specialized cells involved in innate immune responses (Sinenko, 2011).

Abnormal metabolism is often associated with oxidative stress that results in increased production of ROS by mitochondria. Different concentrations of ROS and their derivatives are required for proper maintenance, proliferation, differentiation and apoptosis of stem cells and their committed progenitors. In Drosophila, developmentally regulated levels of ROS are critical for maintenance of haematopoietic progenitors within the medullary zone (MZ) of the lymph gland. In contrast, under normal growth conditions, posterior signaling center (PSC) cells in wild-type larvae had very low levels of ROS expression compared with that in the progenitor population of cells within the MZ. To induce oxidative stress in the PSC ND75, a component of complex I of the electron transport chain (ETC), was inactivated with double-stranded RNA (dsRNA) using the Gal4/UAS misexpression system and the PSC-specific Antp-Gal4 driver. ND75 inactivation causes a readily detectable increase in ROS in the PSC cells, rising to levels similar to those seen in the progenitor cells of the MZ. The phenotypic consequence of inducing oxidative stress in the cells of the PSC was a remarkably robust increase in numbers of circulating lamellocytes. Such an elevated number of lamellocytes was usually observed in wild-type larvae only if they were infested by parasitic wasps. Although Antp-Gal4 is not expressed anywhere in the blood system, except the PSC, this driver is also expressed in other larval tissues. To exclude the possibility that the effect was due to a non-PSC expression of Antp-Gal4, the function of ND75 was also eliminated using the Dot-Gal4 driver normally expressed at high levels in the PSC, and this resulted in an identical lamellocyte response. In contrast, oxidative challenge to various other larval tissues, including the fat body (LSP2-GaI4), the epidermis (A58-GaI4), the neurons (C127-GaI4), the dorsal vessel (Hand-GaI4), the ring gland (5015-GaI4), the wing imaginal disc (ap-Gal4) or the trachea (btl-GaI4), did not have a significant effect on lamellocyte differentiation. Furthermore, high ROS levels generated within the progenitor cells (dome-GaI4) of the lymph gland, which causes autonomous differentiation of this population, also did not have any significant effect on the non-autonomous differentiation of lamellocytes in the circulation. In contrast, oxidative challenge of the PSC caused non-autonomous lamellocyte response in circulation as well as within the lymph gland. The PSC-mediated effect was due to mitochondrial dysfunction and not specifically linked to the product of the ND75 gene, because attenuation of PDSW (another complex I component), cytochrome-c oxidase, subunit Va (CoVa, a component of ETC complex IV) or Marf (mitochondrial assembly regulatory factor) function in the PSC, all induced increases in lamellocyte differentiation. The strength of the lamellocyte response to complex I inactivation depended on the strength of the dsRNA construct used in the experiment. Temporally, induction of the mutation in the second-larval instar caused the lamellocyte response to be seen in the third instar. This correlates well with the timescale of response to parasitic wasp infection. Finally, this oxidative stress elicited a cell-specific response; for example, no significant effect was seen on the differentiation of crystal cells and plasmatocytes in circulation. These results establish that the oxidative status of the PSC has a specific and non-autonomous role in lamellocyte differentiation as an immune response to parasitic invasion (Sinenko, 2011).

The status of the PSC cells on oxidative stress conditions was further analysed in some detail. ND75 dysfunction does not affect proliferation or maintenance of the PSC, because the number of PSC cells, which maintain expression of Antp, remains intact in this mutant background. In addition, no apoptosis is detected in ND75-deficient PSC cells, and also, apoptosis in the PSC alone, specifically induced by overexpression of Hid/Rpr, has no effect on lamellocyte differentiation (Sinenko, 2011).

Overexpression of superoxide dismutase-2 (SOD2) as a scavenger for ROS in ND75-deficient PSC is able to suppress the lamellocyte response significantly. Furthermore, activation of the Forkhead box O (FoxO) transcription factor that positively regulates expression of antioxidant enzymes, including SOD2, completely suppresses the dsND75-induced lamellocyte response. Inactivation of the Akt1 protein kinase in PSC also results in a near-complete suppression of the dsND75-induced lamellocyte response, suggesting a role for the PI3K/Akt pathway in the regulation of FoxO. This is an important issue because FoxO activity can also be controlled by the Jun N-terminal kinase (JNK) pathway, but in the PSC the AKT pathway mediates this effect. The JNK reporter (puc69-lacZ) is not expressed in the PSC, and inactivation of JNK (encoded by the basket gene) using the dominant-negative form (bsk^DN) does not suppress dsND75-induced lamellocyte response. The FoxO reporter (4E-BP-lacZ) is robustly activated in the ND75-deficient PSC; however, loss of translational inhibition mediated by 4E-BP does not mimic this effect. It is important to point out that under wild-type non-stressed conditions, the PSC has relatively low levels of ROS, and therefore inactivation of either Foxo or SOD2 has no phenotypic consequence. These data are interpreted to indicate that metabolic dysfunction induces an oxidatively stressed PSC that causes the activation of this pathway and the lamellocyte response (Sinenko, 2011).

Differentiation of lamellocytes has been associated with the JAK/STAT, JNK and Ras/Erk signalling pathways. These pathways were genetically altered in an ND75-deficient PSC background to identify which, if any, is involved in the lamellocyte response. Inactivation of the unpaired ligands (upd3, upd2 or upd) that activate the JAK/STAT pathway or of eiger (egr), which activates JNK signalling, did not suppress the lamellocyte phenotype. This strongly suggests that these pathways are not involved in the process downstream of ROS in the PSC and is consistent with previous studies showing that components of the JAK/STAT pathway (upd3, dome and Tep4) and JNK (puc69-lacZ reporter) are not involved in the functioning of the PSC. However, these pathways are likely to be involved in direct regulation of lamellocyte differentiation independently of the PSC function. In contrast, inactivation of spitz (spi), encoding the ligand for epidermal growth factor receptor (EGFR), in the context of ND75-deficient PSC significantly suppresses the lamellocyte response. Furthermore, overexpression of the secreted form of Spi (s.Spi), but not the alternative EGFR ligand, Vein (Vn) in the PSC, causes increased differentiation of circulating lamellocytes in an otherwise wild-type larva. EGFR mutant Egfr^TS/Egfr¹⁸ lymph glands develop normally, suggesting that EGFR signalling is not required for normal lymph gland development but rather is involved in the regulation of a cellular immune response as a signalling event from the PSC only when the latter is oxidatively stressed (Sinenko, 2011).

The PSC-dependent parasitic challenge induced by wasp egg infestation and the mechanism described above both give rise to the same cellular response. Therefore, whether parasitization causes oxidative stress to the PSC was examined. Immune challenge caused by wasp infestation was found to induce high levels of ROS in the PSC cells as seen 12 h after invasion. The most prominent effect is on superoxide radicals detected with dihydroethidium staining; a smaller but detectable elevation of peroxide radicals revealed by RedoxSensor staining is also apparent in PSC cells on this immune challenge. Scavenging these ROS types in the PSC by overexpressing SOD2 or catalase (Cat) but not glutathione peroxidase (GPx), which reduces thioredoxin-mediated effects, significantly suppresses the lamellocyte response caused by wasp infestation. These genetic results are consistent with a model in which parasitic infection by wasp eggs raises ROS levels in the PSC, which then causes lamellocyte induction by expressing Spitz. To test this model, spi within the PSC was inactivated in larvae infected by parasitic wasps. This caused a strong suppression of the lamellocyte response; the few remaining L1 marker-positive cells are immature, as indicated by their relatively small cell size and their morphology. In addition, melanotic capsules that are indicative of extensive cellular immune response to parasitic infection do not develop in a spi mutant background during wasp infestation. Inactivation of spitz in the PSC did not affect the increase in ROS triggered by wasp infeststion. Thus spi does not regulate the ROS levels in the PSC; rather, wasp infection raises ROS levels, which leads to release of the s.Spi. Previous studies have shown that s.Spi production requires the function of the trafficking protein Star (S), and the protease Rhomboid (Rho1). This study found that the wasp-induced lamellocyte response and melanotic capsule formation are robustly suppressed on the loss of a single copy of Star. More importantly, parasite-induced immune challenge specifically upregulates Rho1 in the PSC by an as yet unidentified mechanism. These data establish that S and Rho1 are canonically required for processing and releasing the Spitz from the PSC (Sinenko, 2011).

Secreted Spitz is known to bind to EGFR and activate the Ras/Erk pathway. A dominant-negative form of EGFR (Egfr^DN) strongly suppresses the lamellocyte response induced by wasp infestation when it is expressed in the lymph gland and the circulating haemocytes using the pan-haemocyte HHLT Gal4 driver. This phenotype is virtually identical to that seen when spi^RNAi is expressed in the PSC using Antp-Gal4. In addition, compartment-specific drivers were used, and inactivation of the receptor in the cortical zone of the lymph gland and in circulating haemocytes (using lineage-traced Hml^Δ-Gal4 line) was found to prevent Hml-positive cells from becoming lamellocytes on wasp infestation. Importantly, it was also found that a small subset of lamellocytes does not express Hml in the wild-type background and consequently Egfr^DN is not expressed in these cells when Hml^Δ-Gal4 is used as a driver. These Hml⁻,L1⁺ lamellocytes are easily detectable in this genetic background and act as an internal control. Expression of an activated form of EGFR (Egfr^Act) in Hml⁺ haemocytes causes a robust increase in lamellocyte differentiaion. This is also consistent with previous work, which showed that activated Ras induces an increase in the total number of haemocytes, including lamellocytes. Finally, both loss of ND75 in the PSC and wasp infestation cause robust activation of Erk as evident by an increase in dpErk staining in circulating haemocytes including lamellocytes. This indicates that lamellocytes in circulation differentiate from precursor cells on activation of Spi/EGFR/Erk signalling (Sinenko, 2011).

PSC cells have two independent functions: they serve as a haematopoietic niche in the lymph gland, where they orchestrate the maintenance and proper differentiation of haematopoietic progenitors, and they regulate the cellular immune response by controlling lamellocyte differentiation in response to infection. The results presented in this study establish the mechanism for this latter function. Changes in oxidative status, caused by events of parasite invasion or ETC dysfunction, initiates a signal within this immunocompetent compartment causing the secretion of a cytokine ligand, Spitz, that induces differentiation of lamellocyte precursors in the circulatory system of the larva. The identified mechanism is consistent with previously reported studies in mammals, which have shown that mitochondrial ROS can trigger systemic signals that reinforce the innate immune response. These studies raise the possibility that specific populations of cells also exist in mammalian systems that sense oxidative stress due to infection and non-autonomously signal myeloid progenitors to initiate differentiation and enhance the immune response. Whether such populations are to be found within the haematopoietic niche as in Drosophila remains a speculation that can be tested in future studies (Sinenko, 2011).

Coordinated sequential action of EGFR and Notch signaling pathways regulates proneural wave progression in the Drosophila optic lobe.

During neurogenesis in the medulla of the Drosophila optic lobe, neuroepithelial cells are programmed to differentiate into neuroblasts at the medial edge of the developing optic lobe. The wave of differentiation progresses synchronously in a row of cells from medial to the lateral regions of the optic lobe, sweeping across the entire neuroepithelial sheet; it is preceded by the transient expression of the proneural gene lethal of scute [l(1)sc] and is thus called the proneural wave. This study found that the epidermal growth factor receptor (EGFR) signaling pathway promotes proneural wave progression. EGFR signaling is activated in neuroepithelial cells and induces l(1)sc expression. EGFR activation is regulated by transient expression of Rhomboid (Rho), which is required for the maturation of the EGF ligand Spitz. Rho expression is also regulated by the EGFR signal. The transient and spatially restricted expression of Rho generates sequential activation of EGFR signaling and assures the directional progression of the differentiation wave. This study also provides new insights into the role of Notch signaling. Expression of the Notch ligand Delta is induced by EGFR, and Notch signaling prolongs the proneural state. Notch signaling activity is downregulated by its own feedback mechanism that permits cells at proneural states to subsequently develop into neuroblasts. Thus, coordinated sequential action of the EGFR and Notch signaling pathways causes the proneural wave to progress and induce neuroblast formation in a precisely ordered manner (Yasugi, 2010).

Loss of EGFR function in progenitor cells caused failure of L(1)sc expression and differentiation into neuroblasts (see A model of progression of the proneural wave). In addition, elevated EGFR signaling resulted in faster proneural wave progression and induced earlier neuroblast differentiation. The activation of the EGFR signal is regulated by a transient expression of Rho, which cleaves membrane-associated Spi to generate secreted active Spi. This study also demonstrated that Rho expression itself depends on EGFR function, and thus the sequential induction of the EGFR signal progresses the proneural wave. Clones of cells mutant for pnt were not recovered unless Minute was employed, suggesting that the EGFR pathway is required for the proliferation of neuroepithelial cells. However, the progression of the proneural wave is not regulated by the proliferation rate per se (Yasugi, 2010).

The function of the Notch signaling pathway in neurogenesis is known as the lateral inhibition. A revision of this notion has recently been proposed for mouse neurogenesis, in which levels of the Notch signal oscillate in neural progenitor cells during early stages of embryogenesis, and thus no cell maintains a constant level of the signal. The oscillation depends mainly on a short lifetime and negative-feedback regulation of the Notch effecter protein Hes1, a homolog of Drosophila E(spl). This prevents precocious neuronal fate determination. The biggest difficulty in analysis of Notch signaling is the random distribution of different stages of cells in the developing ventricular zone, which is thus called a salt-and-pepper pattern. In medulla neurogenesis, however, cell differentiation is well organized spatiotemporally and the developmental process of medulla neurons can be viewed as a medial-lateral array of progressively aged cells across the optic lobe. Such features allowed the functions of Notch to be precisely analyzed. Cells are classified into four types according to their developmental stages: neuroepithelial cells expressing PatJ, neuronal progenitor I expressing a low level of Dpn, neuronal progenitor II expressing L(1)sc and neuroblasts expressing high levels of Dpn. The Notch signal is activated in neuronal progenitor I and II. The EGFR signal turns on in the neuronal progenitor II stage and progresses the stage by activating L(1)sc expression. Cells become neuroblasts when the Notch and EGFR signals are shut off. Cells stay as neuronal progenitor I when Notch signal alone is activated, whereas cells stay as neuronal progenitor II when the Notch signal is activated in conjunction with the EGFR signal. Although the Notch signal is once activated, it must be turned off to let cells differentiate into neuroblasts. In neuronal progenitor II, E(spl)-C expression is induced by Notch signaling, and the increased E(spl)-C next downregulates Dl expression and subsequent activation of the Notch signal (Yasugi, 2010).

What does Notch do in medulla neurogenesis? It is infered that the Notch signal sustains cell fates, whereas the EGFR signal progresses the transitions of cell fate. This was well documented when a constitutively active form of each signal component was induced. EGFR, or its downstream Ras, induces expression of L(1)sc but does not fix its state, even though the constitutively active form is employed. At the same time, a constitutively active Notch sustains cell fates in a cell-autonomous manner. Constitutively active N receptors, by contrast, autonomously determine cell fates depending on the context: cells become neuronal progenitor I in the absence of EGFR and neuronal progenitor II in the presence of EGFR. The precocious neurogenesis caused by the impairment of Notch signaling suggests that Notch keeps cells in the progenitor state for a certain length of time in order to allow neuroepithelial cells to grow into a sufficient population. In the prospective spinal cord of chick embryo (Hammerle, 2007), the development from neural stem cells to neurons progresses rostrocaudally, during which the transition from proliferating progenitors to neurogenic progenitors is regulated by Notch signaling (Yasugi, 2010).

Although Notch plays a pivotal role in determining cell fate between neural and non-neural cells, the function may be context dependent and can be classified into three categories. (1) Classical lateral inhibition is seen in CNS formation in embryogenesis and SOP formation in Drosophila. Cells that once expressed a higher level of the Notch ligand maintain their cell states and become neuroblasts. (2) Oscillatory activations are found in early development of the mouse brain (Shimojo, 2008). Progenitor cells are not destined to either cell types. (3) An association with the proneural wave found in Drosophila medulla neurogenesis as is described in this study. The Notch signal is transiently activated only once and then shuts off in a synchronized manner. The notable difference in the outcome is the ratio of neural to non-neural cells; a small number of cells from the entire population become neuroblasts or neural stem cells in the former cases (1 and 2), whereas most of the cells become neuroblasts in the latter case (3). The differences between (1) and (2) can be ascribed at least in part to the duration of development. Hes1 expression has been shown to oscillate within a period of 2 hours in the mouse, whereas in Drosophila embryogenesis, selection of neuroblasts from neuroectodermal cells takes place within a few hours. Thus, even if Drosophila E(spl) has a half-life time equivalent to Hes1, the selection process during embryogenesis probably finishes within a cycle of the oscillation. The process of medulla neuroblast formation continues for more than 1 day, but Notch signaling is activated for a much shorter period in any given cell. This raises the possibility that E(spl)/Hes1 may have a similarly short half-life but outcome would depend on the developmental context (Yasugi, 2010).

The functions of EGFR and Notch described in this study resemble their roles in SOP formation of adult chordotonal organ development; the EGFR signal provides an inductive cue, whereas the Notch signal prevents premature SOP formation. In addition, restricted expression of rho and activation of the EGFR signal assure reiterative SOP commitment. Several neuroblasts are also sequentially differentiated from epidermal cells in adult chordotonal organs (Yasugi, 2010).

Unpaired, a ligand of the JAK/STAT pathway is expressed in lateral neuroepithelial cells and shapes an activity gradient that is higher in lateral and lower in the medial neuroepithelium. The JAK/STAT signal acts as a negative regulator of the progression of the proneural wave (Yasugi, 2008). This report has shown that activation of both EGFR and Notch signaling pathways depends on the activity of the JAK/STAT signal. The JAK/STAT signal probably acts upstream of EGFR and Notch signals in a non-autonomous fashion. These three signals coordinate and precisely regulate the formation of neuroblasts (Yasugi, 2010).

Oogenesis

Autocrine signaling through the Epidermal growth factor receptor operates at various stages of development across species. A recent hypothesis suggested that a distributed network of Egfr autocrine loops is capable of spatially modulating a simple single-peaked input into a more complex two-peaked signaling pattern, specifying the formation of a pair organ in Drosophila oogenesis (two respiratory appendages on the eggshell) (Wasserman and Freeman, 1998). To test this hypothesis, genetic and biochemical information about the Egfr network were integrated into a mechanistic model of transport and signaling. The model allows the relative spatial ranges and time scales of the relevant feedback loops to be estimated, the phenotypic transitions in eggshell morphology to be interpreted and the effects of new genetic manipulations to be predicted. It has been found that the proposed mechanism with a single diffusing inhibitor is sufficient to convert a single-peaked extracellular input into a two-peaked pattern of intracellular signaling. Based on extensive computational analysis, it is predicted that the same mechanism is capable of generating more complex patterns. At least indirectly, this can be used to account for more complex eggshell morphologies observed in related fly species. It is proposed that versatility in signaling mediated by autocrine loops can be systematically explored using experiment-based mechanistic models and their analyses (Shvartsman, 2002).

The mechanism that converts a single-peaked Gurken input into a more complex pattern of MAPK signaling in the responding epithelial cells has been analyzed. Interest in this particular event in Drosophila oogenesis mechanism is twofold. First, it provides an excellent example of versatility in signaling, demonstrating how simple stimuli define complex patterns in development. More importantly, this mechanism is arguably one of the best studied at the genetic and biochemical level. It therefore provides a good target for the development of modeling methodologies and experimental tests of modeling predictions (Shvartsman, 2002).

The oocyte-derived signal is modulated by a network of feedback loops in the follicular epithelium. One positive feedback loop is established when the Egfr, which acts through the Ras/MAPK pathway, induces the expression of rhomboid. Rhomboid is an intracellular protease that, together with intracellular protein Star, is responsible for processing and secretion of Spitz, another TGF-beta like ligand of the Egfr. The secreted Spitz directly interacts with the Egfr, further stimulating the intracellular MAPK. Thus, Rhomboid acts as a positive regulator of EGFR signaling. This positive feedback, together with another one mediated by a secreted EGFR ligand Vein, both amplifies and spatially expands MAPK signaling induced by Gurken (Shvartsman, 2002 and references therein).

The amplified and expanded signal is then downregulated by a number of negative feedback loops in the Drosophila Egfr system. Although there are large numbers of negative regulators of Egfr signaling in Drosophila, the original mechanism invokes a single endogenous inhibitor, Argos (Wasserman, 1998). The expression of argos is induced at high levels of MAPK signaling. Argos is a secreted protein that directly interacts with the extracellular domain of the Drosophila Egfr, and inhibits intracellular MAPK signaling induced by Gurken, Vein and Spitz. According to Wasserman, the amplification of the Gurken signal by Spitz and Vein, and its inhibition by Argos first expands the domain of MAPK signaling and then splits it into two smaller domains, establishing in this way the two groups of appendage-producing cells. In this mechanism, the two peaks detected in the MAPK signaling pattern (Wasserman, 1998) are closely linked and co-localized with the two stripes that have been repeatedly observed in the pattern or rhomboid expression (Shvartsman, 2002).

The mechanism of Wasserman links gurken, Egfr, spitz, rhomboid, vein and argos into a system of interconnected feedback loops that are jointly regulated by the EGFR and intracellular MAPK signaling (Wasserman, 1998). Although the mechanism is supported by genetic and biochemical data, several important questions remain unanswered. (1) Is the proposed network actually capable of converting single-peaked inputs into persistent two-peaked outputs? An independent measurement of the MAPK dynamics in oogenesis reports patterns that are more complex, and cannot be as straightforwardly correlated with the number of appendages of the mature egg. (2) Although the necessary role of argos is supported by genetic approaches, the role of other known Egfr inhibitors remains to be clarified. Argos is the only secreted inhibitor of Egfr in Drosophila. If it is the only necessary inhibitor, does this mean that the other negative regulators merely serve to modulate the basic pattern established by the secreted Argos? This must be reconciled with the phenotypic transitions in eggshell morphology induced by changing the levels of other inhibitors. (3) Several papers have indicated that the expression of argos is detected later than the relevant changes in the rhomboid expression. Can this be used to argue against the mechanism with a single secreted inhibitor? To summarize, the sufficiency of the proposed mechanism with a single diffusing inhibitor and its consistency with the large body of genetic data remains to be established (Shvartsman, 2002).

Based on the computational analysis of this model, the proposed mechanism of pattern formation by peak splitting (Wasserman, 1998) has been validated. The proposed network of positive- and negative-feedback loops in the Egfr system can indeed convert a quasistatic, single-peaked input, into stable, two-peaked outputs. Analysis of the parametric dependence of the stationary patterns in the model indicates that the underlying mechanism accounts for a number of experimentally observed phenotypic transitions (Shvartsman, 2002).

Stationary patterns predicted by the model fall into several qualitatively different classes characterized by the number of peaks in the signaling profiles. The transitions between the classes are discontinuous; this might explain why numerous experiments can be classified in terms of a small number of phenotypes. Stable two-peaked patterns not only exist in this model, but are also kinetically accessible from the state of zero stimulation and are realized through inputs with a single maximum. These stable two-peaked patterns are robust for a wide range of network inputs and strengths of the positive and negative feedback loops. The existence of these stationary patterns requires intermediate input amplitudes and widths, intermediate strengths of the positive feedback, as well as a sufficiently strong and long-ranged inhibition. The fact that large parameter changes induce transitions to qualitatively different patterns is consistent with a large body of genetic data (Shvartsman, 2002).

Several of the predictions of the original mechanism are at variance with experiments. The first quantitative disagreement is observed in the behavior of the two-peaked patterns upon increases in the doses of the stimulatory signal or the strength of the positive feedback. In the model, these changes produce a discontinuous transition to the pattern with a single broad peak; this collapse of the two peaks is preceded by their slight separation. In experiments, eggs laid by the females with extra copies of gurken, heat-shock activated rhomboid, or deficient in Cbl (a negative Egfr regulator) have significantly increased inter-appendage distance. However, a broad appendage phenotype has been reported in a recent experiment that had used tissue-specific gene expression to increase the level of oocyte-derived Gurken (Shvartsman, 2002 and references therein).

A more serious problem is related to the role of argos. First, it is unclear why the onset of argos expression is detected much later than the major changes in the patterns of rhomboid and MAPK activity. The second question, based on the analysis of the model, is related to the relative position in the maxima of rhomboid and argos gene expression patterns. According to the model, the maxima in the expression of the two genes should be co-localized; this is an immediate consequence of the fact that both genes require receptor activity for their production. At the same time, several independent measurements find that for a while argos is expressed between the two regions of high rhomboid expression. If the resulting two-peaked signaling patterns are quasi-stationary, as suggested by the analysis of the original mechanism, then it is unclear what maintains argos expression in the region of decreased MAPK activity. One possibility is that the rate of argos expression is much slower than that of rhomboid, and that the experimentally observed patterns should be interpreted as a transient in the model. This mechanism has been computationally explored and rejected; making the generation of Argos much slower than that of Rhomboid generates pathologic oscillatory instabilities of the two-peaked patterns. Another possible explanation for the observed relative location of the maxima in the gene expression patterns might involve an additional feedback loop. In this extended mechanism, a yet undiscovered positive feedback regulates the expression of argos, making it insensitive to decreases in Egfr signaling after peak splitting (Shvartsman, 2002).

In order for the peak-splitting mechanism to work, the differences in the thresholds of Argos and Rhomboid production must not be too large. In the simulations, the difference in these thresholds is 25%. It has been found that in the case when Argos generation is characterized by a significantly higher threshold than that of Rhomboid, only the one-peaked patterns are realized and peak splitting does not occur. In fact, peak splitting requires a rather delicate balance between the spatially distributed stimulation by Gurken and inhibition by Argos (Shvartsman, 2002).

The analysis supports the original hypothesis about the differences in the spatial ranges of Argos and Spitz. The cause of this separation of length scales is still unclear. Argos and Spitz are secreted into the gap between the oocyte and the follicle cells, where their transport is accompanied by binding to Egfrs in the follicular epithelium. The strength of ligand/receptor binding can regulate the range of the secreted signal in this situation. If this is the case, then the binding constant characterizing the Argos/Egfr interaction should be less than that of Spitz. Surprisingly, it was found that Argos has a higher affinity for the Egfr (but, at the same time, an alternative Argos-like EGF mutant has a lower Egfr-binding affinity). Another mechanism regulating the range of secreted ligands relies on their receptor-mediated endocytosis. Based on the fact that all ligands of mammalian Egfrs are rapidly internalized, it is believed that this mechanism can control spatial ranges of Egfr ligands in Drosophila oogenesis. Furthermore, it is known that Argos interaction with Egfr prevents receptor dimerization and phosphorylation. Since these processes are required to initiate receptor-mediated endocytosis of receptor tyrosine kinases, this further supports the mechanism in which the ranges of Argos and Spitz are controlled by the rates of their internalization (Shvartsman, 2002).

The model does not rely on the difference between the diffusivities of Spitz and Argos. This is in contrast to the classical activator-inhibitor mechanism for morphogenesis proposed by Turing (1952). Instead, it relies on differences of ranges of these molecules, which depend on a combination of their diffusivities and rates of degradation. This is also true for the Turing mechanism; however, in the latter, the difference between the time constants of the activator and the inhibitor leads to oscillatory instabilities. Moreover, the model devised in this study is different from the activator-inhibitor models: while Argos plays the role of a long-range inhibitor, the positive feedback is operated by an autocrine switch with a non-diffusing component (Rhomboid) and a short-ranged diffusing messenger (Spitz). Here, it is the time constant of Spitz degradation that determines the range of the positive feedback; however, the existence of the oscillatory instability is determined by the ratio of the time scales of Rhomboid and Argos. Furthermore, it is not difficult to show that with the given relationship between the thresholds of Rhomboid and Argos production the Turing-like instability (as a result of the homogeneous increase of the level of Gurken input) is not realized at all in the current model. Another difference between this model and that of Turing is that in this model, pattern formation occurs as a result of the instabilities, leading to the abrupt formation and transitions between large-amplitude localized patterns. Large amplitude localized patterns, often referred to as autosolitons, are frequently encountered in different nonlinear systems (Shvartsman, 2002).

In addition to patterns with one and two peaks, the mechanism supports more complex patterns. If the number of peaks in the profile of the receptor activity determines the number of respiratory appendages, then this finding predicts that more complex eggshell phenotypes are to be expected upon quantitative variation of the parameters of the Egfr network. At this point there is a single published observation of eggs with four appendages in mutants of D. melanogaster. Occasionally, eggs with three appendages are laid by the mutants with defects in Gurken accumulation. Notably, eggs with four and even six appendages represent wild-type phenotypes of several related fly species: the eggs of D. virilis have four appendages, while the eggs laid by the flies of subgenus Pholadoris have six appendages. According to the current model, eggs with multiple appendages can be generated by increases in the width and amplitude of the stimulatory signal. Further experimental, modeling and computational studies are required to check whether these more complex phenotypes can be realized in oogenesis or whether they manifest a pathological feature of the mechanism with a single diffusing inhibitor (Shvartsman, 2002).

Multiple EGFR ligands participate in guiding migrating border cells

Cell migration is an important feature of embryonic development as well as tumor metastasis. Border cells in the Drosophila ovary have emerged as a useful in vivo model for uncovering the molecular mechanisms that control many aspects of cell migration including guidance. Two receptor tyrosine kinases, epidermal growth factor receptor (EGFR) and PDGF- and VEGF-related receptor (PVR), together contribute to border cell migration. Whereas the ligand for PVR, PVF1 is known to guide border cells, it is unclear which of the four activating EGFR ligands function in this process. An assay was developed to detect the ability of secreted factors to reroute migrating border cells in vivo, and the activity of EGFR ligands was tested compared to PVF1. Two ligands, Keren and Spitz, guided border cells whereas the other ligands, Gurken and Vein, do not. In addition, only Keren and Spitz are expressed at the appropriate stage in the oocyte, the target of border cell migration. Therefore, a complex combination of EGFR and PVR ligands together guide border cells to the oocyte (McDonald, 2006).

Ectopic expression of ligands for the Drosophila EGFR can redirect migratory cells in vivo. The ability of s-SPI and s-KRN to reroute border cells, together with the endogenous expression of spi and Krn mRNA in the oocyte and the strong border cell migration phenotype following reduction of both EGFR and PVR signaling, indicate that signaling through EGFR normally contributes to guiding border cells to the oocyte. VN is unlikely to contribute significantly because VN is ineffective in the misguidance assay and endogenous expression is not detected in the germline. The question then arises as to whether GRK contributes to guiding border cells. It seems unlikely to contribute significantly to their posterior migration because it is ineffective in the misguidance assay, even when the ectopic expression equals or exceeds the endogenous expression and the ectopic expression is closer to border cells. Furthermore, GRK protein is highly localized in a dorsal/anterior crescent within the oocyte, whereas there is no discernable dorsal bias to the path that border cells take to the oocyte, even in the absence of Pvf1 (McDonald, 2006).

Niche appropriation by Drosophila intestinal stem cell tumours

Mutations that inhibit differentiation in stem cell lineages are a common early step in cancer development, but precisely how a loss of differentiation initiates tumorigenesis is unclear. This study investigated Drosophila intestinal stem cell (ISC) tumours generated by suppressing Notch(N) signalling, which blocks differentiation. Notch-defective ISCs require stress-induced divisions for tumour initiation and an autocrine EGFR ligand, Spitz, during early tumour growth. On achieving a critical mass these tumours displace surrounding enterocytes, competing with them for basement membrane space and causing their detachment, extrusion and apoptosis. This loss of epithelial integrity induces JNK and Yki/YAP activity in enterocytes and, consequently, their expression of stress-dependent cytokines (Upd2, Upd3). These paracrine signals, normally used within the stem cell niche to trigger regeneration, propel tumour growth without the need for secondary mutations in growth signalling pathways. The appropriation of niche signalling by differentiation-defective stem cells may be a common mechanism of early tumorigenesis (Patel, 2015).

This paper described a step-wise series of events during the earliest stage of tumour development in a stem cell niche. First, the combination of environmentally triggered mitogenic signalling and a mutation that compromises differentiation generates small clusters of differentiation-defective stem-like cells. Autocrine (Spi/EGFR) signalling between these cells then promotes their expansion into clusters, which quickly reach a size capable of physically disrupting the surrounding epithelium and driving the detachment and apical extrusion of surrounding epithelial cells (that is, ECs). This loss of normal cells seems to involve tumour cell/epithelial cell competition through integrin-mediated adhesion. Subsequently, the loss of epithelial integrity (specifically, EC detachment) triggers stress signalling (JNK, Yki/YAP) in the surrounding epithelium and underlying VM, and these stressed tissues respond by producing cytokines (Upd2,3) and growth factors (Vn, Pvf, Wg, dILP3). These signals are normally used within the niche to activate stem cells for epithelial repair, but in this context they further stimulate tumour growth in a positive feedback loop. It is noteworthy that in this example a single mutation that blocks differentiation is sufficient to drive early tumour development, even without secondary mutations in growth signalling pathways that might make the tumour-initiating cells growth factor- and niche-independent (for example, Ras, PTEN). Thus, tumour cell-niche interactions can be sufficient to allow tumour-initiating cells to rapidly expand, increasing their chance to acquire secondary mutations that might enhance their growth or allow them to survive outside their normal niche. This study highlights the importance of investigating the factors that control paracrine stem cell mitogens and survival signals in the niche environment. Tumour-niche interactions may be important to acquire a sizable tumour mass before the recruitment of a tumour-specific microenvironment that supports further tumour progression. A careful analysis of similar interactions in other epithelia, such as in the lung, skin or intestine could yield insights relevant to the early detection, treatment and prevention of cancers in such tissues (Patel, 2015).

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