EGF receptor



There is a substantial correlation between Egfr expression and the phenotypes associated with a variety of mutant alleles. Of particular note are high levels of expression in the primordia of the mouth parts (the embryonic tissues most sensitive to reductions in Egfr activity), discrete expression in a subset of neural cells essential for construction of the axonal scaffold (a structure that is deformed in mutant embryos), uneven expression in the eye disc (the development of which is abnormal in both mild hypomorphs and hypermorphs), and expression in the follicular epithelial cells of the ovary, ( responsible for producing the eggshell of developing oocytes) and which do so aberrantly in the mildest hypomorphs (Katzen, 1991).

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 Egfr 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 Egfr-expressing midline glia from the nerve cord, even those that already express argos. Egfr expression is not terminated in the midline glia after spitz group signaling triggers changes in gene expression. Paradoxically, although all midline glia express Egfr, argos expression is restricted to the midline glia that do not enter apoptosis. It is therefore likely that an attenuation of Egfr signaling by Argos is integrated with the augmentation of Egfr signaling by Spitz throughout the period of reduction of midline glia numbers, and argos-expressing midline glia depend upon continued Spitz activation of the Egfr 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).

Mesectodermal cells (MEC) give rise to the first nervous system cells to become postmitotic and differentiate into identified cell types. Existing models of MEC lineage determination predict that there are between 2 and 6 midline glia (MG) precursors. A study was undertaken to clarify the origin of supernumerary MGs in embryos that lack reaper, head involution defective and grim, three closely linked proapoptotic genes. Drosophila embryos deficient for programmed cell death produce 9 midline glia (MG) in addition to the wild-type complement of 3.2 MG/segment. More than 3 of the supernumerary MG derive from the MGP (MG posterior) lineage and the remainder from the MGA/MGM (MG anterior and middle) lineage. There is one unidentified additional neuron in the mesectoderm of embryos deficient for apoptosis. The supernumerary MG are not diverted from other lineages nor do they arise from an altered pattern of mitosis. Instead, these MG appear to arise from a normally existing pool of 12 precursor cells, a number larger than anticipated by earlier studies. During normal development, MG survival is dependent upon signaling to the Drosophila EGF receptor. The persistence of supernumerary MG in embryos deficient for apoptosis does not alter the spatial pattern of Drosophila EGF receptor signaling. The number and position of MG that express genes dependent upon EGF receptor function, such as pointed or argos, are indistinguishable from wild type. Thus supernumary MG in H99 mutant embryos express EGF receptor but apparently receive insufficient receptor activation to express genes dependent on EGF receptor signaling. Genes of the spitz group are required for Drosophila EGF receptor function. Surviving MG in spitz group/H99 double mutants continue to express genes characteristic of the MG, but the cells fail to differentiate into ensheathing glia and are displaced from the nerve cord. It remains to be clarified how the MG progenitors are selected from the MEC population (Dong, 1997).

EGF receptor signalling protects smooth-cuticle cells from apoptosis during Drosophila ventral epidermis development

Patterning of the Drosophila ventral epidermis is a tractable model for understanding the role of signalling pathways in development. Interplay between Wingless and EGFR signalling determines the segmentally repeated pattern of alternating denticle belts and smooth cuticle: spitz group genes, which encode factors that stimulate EGFR signalling, induce the denticle fate, while Wingless signalling antagonizes the effect of EGFR signalling, allowing cells to adopt the smooth-cuticle fate. Medial fusion of denticle belts is also a hallmark of spitz group genes, yet its underlying cause is unknown. This phenotype has been studied and a new function has been discovered for EGFR signalling in epidermal patterning. Smooth-cuticle cells, which are receiving Wingless signalling, are nevertheless dependent on EGFR signalling for survival. Reducing EGFR signalling results in apoptosis of smooth-cuticle cells between stages 12 and 14, bringing adjacent denticle regions together to result in denticle belt fusions by stage 15. Multiple factors stimulate EGFR signalling to promote smooth-cuticle cell survival: in addition to the spitz group genes, Rhomboid-3/roughoid, but not Rhomboid-2 or -4, and the neuregulin-like ligand Vein also function in survival signalling. Pointed mutants display the lowest frequency of fusions, suggesting that EGFR signalling may inhibit apoptosis primarily at the post-translational level. All ventral epidermal cells therefore require some level of EGFR signalling; high levels specify the denticle fate, while lower levels maintain smooth-cuticle cell survival. This strategy might guard against developmental errors, and may be conserved in mammalian epidermal patterning (Urban, 2004).

The denticle belt fusion phenotype is one of the distinguishing features of the spitz group genes (Mayer, 1988), yet its developmental basis has remained mysterious, since no function has been known for EGFR signalling in the smooth cuticle, which is the affected tissue. Analysis of this phenotype has revealed its cause and uncovered a previously unrecognized function for EGFR signalling in Drosophila epidermal development. Spitz is the primary EGFR ligand in epidermal patterning, and is activated by proteolysis in three rows of rhomboid-1-expressing cells in the future denticle region. High EGFR signalling is required for cells to adopt the denticle fate, and other signalling pathways are used to elaborate the different denticle morphologies. The Wingless signal emanates from one posterior row of each parasegment and spreads anteriorly, suppressing the denticle fate and thus allowing cells to secrete a smooth cuticle. These future smooth-cuticle cells also require signalling through the EGFR for viability, and its absence results in apoptosis of future smooth-cuticle cells and thus denticle belt fusions. This survival signalling is mediated by low-level stimulation of the EGFR by cooperation between the ligands Vein and Spitz, which is activated by Rhomboid-1, Rhomboid-3 and Star (Urban, 2004).

The ventral epidermis is patterned in multiple stages during development, with cell fate specification occurring late, through antagonism between EGFR and Wingless signalling around stages 12-14. Direct phenotypic analysis indicates that EGFR signalling is required for smooth-cuticle cell survival during these fate specification stages and not earlier or later: epidermal cell apoptosis is greatly elevated in mutant embryos at stages 12-14, and the fusion phenotype first becomes apparent around stage 15 as curvature of Engrailed stripes (Urban, 2004).

This direct phenotypic analysis is also supported by several independent genetic observations. EGFR signalling is not required for survival in future smooth-cuticle cells early, when the ventrolateral fates are being specified (stage 10/11) since removing Rhomboid-1 expression at only this stage using the single-minded mutation never results in denticle belt fusions (Mayer, 1988). Defects at this early stage also cause ventral narrowing in spitz group genes (Mayer, 1988), and since rhomboid-3 does not enhance this phenotype, this suggests that rhomboid-3 cooperates with rhomboid-1 only later in development. Vein acts independently of spitz group genes to suppress denticle belt fusions, and this cannot occur at stage 10/11 since at this early stage Vein expression is dependent on EGFR signalling through a positive feedback loop. Finally, the fusion phenotype itself suggests that it forms late since denticle cells are being pulled into smooth cuticle regions and, as such, their denticle fate must have already been determined and cannot be altered by receiving signals from these smooth domains (Urban, 2004).

Thus, two thresholds with different outcomes exist for EGFR signalling in patterning the ventral epidermis. The level of EGFR signalling that a cell receives is presumably dependent on its distance from the Spitz-processing cells; activated MAPK staining indicates that these rows of cells receive high levels of EGFR signalling. High levels of EGFR signalling are required to induce the denticle fate, while lower levels that reach smooth-cuticle cells are sufficient to suppress apoptosis. All ventral epidermal cells therefore require EGFR signalling, but the exact level, together with antagonism of shavenbaby transcription by Wingless signalling, determines the biological outcome. Importantly, these functions may be separate, since Wingless signalling is known to antagonize shavenbaby transcription to repress the denticle fate, but may not repress EGFR signalling itself in smooth-cuticle cells: activated MAPK staining suggests that some smooth-cuticle cells in the midline may also receive higher levels of EGFR signalling (Urban, 2004).

These results indicate that cells require EGFR signalling for their survival only when they are starting to differentiate. A similar pattern was also observed in the developing eye imaginal disc where removing the EGFR resulted in cell death only once the morphogenetic furrow had passed. These observations raise the intriguing possibility that establishing a requirement for survival signals may be inherent in the differentiation program itself, perhaps for protecting against developmental errors. However, the observation that the requirement for survival signalling is restricted to the central region of the ventral epidermis implies that either this requirement is not ubiquitous, or that another signal is also involved (Urban, 2004).

Pointed is an Ets domain-containing transcription factor that is responsible for transducing most known instances of EGFR signalling. Although it was previously clear that pointed mutant embryos rarely display denticle belt fusions (Mayer, 1988), analysis of a more recent null allele that removes both P1 and P2 transcripts demonstrates that even complete loss of pointed leads only to a very low frequency of denticle belt fusions. This is also consistent with the milder effects of pointed clones in the developing eye, and in particular the late onset of their apoptosis. These observations raise the possibility that EGFR-mediated survival signalling in general occurs primarily at a non-transcriptional level. Consistent with this model, EGFR signalling has been shown to reduce Hid protein stability, thus directly inhibiting apoptosis (Urban, 2004).

Rhomboid exists as a seven-member family in Drosophila, and at least four of these members are intramembrane serine proteases that can cleave all Drosophila membrane-tethered EGFR ligands and specifically activate EGFR signalling in vivo. Although the precise role of the rhomboid protease family in EGFR signalling and in other biological contexts has been unclear, mutations have now been isolated for both Rhomboid-2 and -3. Genetic analysis with null alleles has revealed that both act as tissue-specific activators of EGFR signalling much like Rhomboid-1. Rhomboid-2 is the only rhomboid known to be expressed early in gametogenesis, and is involved in sending EGFR signals from the germline to the soma to guide its encapsidation by somatic cells. In this context, Rhomboid-2 appears to act alone. Rhomboid-3 displays strong expression in the developing eye imaginal disc, and is allelic to roughoid, one of the first Drosophila mutants described. Rhomboid-3 is the dominant rhomboid protease during eye development, but does not act alone: Rhomboid-3 cooperates with Rhomboid-1 in the developing eye (Urban, 2004).

Despite the power of these genetic approaches, it should be noted that rhomboid-1, -2 and -3 exist as a gene cluster on chromosome 3L and, as such, combined mutations are difficult to generate by recombination. Analysis of epidermal patterning using RNAi to overcome this limitation is the first implication of a rhomboid homolog function in embryogenesis. Interestingly, the rhomboid involved is Rhomboid-3, the rhomboid that was previously thought to be eye-specific. However, unlike in the developing eye where Rhomboid-3 has the dominant role, and removing Rhomboid-1 by itself has no effect, the exact opposite is true in embryogenesis: Rhomboid-1 is the main protease in epidermal patterning while removing Rhomboid-3 alone does not result in detectable defects. This analysis suggests that different rhomboid proteases function predominantly to activate EGFR signalling in distinct tissues, but often act cooperatively or with a degree of redundancy (Urban, 2004).

The requirement for high levels of signalling for fate specification and lower levels for viability in developing tissues may not be limited to the EGFR pathway. Intriguingly, analysis of cell death in wingless mutant embryos suggests that a reciprocal signalling function may also be required to maintain cell viability in denticle regions of the ventral epidermis: in conditions of reduced Wingless signalling, specifically during the stage of epidermal fate specification (but not earlier), cells corresponding to two denticle rows were observed to undergo apoptosis. Therefore, as with EGFR signalling, high levels of Wingless signalling induce the smooth-cuticle cell fate, while lower levels may be required for survival of a subset of denticle cells. Thus, the Wingless and EGFR signalling pathways may act antagonistically in specifying cell fate, while having complementary and reciprocal functions in maintaining cell viability in the developing epidermis of Drosophila. These survival functions may be conserved since EGFR signalling also has multiple roles in mammalian epidermal development, while some mammalian epidermal tumors are also specifically dependent on EGFR signalling for cell survival. Wnt signalling has also been linked to maintaining cell viability in certain developmental contexts (Urban, 2004).

Recruitment of ectodermal attachment cells via an EGFR-dependent mechanism during the organogenesis of Drosophila proprioceptors

Drosophila proprioceptors (chordotonal organs) are structured as a linear array of four lineage-related cells: a neuron, a glial cell, and two accessory cells, called cap and ligament, between which the neuron is stretched. To function properly as stretch receptors, chordotonal organs must be stably anchored at both edges. The cap cells are anchored to the cuticle through specialized lineage-related attachment cells. However, the mechanism by which the ligament cells at the other edge of the organ attach is not known. The identification of specialized attachment cells is reported that anchor the ligament cells of pentascolopidial chordotonal organs (lch5) to the cuticle. The ligament attachment cells are recruited by the approaching ligament cells upon reaching their attachment site, through an EGFR-dependent mechanism. Molecular characterization of lch5 attachment cells demonstrates that they share significant properties with Drosophila tendon cells and with mammalian proprioceptive organs (Inbal, 2004).

In an attempt to characterize the origin and fate of ch attachment cells, the distribution was examined of alpha85E-tubulin (alpha85E-tub) in ch organs. This minor alpha-tub variant is known to be expressed in the cap cells and the adjacent attachment cells, as well as in the ligament cells of lch5 organs. Close inspection of the distribution of this protein in mature embryos and first instar larvae revealed another alpha85E-tub-expressing cell in close proximity to the ventral edge of the ligament cells. Rarely, two such cells were observed. These large cells appeared to be good candidates to function in the attachment of ligament cells. Indeed, further analysis demonstrated that these cells are localized within the epidermal layer and are connected to the ventral edges of the ligament cells via Integrin-mediated adhesion, as suggested by the high concentration of the Integrin ßPS subunit in the contact site between these two cell types. In addition, these cells possess many features that are typical of other types of attachment cells. To avoid confusion, the attachment cells that anchor the cap cells are referred to as CA (cap attachment) cells and to the attachment cells that anchor the ligament cells as LA (ligament attachment) cells (Inbal, 2004).

Lineage-tracing experiments have shown that the CA cells originate from the ch organ lineage. This observation predicts the formation of five CA cells in each of the lateral pentascolopidial organs. However, only two CA cells can be identified in each of these organs. It has been suggested that the other three cells either degenerate or migrate away from the cluster. In order to identify which of the five ch organs generate a CA cell, spitz (spi) and rhomboid (rho) mutant embryos were examined, in which only the first three ch organs are formed. In both types of mutants, only one CA cell was detected in each pentascolopidial organ. This observation suggests that one of the CA cells originates from one of the first three ch organs, whereas the second cell is formed by one of the two organs that are recruited later. The LA cell was not detected in the lineage-tracing experiments mentioned above, suggesting that this cell is not related to the ch lineage (Inbal, 2004).

The function of ch organs as stretch receptors requires the stable attachment of both their edges to fixed positions. The identification of LA cells provides an answer to the question of how lch5 organs attach through their ligament cells. However, this does not seem to be the case for all ch organs in the embryonic PNS. For example, the ventral ch organs A and B (vchA and vchB) have no apparent ligament cells and LA cells. Thus, different subtypes of ch organs exist, which differ in their structure as well as attachment mechanisms (Inbal, 2004).

To learn more about the structural and molecular features of ch attachment cells, tests were performed to see whether these cells share any molecular properties with tendon cells, which attach muscles to the cuticle. Tendon cells have been extensively studied, and several genes that are involved in their differentiation have been identified. Since both tendon and ch attachment cells are designed to resist mechanical strain, whether the ch attachment cells express tendon cell-typical markers was examined. The formation of tendon cells requires the expression of Stripe (Sr), an early growth response (EGR)-like transcription factor. Sr induces the expression of an array of tendon cell-specific proteins, which are required for tendon cell differentiation. Double labeling wild-type embryos for alpha85E-tub and Sr reveal that Sr is expressed in ch organs in the CA, LA, and ligament cells. Sr expression was first detected in the CA cells at stage 13. CA cells are the first to express Sr in the embryo and seem to express the highest levels of Sr throughout embryonic development. The ligament cells express lower levels of Sr from late stage 14 onward, and the LA cells express Sr in stage 16 or older embryos (Inbal, 2004).

Two other genes that are implicated in tendon cell terminal differentiation are delilah (dei), which encodes a bHLH transcription factor, and ß1-tubulin (ß1-tub). Expression of both genes has been reported in ch organs; however, their exact distribution within these organs has not been described. Double labeling wild-type embryos for alpha85E-tub and Dei reveals expression of Dei in the CA and LA cells and in the cap and ligament cells. In situ hybridization reveals that ß1-tub is expressed similarly to Dei. Very low levels of ß1-tub transcripts were observed in addition in lch5 neurons. Work done in tendon cells has shown that the expression of Dei and ß1-tub is induced by Sr in a cell-autonomous manner. The fact that in lch5 organs the expression of Dei and ß1-tub is not limited to Sr-expressing cells suggests that additional mechanisms control the expression of these genes. Thus, the differential distribution of alpha85E-tub, Sr, Dei, and ß1-tub in the cells of lch5 organs adds a new dimension of complexity to these organs and raises new questions regarding the regulation of gene expression, cell fate determination, and differentiation in each cell type (Inbal, 2004).

Despite the similarities between tendon and ch attachment cells, muscles and ch organs do not share the same attachment sites, and the CA and LA cells serve for the anchoring of lch5 organs only. One prominent difference between CA, LA, and tendon cells is the expression of the alpha85E-tub protein in ch attachment cells but not in tendon cells. This suggests that alpha85E-tub has a unique function that is required in ch organs. It has been suggested that this isoform of alpha-tubulin, which is expressed specifically in ch organ accessory cells, developing muscles, and testis cyst cells, is likely to function in cells that must elongate extensively. Thus, the contribution of the alpha85E-tub to the organization of the microtubule cytoskeleton in the ch organ accessory cells are likely to affect the elasticity of the cells and their ability to withstand tension (Inbal, 2004).

Sr functions at the top of the hierarchy to direct tendon cell differentiation. In the absence of Sr, tendon cells do not develop, and the muscles fail to attach to the ectoderm. To test the role of Sr in the formation of lch5 attachment cells, how sr loss of function affects these cells was examined. Staining sr mutant embryos with anti-alpha85E-tub reveals a loss of LA cells in the absence of Sr. The CA cells were only occasionally missing; however, their morphology appeared to be abnormal. The lch5 organs were not properly stretched and appeared to be somewhat collapsed, possibly as a result of their failure to form stable attachments to the ectoderm. Thus, Sr is required for the generation of functional lch5 organs by playing a role in the formation of LA cells and in the differentiation of CA cells (Inbal, 2004).

It is not surprising that the two types of lch5 attachment cells are affected differently by the loss of Sr function. The earliest expression of Sr in the CA cells is observed in stage 13 embryos, after all cells of lch5 organs have already formed. Thus, Sr is not expressed early enough to affect primary decisions of cell fate in the lch5 lineage, but it may represent the earliest marker of the fate acquired by CA cells. As for the LA cells, their identity is defined very late in embryonic development, and Sr expression seems to be the earliest sign of their existence. Thus, Sr is likely to play a role in their induction as well as their differentiation into attachment cells (Inbal, 2004).

Sr is a member of the EGR family of transcription factors. In mammals, EGR proteins are involved in multiple developmental processes. Egr3, which shows a significant sequence similarity to Sr, is expressed in differentiating muscle spindles, a subgroup of proprioceptors. In Egr3 null mice, these proprioceptors are missing as a result of their failure to differentiate. Thus, an intriguing molecular parallelism might exist between the formation and differentiation of Drosophila and mammalian proprioceptive organs, despite the significant differences in their structure (Inbal, 2004).

The fact that the LA cells do not belong to the ch lineage raises the question of what triggers their formation. lch5 organs are initially formed with their ligament cells in a relatively dorsal position; subsequently, these cells descend until they reach their final position in the lateral cluster. Thus, the late appearance of the LA cells presents two possibilities with regard to their induction: these cells could form at a late embryonic stage independently of the ligament cells, or they could be recruited by the approaching ligament cells. To find which of these possibilities is correct, embryos were examined in which the ligament cells were ablated, relatively late in development, by expressing in them the apoptosis-inducing gene rpr, or mutant embryos were examined in which ligament cells do not form due to mutation in the gcm or repo genes. In the absence of ligament cells, the LA cells could not be detected, suggesting that their formation depends on the presence of ligament cells. When the ligament and LA cells are missing, the lch5 organs are not fully stretched, and the cap cells appear shorter than normal. However, different types of connections between the lch5 cells and their environment (e.g., the fasciculation of the lch5 axons with the intersegmental nerve) prevent a complete collapse of these organs in the absence of their ventral anchor (Inbal, 2004).

To find whether the presence of ligament cells is sufficient to induce the formation of LA cells regardless of their position, embryos were examined in which the ligament cells were abnormally localized. Mutations in abdominal-A (abd-A), homothorax (hth), and ventral veinless (vvl) result in frequent dorsal localization of lch5 organs. lch5 organs that fail to localize to their correct position in these mutants do not have LA cells. However, since the protein products of abd-A, hth, and vvl are normally expressed in the ectoderm, it is possible that, in their absence from the ectoderm of mutant embryos, LA cells cannot develop, regardless of the positioning of ligament cells. To assess specifically the influence of ligament cell positioning, an inducible Hth antimorph (En-Hth1-430) was used that can phenocopy hth loss of function. Expression of this antimorph in ch organs under the regulation of ato-Gal4 results in a high percentage of abnormally oriented lch5 organs. Except for their abnormal positioning, lch5 organs in these embryos appear to be fully differentiated, as judged by their ability to express typical markers, such as Repo, alpha85E-tub, and Sr. In ato-Gal4/UAS-En-Hth1-430 embryos, no LA cells could be observed in abdominal segments that exhibited abnormally oriented lch5 organs. Altogether, these data suggest that lch5 ligament cells recruit their attachment cells and that this process is restricted spatially, perhaps due to competence of cells in the attachment site region (Inbal, 2004).

The recruitment of LA cells by ligament cells resembles the recruitment of tendon cells by myotubes. In the case of tendon cells, the leading edges of myotubes approach preexisting clusters of Sr-expressing cells, and upon reaching their target they induce the terminal differentiation of a single tendon cell. The expression of Sr in the tendon precursor clusters also serves to attract the myotubes. In the case of ligament cells, however, no clear expression of Sr could be detected in the prospective site of their attachment prior to the appearance of the LA cell: this occurs only when the ligament cells are in their final position. Thus, despite the high similarity between the two processes, differences seem to exist in the mechanisms that guide ligament cells and myotubes to their attachment sites (Inbal, 2004).

Tendon cells are induced by the approaching myotubes, which secrete the EGFR ligand Vein and activate the EGFR pathway in the tendon precursor cells that they contact. This activation results in the expression of tendon cell-typical markers and terminal differentiation of tendon cells. Since the approaching ligament cells seem to induce the LA cells that share many properties with tendon cells, whether the EGFR pathway plays a role in the process of LA cell induction was tested. Activation of the EGFR pathway within the developing LA cells was tested by costaining wild-type embryos for the activated form of MAP kinase (dp-ERK) and for alpha85E-tub. In stage 16 embryos, low levels of dp-ERK could be detected in the LA cells but not in any of the other lch5 cells. Higher levels of dp-ERK were detected in the LA cells of stage 17 embryos. These observations demonstrate that the MAP-kinase pathway is activated in the LA cells at the time of their formation. To establish whether this pathway is necessary for the induction of these cells and whether it is mediated through EGFR activation, the EGFR pathway was blocked specifically by expressing a dominant-negative form of the receptor (DN-DER). The DN-DER was expressed throughout the ectoderm using the 69B-Gal4 driver or, in all of the Sr-expressing cells, including the LA cells, using a sr-Gal4 driver. In both cases, the expression of DN-DER abolished the formation of LA cells, indicating that activation of the EGFR pathway is necessary for LA cell development. To establish whether activation of the pathway plays a permissive or an instructive role in the formation of LA cells, whether higher levels of EGFR activation can lead to the formation of supernumerary LA cells was tested. To elevate the level of EGFR activation locally, the EGFR ligand Vein or a secreted form of the ligand Spitz (sSpi) was expressed in the ligament cells under the regulation of repo-Gal4. This excessive activation results in the formation of increased numbers of LA cells, indicating that the EGFR pathway plays an instructive role in the induction of lch5 LA cells. Expression of sSpi throughout the ectoderm led to the induction of multiple ectopic Sr-expressing cells; however, these cells did not express the alpha85E-tub protein, suggesting that EGFR pathway activity is required but not sufficient to determine the identity of an LA cell (Inbal, 2004).

While EGFR pathway activity is clearly required for the generation of LA cells, CA cells did not seem to be affected significantly by localized blocking of EGFR signaling. Moreover, CA cells appear to be almost the only cells that continue to express high levels of Sr when the EGFR pathway is blocked, suggesting that Sr expression in these cells is controlled by a different mechanism than in LA and tendon cells (Inbal, 2004).

The data suggest that Vein is expressed in the lch5 ligament cells in late stages of embryogenesis and that Vein is the major ligand responsible for EGFR activation in the prospective LA cells. The ability of Vein to induce supernumerary LA cells when overexpressed in the ligament cells is consistent with this conclusion. The role of Vein in the induction of LA cells extends the molecular similarity between the development of lch5 organs and mammalian proprioceptors. It has been shown that Neuregulin1, a Vein homolog, is secreted from proprioceptive afferent nerve endings and is required for the expression of Egr3 and differentiation of muscle spindles in the mouse (Inbal, 2004).

The jing and ras1 pathways are functionally related during CNS midline and tracheal development

The Drosophila jing gene encodes a zinc finger protein required for the differentiation and survival of embryonic CNS midline and tracheal cells. There is a functional relationship between jing and the Egfr pathway in the developing CNS midline and trachea. jing function is required for Egfr pathway gene expression and MAPK activity in both the CNS midline and trachea. jing over-expression effects phenocopy those of the Egfr pathway and require Egfr pathway function. Activation of the Egfr pathway in loss-of-function jing mutants partially rescues midline cell loss. Egfr pathway genes and jing show dominant genetic interactions in the trachea and CNS midline. Together, these results show that jing regulates signal transduction in developing midline and tracheal cells (Sonnenfeld, 2004).

The effect of a reduction in EGFR signaling on the jing gain-of-function phenotype was examined in the midline glia. sim-Gal4 and sli-Gal4 drivers were used to over-express jing specifically in the CNS midline in heterozygous and homozygous spi and S mutant backgrounds. The number of sli-lacZ-expressing midline glia in each nerve cord segment was quantified during stage 13 and compared to that in wild-type embryos over-expressing jing. Expression of two copies of the UAS-jing transgene in the midline glia of wild-type or heterozygous spi and S embryos resulted in an average of 12 midline glia instead of the normal 8 during stage 13. In contrast, UAS-jing transgene expression was unable to induce 12 midline glia in homozygous spi and S mutant backgrounds. In these embryos, there was an average of 1.5 midline glia in each nerve cord segment after jing over-expression; this is similar to the number of midline glia present in homozygous spi and S mutant embryos during stage 13 (Sonnenfeld, 2004).

To test the independent activity of jing, the effects of ectopic jing expression were examined in the Drosophila eye, which is a system that is functional for the Egfr pathway but not for jing or upstream regulators including single-minded (sim) or trachealess (trh). Analysis of jing01094 enhancer trap lacZ expression and of endogenous mRNA expression by in situ hybridization shows that jing is not expressed in third instar larval eye imaginal discs. Expression of wild-type jing in the eye, under regulation of the glass promoter (P[GMR-Gal4]), was associated with a rough appearance compared to P[GMR-Gal4] heterozygotes or wild-type. The rough eye consisted of highly disorganized ommatidia and mechanosensory bristles in 45% of flies and the number of ommatidia was reduced by 50% from that in wild-type and P[GMR-Gal4] heterozygous eyes. Therefore, the gain-of-function phenotypes of jing and Egfr both result in a significant reduction in ommatidia. Consistent with similar pathways, the rough eye phenotype of Egfr gain-of-function was not enhanced by that of jing. Out of 1000 flies scored, carrying P[GMR-Gal4] and both UAS-jing and UAS-ellipse, 100% showed the same eye phenotype as flies carrying only P[GMR-Gal4] and UAS-ellipse (Sonnenfeld, 2004).

The jing ectopic expression phenotype was dominantly suppressed by a 50% reduction in the levels of spi(spi1) and Df(2L)TW50 or Egfr deficiency [Df(2R)Egfr5]. After spi reduction, ommatidia were more organized and more abundant, although the position of the photoreceptors was not like that in controls. This interaction was not influenced by activation of the glass promoter in the heterozygous spi background (P[GMR-Gal4]/spi1). These results suggest that there is a dosage-sensitive interaction between the Egfr pathway and jing function in the eye, where increased jing activity can be suppressed by a reduction in downstream components such as spi and Egfr. Given that sim and trh are not expressed in third instar larval eye discs, these experiments suggest that jing can have an effect on the Egfr pathway in the absence of sim or trh and support the model that jing works as an independent regulator in bHLH-PAS pathways (Sonnenfeld, 2004).

Gene dosage experiments were used to determine the effects of simultaneously altering the levels of jing and genes of the Egfr pathway. Mutations in spi and its regulator Star, have been characterized for their midline and tracheal phenotypes. To determine whether jing and Egfr function is inter-dependent, the development of the CNS midline and trachea was analyzed in double heterozygotes of jing and S or spi. The basis for this experiment is that if the Egfr and jing pathways are inter-dependent then simultaneous reduction of only one copy of each gene should alter CNS midline and tracheal function. Multiple jing alleles balanced with wg-lacZ Cyo were crossed to SIIN23/wg-lacZ Cyo or spi1/wg-lacZ Cyo flies and their progeny were double stained with anti-Sim or anti-Trh and anti-β-Gal (Sonnenfeld, 2004).

The number of CNS midline cells was reduced from wild-type in embryos homozygous and double heterozygous for jing, spi or S and stained with anti-Sim. Since some of the Sim-positive nuclei appeared to be fragmenting, their fate was determined by TUNEL labeling to identify apoptotic cells. In wild-type embryos, cell death is uncommon in the CNS midline during stage 12 with an average of 6(±2) Sim-positive apoptotic nuclei per embryo. In contrast, in homozygous jing stage 12 mutant embryos, there was an average of 35(±3) Sim-positive apoptotic nuclei per embryo, therefore, displaying a significant increase over that in wild-type embryos. In embryos double heterozygous for mutations in jing and S or spi there was an average of 25(±2) and 30(±3) SIM-positive apoptotic nuclei per embryo during stage 12, respectively. This is consistent with the time period for the requirement of Egfr function in CNS midline glia. Embryos heterozygous for either jing, spi or S mutations did not alter the normal events of midline cell apoptosis. In summary, these results suggest that proper dosage of both jing and spi group gene function is required for midline cell survival (Sonnenfeld, 2004).

The jing gene was identified in two independent genetic screens for regulators of CNS midline development and border cell migration, two processes which are regulated by the EGFR. RTK signaling pathways have been implicated in multiple cell biological processes including proliferation, migration, differentiation and survival. How one MAPK pathway controls such different outcomes is a major area of research. Studies of Egfr function in the Drosophila adult eye suggest that signaling levels dictate the multiple cellular responses to the EGFR, such that differentiation requires the highest levels of signaling while mitosis and cell survival require less. Therefore, it is important to understand the mechanisms that control the expression of positive and negative regulators of this family of signaling molecules (Sonnenfeld, 2004).

Prior work has established the important role that the Egfr plays during the differentiation of midline glia (MG) and tracheal cells. Several lines of supportive evidence show that jing regulates Egfr signaling in the MG and trachea. (1) jing mutant embryos fail to maintain MAPK activity and Egfr expression in cells that clearly have midline and tracheal identities. (2) jing is required for and can induce Egfr pathway transcription in the CNS midline and trachea. (3) jing over-expression promotes midline glial survival in a similar fashion as over-expression of Egfr pathway genes. (4) jing-mediated over-expression phenotypes require Egfr pathway function in CNS midline glia and the adult eye. (5) A transgenic copy of either activated ras1, secreted spi or gain-of-function Egfr can partially rescue midline cell death in homozygous jing mutants. (6) Proper dosage of both pathways is essential for survival of midline glia and for proper tracheal morphogenesis. Together, these findings suggest that jing functions upstream in the Egfr/ras1 pathway. Future studies will be aimed at elucidating the nature of the relationship between jing and Egfr pathway genes and may help in the design of therapeutics to regulate over-active RTK pathways in oncogenic cells (Sonnenfeld, 2004).

jing is the only gene, other than those already characterized in the Egfr pathway, that can promote midline glial survival. jing over-expression, as driven by the sim and sli promoters, induces extra midline glia that express Egfr, slit and sim, and these glia appear to be rescued from apoptotic fates. The extra glia are observed during stage 13 which is consistent with the timing of Egfr pathway-induced extra glia. The absence of apoptotic glia and the wild-type midline neuronal numbers after jing over-expression suggest that the supernumerary glia are not likely recruited from neuronal populations and may represent glia rescued from death due to inappropriate Egfr expression. This effect phenocopies gain-of-function phenotypes in EGFR signaling in the CNS midline and suggests that jing-mediated cell survival may be carried out by the EGFR/RAS1 signaling pathway. In support, jing over-expression phenotypes in the CNS midline and eye are suppressed by reductions in Egfr function (Sonnenfeld, 2004).

The results suggest that jing is involved in both the differentiation and survival of cells in the embryonic CNS midline and trachea. In wild-type embryos, early MAPK activity controls midline glial (MG) differentiation through activation of the downstream Ets-type transcription factor pointed (pnt). The reductions in early MAPK activity and Egfr expression in the midline of jing mutants, therefore, reveals the requirement for jing function in MG differentiation. The similarities in gain- and loss-of-function midline glial phenotypes between pnt and jing are consistent with this model. In jing mutants, reduced MAPK activity occurs in midline and tracheal cells that express the sim and trh genes, respectively, indicating that the reductions in MAPK activity are not due to a general failure in cellular differentiation (Sonnenfeld, 2004).

It is possible that improper MG differentiation in jing mutants could be due to cells being committed to death. However, a loss of MAPK activity is detected prior to apoptosis in the CNS midline of homozygous jing mutants, suggesting that early MAPK inactivity in the CNS midline is independent of the apoptotic machinery. In support, the MG initially form in MAPK mutants and it is not until later stages, which are dependent on repression of hid, that the MG die. MG death in jing mutants may be due to a combined lack of the axon-glial contacts that are necessary for MAPK-mediated inactivation of hid as well as from reduced MAPK activity within the MG (Sonnenfeld, 2004).

During stage 10, EGFR signaling is activated in the central region of the tracheal placode by transcription of rhomboid resulting in the formation of anteroposterior branches including the dorsal trunk and visceral branch. Wingless (Wg) signaling originates in ectodermal cells adjacent to the tracheal placodes and causes Egfr-induced cells to form the dorsal trunk. jing is expressed in most tracheal cells and its protein product is localized to their nuclei suggesting that this C2H2-type zinc finger may have a regulatory role directly within these cells. Additional evidence that jing may have a role directly in tracheal cells comes from its perturbation of tracheal morphogenesis and alteration of Egfr/ras pathway gene expression profiles when over-expressed specifically in the trachea (Sonnenfeld, 2004).

jing affects branching morphogenesis and cellular survival in the tracheal system and its expression in the tracheal placodes coincides with that of Egfr pathway genes. jing and Egfr pathway mutants have similar tracheal phenotypes which include breaks in the dorsal trunk and reduced visceral branch formation. The reductions in Egfr-induced cells may explain the defects in dorsal trunk formation in jing homozygous mutant embryos and possibly in jing and Egfr pathway double heterozygotes. Alternatively, the dorsal trunk defects may arise from perturbations in Wg signaling in the ectoderm of jing mutants. spitz group tracheal mutant phenotypes do not reflect ectodermal patterning defects but this remains to be analyzed in more detail in jing mutants (Sonnenfeld, 2004).

The results indicate that proper Egfr pathway and jing function is required for midline and tracheal cell survival. This is the first evidence of such a survival role in the trachea and requires further investigation. However, this does not rule out the possibility that other processes involved in tracheal morphogenesis are not affected in double heterozygotes and jing homozygotes. Furthermore, in jing homozygotes and hemizygotes, truncated tubules are present in the transverse connectives, suggesting that the requirement for jing function is more global than that of Egfr/ras1. In support, jing is expressed in embryonic tissues that are not active in MAPK, suggesting that jing has additional functions (Sonnenfeld, 2004).

Compared to other midline and tracheal-expressed genes, those of the Egfr pathway are more highly expressed after jing over-expression (but not more than three-fold). Nevertheless, the effects of jing over-expression in the CNS midline can be seen by extra glia and Egfr expression establishing the importance of regulating jing expression during embryogenesis. Ectopic expression analyses suggest that jing is not sufficient to activate Egfr pathway gene expression. Therefore, these results suggest that in order to induce gene expression jing may require another protein, such as a cell-specific chromatin remodeling protein, that is not present in prd stripes but is present in the CNS midline, trachea and eye. The exact relationship between jing and Egfr pathway genes requires further analysis (Sonnenfeld, 2004).

EGF receptor signaling regulates pulses of cell delamination from the Drosophila ectoderm

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, svplacZ, and svplacZΔ18) were analyzed. In wild-type embryos, the high-threshold EGFR outputs of argos and svplacZ expression, detectable Rolled activation, and strong Yan downregulation are all inner ring specific, whereas lower-threshold outputs such as Sal upregulation and svplacZΔ18 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 svplacZ 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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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).

Egfr is essential for maintaining epithelial integrity during tracheal remodelling in Drosophila

A fundamental requirement during organogenesis is to preserve tissue integrity to render a mature and functional structure. Many epithelial organs, such as the branched tubular structures, undergo a tremendous process of tissue remodelling to attain their final pattern. The cohesive properties of these tissues need to be finely regulated to promote adhesion yet allow flexibility during extensive tissue remodelling. This study reports a new role for the Egfr pathway in maintaining epithelial integrity during tracheal development in Drosophila. The integrity-promoting Egfr function is transduced by the ERK-type MAPK pathway, but does not require the downstream transcription factor Pointed. Compromising Egfr signalling, by downregulating different elements of the pathway or by overexpressing the Mkp3 negative regulator, leads to loss of tube integrity, whereas upregulation of the pathway results in increased tissue stiffness. Regulation of MAPK pathway activity by Breathless signalling does not impinge on tissue integrity. Egfr effects on tissue integrity correlate with differences in the accumulation of markers for cadherin-based cell-cell adhesion. Accordingly, downregulation of cadherin-based cell-cell adhesion gives rise to tracheal integrity defects. These results suggest that the Egfr pathway regulates maintenance of tissue integrity, at least in part, through the modulation of cell adhesion. This finding establishes a link between a developmental pathway governing tracheal formation and cell adhesiveness (Cela, 2006).

This study documents a new role for the Egfr pathway in the regulation of tissue integrity. This new requirement could depend on the described early peak of Egfr activity, which would be sufficient to prevent defects at later stages. However, it is proposed that Egfr-promoted epithelial integrity depends on a later, or continuous but lower, or basal activity of the pathway that does not correlate with detectable ERK phosphorylation. Consistent with this hypothesis, it was found that downregulation of the pathway by overexpressing 801 or UAS-EgfrDN with btlGal4, which is expressed after the early peak of ERK phosphorylation, produces a conspicuous branch integrity phenotype. In any case, tissue integrity defects are mainly observed in the most dorsal and ventral tracheal branches, which are subjected to stronger pulling forces as development proceeds, and, therefore, it is precisely at late stages when defects in tissue integrity are expected (Cela, 2006).

AJs connecting epithelial cells dynamically disassemble and reassemble, thereby allowing tissue remodelling. Tracheal tissue remodelling might require the fine-tuning of cell adhesion properties, since tracheal cells need to be able to change their relative position (probably by loosening cell adhesion) while maintaining epithelial continuity. The data indicates that the Egfr pathway is a modulator of this balance, not only in the tracheal system, but also in other tissues undergoing extensive remodelling, such as the salivary glands, where a similar regulation of DE-cad and actin levels is found upon modulation of Egfr signalling. Conversely, no such a regulation was found in more static tissues, like the ectoderm, whose maintenance was proposed to depend on the maternally provided DE-cad protein. It is suggested that the Egfr pathway plays a role in the modulation of cell adhesion in tissues that undergo dramatic morphogenetic events, which might need the zygotic DE-cad contribution and a more dynamic regulation of cell adhesion. The results indicating a modulation of junctional complexes and/or the actin cytoskeleton by the Egfr pathway establish a link between a developmental pathway required for many biological events and cell biology in terms of cell adhesiveness and cell shape (Cela, 2006).

The results show that downregulation of several intracellular elements of the MAPK pathway produce defects in branch integrity, whereas a constitutively activated form of rl (rlsem) rescues the phenotype of btlGal4 801 embryos. This suggests that the conserved MAPK cassette is required to maintain branch integrity (Cela, 2006).

Two tyrosine kinase receptors, Egfr and Btl, activate the MAPK pathway during embryonic tracheal development. However, the two receptors, acting through the same intracellular cascade, elicit different responses. The MAPK pathway requirement in primary branching is likely to depend on input by btl, whereas the tissue integrity requirement is likely to depend on input by Egfr. How does the same MAPK pathway trigger distinct outcomes depending on the receptor that activates it? A temporal and/or spatial differential activation of the MAPK pathway could account for the different outcome. In addition, differences in the composition of the intracellular cascade due to specific transducers for one type of receptor, such as downstream of FGFR (dof; stumps-FlyBase), could contribute. Finally, quantitative and/or qualitative differences in the activation of the intracellular transducers by the different receptors could also underlie the outcome diversity (Cela, 2006).

Similar to these observations, air sac development in Drosophila has been recently reported to require both Btl and Egfr, and each receptor seems to elicit different responses. Furthermore, since during embryonic tracheal development, an uncoupling of the MAPK cassette and pnt has been observed during air sac development. These parallels suggest a common mechanism for generating different responses from the same intracellular transduction pathway (Cela, 2006).

The loss of tissue continuity and cell detachment observed in Egfr downregulation conditions may be due, at least in part, to a decrease in cell adhesion. Accordingly, a mild, but reproducible, decrease is observed in the accumulation of DE-cad and cortical actin. As inferred from the phenotypes, such a mild decrease could cause a loss of cell adhesion during tracheal remodelling, while not grossly affecting other processes requiring DE-cad-based cell adhesion, such as branch fusion. As expected, it was found that compromising AJ assembly or the actin cytoskeleton also gives rise to defects in tracheal tissue integrity (Cela, 2006).

Cadherins have been shown to support cell cohesion and participate in morphogenetic events. The actin cytoskeleton also plays an important role in shaping the cell architecture and in many morphogenetic processes. AJs and the actin cytoskeleton are intimately coupled, and their formation and maintenance is interdependent. Such interdependence is also observed in the tracheal system (Cela, 2006).

Cadherin-based cell-cell adhesion can be regulated at transcriptional and posttranscriptional levels. The modulation of a DE-cadGFP chimaera driven by heterologous promoters shows that, in the current case, DE-cad regulation is posttranscriptional. Several posttranscriptional mechanisms of DE-cad regulation have been proposed, and a role for the Egfr pathway can be envisaged in each of them. A first mechanism is at the level of DE-cad endocytic trafficking. In this context, the Egfr pathway could modulate the balance between recycling to the plasma membrane of internalised DE-cad or lysosomal targetting and degradation. A second mechanism of cell-cell adhesion regulation is posttranslational modifications of AJ components, such as phosphorylation or ubiquitination. Finally, another possible mechanism of regulation is through the cytoskeleton. The Rho family of small GTPases plays a key role in actin cytoskeleton regulation, and growth factor receptors such as Egfr have been reported to regulate their activity. Remarkably, the Egfr pathway has been recently shown to regulate the expression of the rhoGAP cv-c in the tracheal placodes, and it was found that cv-c mutants display tracheal integrity defects, although they are milder than those seen upon downregulation of the Egfr signal. It is therefore proposed that cv-c is at least one of the effectors of Egfr-mediated modulation of DE-cad levels and tracheal tissue integrity. Further analysis will be needed to disentangle the exact molecular mechanisms and to find other possible mediators of the Egfr signal (Cela, 2006).

The decrease of cadherin activity upon activation of the Egfr pathway has been extensively reported in the literature. This study reports the opposite: that Egfr pathway downregulation correlates with a decrease of cadherin-based cell adhesion. Although this is not the first example of such a relationship, it illustrates the versatility and complexity of the interactions occurring between signalling pathways and adhesion molecules, and establishes another model with which to analyse how cell adhesion is modulated (Cela, 2006).

The RhoGAP crossveinless-c links trachealess and EGFR signaling to cell shape remodeling in Drosophila tracheal invagination

A major issue in morphogenesis is to understand how the activity of genes specifying cell fate affects cytoskeletal components that modify cell shape and induce cell movements. This study approaches this question by investigating how a group of cells from an epithelial sheet initiate invagination to ultimately form the Drosophila tracheal tubes. Tracheal cell behavior is described at invagination; it is show to be associated with, and requires, a distinct recruitment of Myosin II to the apical surface of cells at the invaginating edge. This process is achieved by the activity of crossveinless-c, a gene coding for a RhoGAP and whose specific transcriptional activation in the tracheal cells is triggered by both the trachealess patterning gene and the EGF Receptor (EGFR) signaling pathway. These results identify a developmental pathway linking cell fate genes and cell signaling pathways to intracellular modifications during tracheal cell invagination (Brodu, 2006).

Tracheal cells are singled out as cell clusters in the ectodermal unicellular layer, one at each side of 10 central embryonic segments. This study focused on the central tracheal placodes because the first and last one have distinct features. By stage 10, tracheal cells form a flat epithelium with their neighboring ectodermal cells. Longitudinal optical sections (1 microm apart) show the apical cell membrane, visualized by PKC, in a more exterior plane and the tracheal nuclei in a deeper one. A transverse optical section across the middle of the placode reveals its straight surface. By early stage 11, a group of around six cells reduces its apical cellular perimeter; this is the earliest indication of tracheal invagination since the constricted apical surface of those cells can be detected deeper inside. Local constriction is associated with cell shape changes; those cells pinch at their apical surface while their basal surface and nuclei appear deeper than those of the other tracheal cells. By middle stage 11, the invagination proceeds further; now the apical marker of the cells can be detected in an even deeper position. In addition, at this stage a significant change is observed in the invagination behavior of these cells. On the dorsal side, cells begin a rotation-like movement folding to form a new layer of cells below the epidermal surface. On the ventral side, cells slide below the invaginating dorsal cells. As a result, a finger-like structure originates in a process that has evolved from a cell monolayer to a 'three-layer organization' (two cell layers initiating a tube below the epidermis layer). As development proceeds, this finger-like structure elongates dorsally incorporating more tracheal cells from the embryonic surface toward the inside (Brodu, 2006).

The results suggest a two-step model by which trh induces and organizes tracheal invagination. First, trh activity appears to outline an invagination field, a region of cells that acquire the competence to invaginate. This effect can be clearly observed in mutants that impair EGFR signaling; in those embryos, trh activity is still able to promote a broad depression of the trh-expressing cells that will only further reorganize due to their ability to migrate in response to FGFR signaling. In this regard, there are clearly some consequences of trh that are independent of EGFR signaling and could be connected with the potential of trh to induce a general depression. For instance, it was found that the microtubule network is highly enriched and polarized apically at the site of invagination; while this arrangement is absent in trh mutants, it remains present in the abnormal invaginating tracheal placodes in the absence of both FGF and EGFR signaling (Brodu, 2006).

A second outcome of trh is accomplished by the triggering of EGFR signaling, which leads to the spatial and temporal organization of tracheal invagination. It is the activity of the EGFR pathway that converts the tracheal cell potential to invaginate into the organized process, resulting in a 'three-layer organization' and initiation of tube formation. A partner required for the organization of tracheal invagination is sal, which is expressed in the dorsal half of the tracheal placode and is responsible for the different morphology and behavior of the cells between the two sides of the placodes. The role of sal is, at least in part, achieved through down-regulation of EGFR signaling activity. However, it is not clear how this modulation is translated into differences in invaginating behavior. For example, no differences have been detected in level or distribution of cytoskeletal components along the sal expression border. An intriguing possibility would be that down-regulation of EGFR signaling gives rise to cells with different forces or stiffness (perhaps due to different levels of actin–myosin contractility), and the resulting apposition of two invaginating cell populations with different properties could force one of them to fold and initiate dorsal-oriented rotation, while the other would slide down under the former (Brodu, 2006).

It is worth noting that a well-organized invagination is an absolute requirement for tracheal morphogenesis. All the mutants that cause an abnormal invagination give rise to an impaired tracheal system in which some branches do not develop or develop deficiently. Thus, for example, rho mutants, which were originally thought to affect specifically the formation of two branches, have a general defect in invagination, and many tracheal cells remain clustered at the embryonic surface. In this regard, an important outcome of proper tracheal invagination appears to be that the tracheal cells reach the appropriate position with respect to the cues that will direct their subsequent migration. It has been suggested that the wild-type organization of the tracheal tree depends on having the appropriate number of cells at the correct position facing those signals, such that a specific number of cells contributes to the formation of the different branches (Brodu, 2006).

In many cases, cell fate commitment leads to cell shape modifications and rearrangements. The results of this study depict a developmental pathway that is initiated by the activity of a gene specifying cell fate (trh), which triggers a cell signaling pathway (EGFR) that, in turn, organizes cell invagination. A key step in this pathway is the transcriptional activation of a gene coding for a RhoGAP enzyme, cv-c, that affects actin–myosin apical distribution, likely by regulation of Rho1 activity (Brodu, 2006).

Regulation of RhoGTPases, either by RhoGAPs or RhoGEFs, appears to be a common trait in the control of morphogenesis. Indeed, RhoGAPs and RhoGEFs have been shown to act in different manners to affect actin and myosin. In this regard, some parallelisms can be found between tracheal cell invagination and other morphogenetic events such as gastrulation and neurulation. In particular, clear similarities can be seen with the mechanism of myosin regulation in Drosophila gastrulation. In this case, it is also the activity of a patterning gene (twist) that gives rise to the expression of a signaling molecule (folded gastrulation) that is thought to elicit a signaling pathway requiring a G-protein alpha subunit (concertina) and a RhoGEF (RhoGEF2). Then, RhoGEF2 ultimately leads to phosphorylation of myosin, which then activates actin binding by myosin and increases actomyosin contractility. However, in tracheal invagination, the remaining colocalization of myosin and actin in cv-c mutants suggest that cv-c is not necessary for the interaction between actin and myosin but instead for the proper localization of the actin–myosin complex. This observation fits well with a recent report that indicates that the cv-c RhoGAP acts on the actin apical accumulation in Malpighian tube morphogenesis and during epithelial dorsal closure (Brodu, 2006).

Different RhoGTPases act as substrates of the cv-c RhoGAP enzyme in different tissues. The results indicate that Rho1 is the substrate for cv-c in tracheal invagination. Notably, there appear to be more RhoGAPs and RhoGEFs molecules than RhoGTPases, which has been interpreted as an indication of the importance of a precise regulation of the transition between active and inactive states of RhoGTPases for different cell processes. Additionally, the fact that mutants for cv-c, a negative regulator of Rho1 activity, and Rho1 both impair actin apical organization and cell invagination in the tracheal placodes illustrates the importance of an appropriate regulation of RhoGTPase activity to achieve proper actin organization and cell behavior. In this regard, the fact that the cv-c RhoGAP has a pivotal role in tracheal invagination does not rule out that additional regulatory mechanisms that act on RhoGTPases could also be in place in tracheal invagination. The variable penetrance of null cv-c RhoGAP phenotypes suggests the possible existence of other invagination-regulating molecules under the control of trh. Additionally, EGFR signaling is only one of the programs elicited by the activity of trh. Altogether, these observations indicate that the developmental pathway that induces and organizes tracheal invagination must have diverse branches with additional target outcomes. It is suggested that many morphogenetic events share the same basic operational logic; leading from patterning genes and cell signaling pathways to cell shape changes, although each case may involve diverse target molecules acting at different steps in the regulation of the actin–myosin complex (Brodu, 2006).

Mitotic cell rounding accelerates epithelial invagination

Mitotic cells assume a spherical shape by increasing their surface tension and osmotic pressure by extensively reorganizing their interphase actin cytoskeleton into a cortical meshwork and their microtubules into the mitotic spindle. Mitotic entry is known to interfere with tissue morphogenetic events that require cell-shape changes controlled by the interphase cytoskeleton, such as apical constriction. However, this study shows that mitosis plays an active role in the epithelial invagination of the Drosophila tracheal placode. Invagination begins with a slow phase under the control of epidermal growth factor receptor (EGFR) signalling; in this process, the central apically constricted cells, which are surrounded by intercalating cells, form a shallow pit. This slow phase is followed by a fast phase, in which the pit is rapidly depressed, accompanied by mitotic entry, which leads to the internalization of all the cells in the placode. It was found that mitotic cell rounding, but not cell division, of the central cells in the placode is required to accelerate invagination, in conjunction with EGFR-induced myosin II contractility in the surrounding cells. It is proposed that mitotic cell rounding causes the epithelium to buckle under pressure and acts as a switch for morphogenetic transition at the appropriate time (Kondo, 2013).

The invagination of epithelial placodes converts flat sheets into the three-dimensional structures that form complex organs, and it is a key morphogenetic process in animal development. A major mechanism of invagination is apical constriction, which is driven by actomyosin contraction. However, not all constricted cells invaginate, and some cell internalization occurs without apical constriction, suggesting that additional mechanisms of inward cell movement contribute to invagination (Kondo, 2013).

To obtain three-dimensional information about cell behaviour during invagination, live imaging was performed of the Drosophila tracheal placode. Ten pairs of tracheal placodes, each of which is composed of about 40 cells, are formed in the ectoderm at mid-embryogenesis, and each placode initiates invagination simultaneously. Using an adherens junction marker, DE-cadherin-green fluorescent protein (E-cad-GFP), it was found that the adherens junctions of the central placode cells slowly created a depression by apical constriction, which became the tracheal pit. After 30 to 60 min of slow movement (slow phase), the tracheal pit was suddenly enlarged, and the tracheal cells were rapidly internalized (fast phase) and eventually formed L-shaped tube structures (Kondo, 2013).

After the fast transition, all the tracheal cells and surrounding epidermal cells entered mitosis 16, the final round of embryonic mitosis. It was noticed that the fast invagination was always associated with the mitotic entry of central cells that were frequently the first to enter mitosis 16. Intriguingly, mitotic rounding of the central constricted cells occurred simultaneously with the rapid depression of their apices, followed by chromosome condensation 10 min later. In this study, this atypical mitotic rounding associated with apical depression in an internalized cell is called 'rounding', to distinguish it from canonical surface mitosis (surface cell rounding) (Kondo, 2013).

To determine whether cell rounding is required for invagination, zygotic mutants were examined of the cell-cycle gene Cyclin A (CycA), which fail to enter mitosis 16, and double parkeda3 (dupa3), which show a prolonged S phase 16 and delayed entry into mitosis 16. Tracheal invagination was initiated normally in the CycA and dupa3 mutants, but proceeded more slowly than in controls, indicating that entry into mitosis 16 is required for proper timing of the fast phase (Kondo, 2013).

Although delayed, the accelerated invagination in the CycA or dupa3 mutants eventually occurred, allowing the formation of tube structures and suggesting that additional mechanisms are involved. After invagination, fibroblast growth factor (FGF) signalling is activated in the tracheal cells to induce branching morphogenesis through chemotaxis. To examine the contribution of FGF signalling to invagination, mutants of the FGF ligand branchless (bnl) or the FGF receptor breathless (btl) were analyzed. These mutants invaginated normally, indicating that chemoattraction to FGF is dispensable for invagination (Kondo, 2013).

Next, to assess FGF's role in the mitosis-defective condition, double mutants were analyzed for CycA and bnl or CycA and btl, and it was found that they showed slower invagination than CycA single mutants. Furthermore, the invagination in these double mutants was incomplete, in that the cells failed to form L-shaped tubular structures. Therefore, FGF signalling is critical for invagination when mitosis is blocked, serving a back-up role. Tracheal-specific CycA expression rescued the defects in invagination speed and tube structure in the CycA btl mutants. In addition, mitosis of cells outside the pit was occasionally observed that occurred before the mitosis of the central apically constricted cells and was not correlated with the fast invagination phase. Thus, mitosis of the surrounding epidermal cells is dispensable for tracheal invagination. Taken together, it is concluded that mitotic entry of central cells is a major mechanism for accelerating tracheal invagination (Kondo, 2013).

To distinguish the role of cell rounding from that of cell division in the fast phase, the microtubule inhibitor colchicine was used to arrest the cell cycle after cell rounding. Colchicine treatment after mitosis 15 induced M-phase arrest at mitosis 16, but the fast invagination movement accompanied by cell rounding was not affected. This result indicates that cell rounding, but not cell division, is responsible for the acceleration phase of the tracheal invagination (Kondo, 2013).

Mitosis of cells in the columnar epithelium normally occurs at the apical surface after surface rounding. It was next asked how the apical surface of the central cells becomes depressed during internalized cell rounding. One possible model explains internalized cell rounding as cell-autonomously controlled by the association of the cells with the basement membrane or underlying mesodermal cells. However, genetic removal of basement-membrane adhesion by the maternal and zygotic mutation of βPS-integrin (also known as mys) did not compromise the speed of invagination, and snail-twist double-mutant embryos, which lack mesodermal cells, still showed tracheal invagination with internalized cell rounding. These results suggest that anchoring to the basal side is probably not required (Kondo, 2013).

A second model proposes that the apical depression of the rounding cells is driven by local planar interactions among the tracheal cells. Before and during tracheal invagination, myosin II is enriched at the cell boundaries tangential to the centre of the placode and regulates cell intercalation. It was noted that the myosin II level in the central cells was lower than in the surrounding, intercalating cells. Nevertheless, the apices of the central cells were constricted during the slow phase, strongly suggesting that the surrounding cells exerted centripetal pressure on the central cells through myosin II cables. Myosin II cables fail to form in EGFR signalling mutants (such as rho, the rhomboid endopeptidase required for EGF ligand maturation, and Egfr), and apical constriction is impaired in these mutants. The first few cells undergoing mitosis 16 in the tracheal placode of rho or Egfr mutants showed surface cell rounding with expanded apices, indicating that EGFR signalling is required to couple the mitotic cell rounding with fast apical depression. It is speculated that the columnar shape of the central cells resists centripetal movements, resulting in the accumulation of inward pressure during the slow phase. The existence of such resistance was supported by the results of a physical perturbation experiment using a pulsed ultraviolet lase. The cell rounding associated with mitotic entry would release the stored inward pressure by means of cytoskeletal remodelling that causes rapid depression of apical surface together with the active shortening of cell height, leading to rapid buckling of the apical surface and the fast phase of invagination (Kondo, 2013).

Even with the loss of both EGFR and FGF signalling, the tracheal placodes form moderately invaginated structures, compared to the flat tracheal placode observed in the rho-bnl-CycA triple mutant at the same stage, indicating that cells needed to undergo mitosis 16 to induce invagination, independent of EGFR and FGF signalling. In rho bnl double mutants, although the cells undergoing the earliest mitoses showed surface cell rounding, some of the subsequent mitotic events were coupled to apical depression and internalized cell rounding. Unlike the earlier mitotic events on the surface, the internalized rounding cells in the rho bnl embryos showed constricted apices and were surrounded by apically rounded cells before mitosis. Internalized rounding with a constricted apical surface were shared properties of cells in mitoses leading to invagination, in both control and rho bnl embryos. It is suggested that the first few cells undergoing surface cell rounding compress the adjacent interphase cells and restrict their apical area, so that they are forced to move internally after rounding, causing the epithelial layer to buckle and invaginate (Kondo, 2013).

Although invagination was largely blocked in the rho-bnl-CycA triple mutants, any double mutant combination permitted invagination to some degree, indicating that three qualitatively distinct mechanisms, mitotic cell rounding, myosin II contractility (EGFR) and active cell motility (FGFR), can independently trigger invagination. In the normal context of wild-type development the combination of cell rounding and EGFR signalling may optimize the timing and speed of invagination, and then invaginated tracheal sacs subsequently respond to FGF emanating from several target tissues guiding branching morphogenesis (Kondo, 2013).

These observations demonstrates a new role for mitosis in tissue morphogenesis to generate mechanical force through cell rounding, independent of cell division. This is distinct from previously described invagination mechanisms involving cell-autonomous constriction by the apical activation of actomyosin contractility, which is incompatible with mitosis. Mitosis 16 outside the tracheal placode occurs in clusters on the ectoderm surface, but does not lead to invagination, suggesting that the tracheal placode is sensitized to invaginate upon mitosis, independent of EGFR and FGFR signalling. Future research to uncover the properties of the tracheal placode that enables it to respond to clustered mitosis will explain not only this new mode of morphogenesis, but also the homeostasis mechanisms of epithelial architecture (Kondo, 2013).

Serial specification of diverse neuroblast identities from a neurogenic placode by Notch and Egfr signaling

The brain insulin-producing cell (IPC) lineage and its identified neuroblast (IPC NB) was used as a model to understand a novel example of serial specification of NB identities in the Drosophila dorsomedial protocerebral neuroectoderm. The IPC NB was specified from a small, molecularly identified group of cells comprising an invaginated epithelial placode. By progressive delamination of cells, the placode generated a series of NB identities, including the single IPC NB, a number of other canonical Type I NBs, and a single Type II NB that generates large lineages by transient amplification of neural progenitor cells. Loss of Notch function caused all cells of the placode to form as supernumerary IPC NBs, indicating that the placode is initially a fate equivalence group for the IPC NB fate. Loss of Egfr function caused all placodal cells to apoptose, except for the IPC NB, indicating a requirement of Egfr signaling for specification of alternative NB identities. Indeed, both derepressed Egfr activity in yan mutants and ectopic EGF activity produced supernumerary Type II NBs from the placode. Loss of both Notch and Egfr function caused all placode cells to become IPC NBs and survive, indicating that commitment to NB fate nullified the requirement of Egfr activity for placode cell survival. The surprising parallels between the serial specification of neural fates from this neurogenic placode and the fly retina are discussed (Hwang, 2011).

These observations provide a framework for understanding several key features of placodal neurogenesis in the placodes in the head midline dorsomedial procephalic NE (Pdm); the steps in placode development are summarized in Model for serial specification of NB identities from the Drosophila medial pars intercerebralis primordium (pPIm). The NE placode, comprising roughly eight cells, with its underlying gene regulatory network, appears to be highly specialized to serial specify a range of distinct neural stem cell identities, beginning from an initial state of equivalent developmental potential. In the case of the pPIm, the initial state of competence is to form IPC NBs. The first indication that the pPIm had acquired prepattern identity was the synchronized round of cell division observed before placode morphogenesis at the 4-cell pPIm stage. After the expansion to the 8-cell stage, the cells entered a cell cycle arrest and formed a neurogenic placode; the pPIm then initiated a lengthy proneural competence period as the various NB identities were produced. Mutant and temperature shift analysis of Notch signaling suggested that a window of competence for the IPC NB fate exists from the time the pPIm expanded to eight cells and acquired proneural competence until the time that IPC NB fate was normally specified (the time that it became Dac+). At roughly this point, the pPIm became dependent on Spi/Egfr activity to promote the survival of NE cells not yet specified as NBs. This Egfr-dependent specification period then extended through stage 15, while alternative NB identities were specified. Neurogenesis then ended with the specification of the single Type II NB fate (Hwang, 2011).

Among the well-studied examples of Drosophila neurogenesis, perhaps the most intriguing parallels are found between serial fate specification in the placodal pPIm and in the developing facets of the Drosophila retina, particularly between specification of the IPC NB and the R8 photoreceptor within each developing ommatidium. The R8 photoreceptor is the first of a series of photoreceptor and cone cell fates to be specified by progressive recruitment to an apically constricted cluster of twelve cells. Each R8 cell, the ommatidium founder, is specified from a proneural R8 fate equivalence group generated by the activity of the bHLH factor Atonal (Ato), and is singled out by Notch-mediated lateral inhibition. Although parallel with respect to specification from a proneural fate equivalence group, the pPIm NE requires activity of the AS-C for IPC NB specification whereas Ato is not essential. In contrast to R8 specification from a proneural group, the photoreceptor R1-7 and the cone cell fates are locally recruited within an ommatidium through inductive and serial Notch and Egfr/receptor tyrosine kinase (RTK) signaling from R8. Analogous to the pPIm and IPC NB specification, Egfr activity is not essential for proneural competence and specification of R8, but is essential for survival of all photoreceptor precursors, except for R8. Egfr-mediated cell survival in the developing retina requires that the pathway activates MAPK by phosphorylation and that pMAPK in turn phosphorylates the proapoptotic factor Head involution defective (Hid). In normal Egfr signaling, phosphorylated Hid is targeted for degradation, which permits survival of the developing ommatidium; cell survival is also promoted by the activity of Pnt1/2, which repress Hid expression. It was previously reported that hid mRNA accumulates in a pan-placodal Pdm NE pattern in stage 12-13 embryos, yet this study found that Pnt1/2 was not obviously essential for survival to stage 14. This suggests that, in contrast to the retina, most of the anti-apoptotic activity of Egfr signaling is relatively independent of Pnt1/2; hence, it might act primarily through the action of pMAPK in the turnover of Hid. This hypothesis will need to be tested more fully in future studies (Hwang, 2011).

Although there are many examples of systems in which Notch and Egfr activities are mutually antagonistic or cooperate in promoting cell fate decisions, no evidence was found of a mutual dependence between Notch and Egfr activity states; either pathway became active in the absence of the other. Admittedly, from these experiments cross-talk between the two pathways could not be definitively ruled out. By contrast, other instances of neurogenesis in Drosophila, such as in the optic lobe and notum macrochaete, depend on Egfr activity to promote neurogenesis by activating as-c genes. In these contexts, the Egfr-dependent proneural state is antagonized by Notch activity. In the pPIm proneural region, it remains unclear from these experiments whether Egfr is essential for neurogenesis subsequent to the IPC NB, since the pPIm cells are lost with the loss of Egfr activity. Indeed, there is a potential parallel with neurogenesis of the abdominal chordotonal precursors, which do form in the absence of Egfr activity but then signal back to the epithelium via Spi to activate ato and extend neurogenesis, thereby recruiting additional chordotonal precursors (Hwang, 2011).

In conclusion, the parallels between serial neural fate specification in the Pdm placode and in eye development raise the interesting possibility that some aspects of the two underlying gene regulatory networks have points of overlap. If so, it is intriguing to consider whether distinct regions of proneural epithelia that express placode genes such as sine oculis, Optix, Six4 and eya, were derived in evolution from a common ancestral neuroepithelial patterning circuit that was capable of serial specification. If this were the case, the implication would be that this mode of fate specification, in which diverse neuronal or neural stem cell identities are generated through local interactions in prepatterned cell groups, is more widely distributed than currently appreciated across animal phylogeny and vertebrate species. Hence, a deeper understanding of such a neural stem cell diversification mechanism will certainly aid efforts to control the differentiation of specific neural progenitor fates in vitro (Hwang, 2011).

The convergence of Notch and MAPK signaling specifies the blood progenitor fate in the mesoderm

Blood progenitors arise from a pool of pluripotential cells ('hemangioblasts') within the Drosophila embryonic mesoderm. The fact that the cardiogenic mesoderm consists of only a small number of highly stereotypically patterned cells that can be queried individually regarding their gene expression in normal and mutant embryos is one of the significant advantages that Drosophila offers to dissect the mechanism specifying the fate of these cells. This paper shows that the expression of the Notch ligand Delta (Dl) reveals segmentally reiterated mesodermal clusters ('cardiogenic clusters') that constitute the cardiogenic mesoderm. These clusters give rise to cardioblasts, blood progenitors and nephrocytes. Cardioblasts emerging from the cardiogenic clusters accumulate high levels of Dl, which is required to prevent more cells from adopting the cardioblast fate. In embryos lacking Dl function, all cells of the cardiogenic clusters become cardioblasts, and blood progenitors are lacking. Concomitant activation of the MAPK pathway by EGFR and FGFR is required for the specification and maintenance of the cardiogenic mesoderm; in addition, the spatially restricted localization of some of the FGFR ligands may be instrumental in controlling the spatial restriction of the Dl ligand to presumptive cardioblasts (Grigorian, 2011).

In this study pursued two goals: to elucidate the precise location and cellular composition of the cardiogenic mesoderm, and to analyze the mechanism by which Notch becomes activated in the restricted subset of these cells that become blood progenitors. The findings show that the cardiogenic mesoderm is comprised of segmentally reiterated pairs of clusters (cardiogenic clusters) defined by high expression levels of Dl, L'sc and activated MAPK. The MAPK pathway, activated through both EGFR and FGFR signaling, is required for the specification (EGFR) and maintenance (EGFR and FGFR) of all cardiogenic lineages. As shown previously, the default fate of all cardiogenic cells is cardioblasts. Notch activity triggered by Dl is required for the specification of blood progenitors (thoracic cardiogenic clusters) and nephrocytes (abdominal cardiogenic clusters), respectively. One of the downstream effects of MAPK signaling is to maintain high levels of Dl in the cardiogenic clusters, and to help localize Dl expression toward a dorsal subset of cells within these clusters, which will become cardioblasts. Dl stimulates Notch activity in the surrounding cells, which triggers the blood progenitor/nephrocyte fate in these cells (Grigorian, 2011).

The cardiogenic clusters form part of a larger population of mesodermal cells defined by high expression levels of l'sc. Based on l'sc in situ hybridization, these authors mapped 19 clusters of l'sc expressing cells within the somatic mesoderm. Many of these clusters (called 'myogenic clusters' in the following) give rise to one or two cells that transiently maintain high levels of l'sc, whereas the remaining cells within the cluster lose expression of l'sc. The l'sc-positive cells segregate from the mesoderm to a more superficial position, closer to the ectoderm, undergo one final mitotic division, and differentiate as muscle founder cells. Dl/Notch mediated lateral inhibition was shown to act during the singling-out of muscle founders from the myogenic clusters. Loss of this signaling pathway caused high levels of L'sc to persist in all cells of the myogenic clusters, with the result that all cells developed as muscle founders. Interestingly, loss of l'sc had only a mild effect, consisting of a slight reduction in muscle founders. This is similar to what was find in this paper in l'sc-deficient embryos, which show only a mild reduction in cardioblasts and other cardiogenic lineages (Grigorian, 2011).

The developmental fate of most of the L'sc-positive clusters within the dorsal somatic mesoderm is different from that of the ventral and lateral myogenic clusters discussed above, even though several parallels concerning the morphogenesis, proliferation, and dependence on Dl/Notch signaling are evident. The somatic (anterior, Wg-positive) mesoderm is divided into a dorsal and ventral domain based on the expression of Tin. Initially expressed at high levels in the entire mesoderm, this gene is maintained only in the dorsal mesoderm, as a result of Dpp signaling from the dorsal ectoderm. The dorsal somatic mesoderm, which is called 'early cardiogenic mesoderm', includes four L'sc-positive clusters, C2 and C14-C16. The development of C2 has been described in detail. C2 gives rise to a progenitor that divides twice; two of the progeny become the Eve-positive pericardial cells. Meanwhile, C15 which appears later at the same position as C2, seems to behave like a 'normal' myogenic cluster. It produces a progenitor that divides once and forms the founders of the dorsal muscle DA1. As shown in this paper, the two remaining dorsal clusters, C14 and C16, give rise to the cardioblasts. It is noted that the Eve-positive progenitors, as well as the cardioblasts, resemble the muscle founders derived from the typical myogenic clusters in three aspects. First, they segregate toward a superficial position, close to the ectoderm, relative to the remainder of the cells within the clusters. Secondly, they undergo one (in case of C2: two) rounds of division right after segregation. And third, they are restricted in number by Dl/Notch signaling: in all cases, they are increased in number following Dl or Notch loss of function (Grigorian, 2011).

Cardiogenic clusters, like myogenic clusters, also depend on the MAPK signaling pathway. Past studies have shown that in the Eve-positive C2 and C15 clusters, Ras, is capable of inducing the formation of additional Eve-positive progenitors. Ras is a downstream activator of both the EGFR and FGFR tyrosine kinase pathways, both of which have been seen to be important for the formation of the Eve-positive progenitors. With a loss of the FGFR pathway, Eve-positive progenitors of both the C2 and C15 cluster are lost; by contrast, the EGFR pathway affects only C15. The balanced activity of MAPK and Notch, which in part depends on reciprocal interactions between these pathways, determines the correct number of C2/C15 derived progenitors. Ras-induced MAPK activation upregulates the expression of other MAPK signaling pathway members (autoregulatory feed-back loop), but also stimulates the antagonist Argos, as well as Dl. Dl-activated Notch, in turn, inhibits MAPK signaling (Grigorian, 2011).

Both Dl/Notch and MAPK signaling are active in the C14 and C16 clusters, which constitute the definitive cardiogenic mesoderm. MAPK activity is required for the maintenance of all lineages derived from these clusters, as shown most clearly in the EGFR LOF phenotype that entails a lack of cardioblasts, blood progenitors, and pericardial nephrocytes. Overexpression of Ras results in an increased number of all three cell types, which indicates that the C14/C16 clusters attain a larger size, possibly by an additional round of mitosis. The phenotype seen in embryos suffering from loss- or overexpression of Dl/Notch pathway members can be interpreted in the framework of a classical lateral-inhibition mechanism: Dl is upregulated in the C14/C16 derived cardioblast progenitors (analogous to the Eve-progenitors of C2/C15), from where it activates Notch in the remainder of the C14/C16 cells; these cells are thereby inhibited from forming cardioblasts, and instead become nephrocytes/blood progenitors. The level of Notch activity affects the expression of tin (low Notch) and the GATA homolog srp (high Notch), which triggers the fate of cardioblasts and blood progenitors/nephrocytes, respectively (Grigorian, 2011).

MAPK is required for the initial activation of Dl in the cardiogenic clusters (just as in the myogenic clusters). Input from the pathway is most likely also instrumental in the subsequent restriction of Dl to the cardioblast progenitors. The positive interaction between MAPK and Notch signaling could occur at several levels. A mechanism shown for the ommatidial precursors of the eye disc involves Ebi and Strawberry notch (Sno), which are thought to act downstream of EGFR signaling and lead to an upregulation of Dl through the Su(H) and SMRTER complex (Grigorian, 2011).

Generally, when one progenitor cell is seen to give rise to two different cell types it is accomplished in one of two ways. One: there is an asymmetric division, where a factor expressed by the progenitor is segregated into only one daughter cell; two: a non-uniformly expressed extrinsic signal effects one cell, but not its neighboring sibling. In the posterior (abdominal) segments of the Drosophila cardiogenic mesoderm, inhibition of Notch by Numb accounts for the asymmetric activity of Notch in a small set of cardiogenic mesoderm cells, the Svp-positive cells. If Numb function is removed, these cells, which normally produce two cardioblasts and two pericardial nephrocytes, instead give rise to four cardioblasts. However, multiple nephrocytes per segment remain in numb loss-of-function mutations; furthermore, loss of numb does not cause any defect in the blood progenitors, where asymmetrically dividing Svp-positive cells are absent. This suggests that in addition to the numb-mediated mechanism, directional activation of Notch by one of its ligands is required for the majority of nephrocytes and all of the blood progenitors. It is proposed in this study that the spatially restricted upregulation/maintenance of Dl in nascent cardioblasts acts to activate Notch in the remainder of the cells within each cardiogenic cluster, which promotes their fate as blood progenitors and nephrocytes (Grigorian, 2011).

The Notch signaling pathway is typically associated with members of two different types of bHLH transcription factors. One type act as activators, while the other act as repressors. In the context of lateral inhibition, best studied in Drosophila neurogenesis, activating bHLH transcriptions factors, including genes of the AS-C like l'sc, are expressed at an early stage in clusters of ectodermal or mesodermal cells, where they activate genetic programs that promote differentiative pathways such as neurogenesis, or myogenesis/cardiogenesis. Subsequently, Notch ligands initiate the Notch pathway in these clusters; cells with high Notch activity turn on members of the Hairy/E(spl) (HES) family of bHLH genes which act as repressors and abrogate the transcriptional programs that had been set in motion by the activating bHLH factors. This paper shows that the gene cassette consisting of the Notch signaling pathway, as well as activating and repressing bHLH factors, operates in the cardiogenic mesoderm to determine the balance between cardioblasts and blood progenitors/nephrocytes. As discussed in the following, the same cassette also appears to be centrally involved in the specification of vascular endothelial cells and hematopoietic stem cells in vertebrates, which adds to the list of profound similarities between Drosophila and vertebrate blood/vascular development (Grigorian, 2011).

Even prior to the appearance of hemangioblasts, the lateral mesoderm of vertebrates is prepatterned by sequentially activated signaling pathways and transcriptional regulators similar to those that act in flies. The Wnt/Wg pathway, for example, separates subdomains of the mesoderm in vertebrates and Drosophila, as well as more ancestral ecdysozoans. Notch signaling plays an essential role in generating boundaries between segmental, as well as intra-segmental, subdomains within the ectoderm and mesoderm. The FGF signaling pathway predates the appearance of Bilaterians and plays a highly conserved role in early mesoderm patterning. Likewise, specific sets of transcriptional regulators are the targets of these signaling pathways (e.g., twist, zfh and myostatin) and play a role during the establishments of cell fate in the mesoderm. It appears, therefore, that the bilaterian ancestor featured a mesodermal subdomain, the 'cardiogenic/lateral mesoderm, in which signals of the Wg, BMP, Notch, and FGF pathways and conserved sets of transcriptional regulators established boundaries and cell fate in the mesoderm (Grigorian, 2011).

The vertebrate gene encoding an activating bHLH factor with sequence similarity to the Drosophila AS-C genes is SCL. SCL expression in the lateral mesoderm marks the first appearance of hemangioblasts; note that SCL is also expressed widely in the developing vertebrate CNS. In Zebrafish, from their site of origin in the lateral mesoderm, SCL-positive hemangioblasts migrate dorso-medially and form the intermediate cell mass (ICM). The ICM is the site of primitive endothelial blood vessel and hematopoietic cell specification. Gain of function studies carried out in zebrafish embryos have shown SCL to be one of the genes important in specifying the hemangioblast from the posterior lateral plate mesoderm. The specification of hemangioblast here comes at the expense of other mesodermal cell fates, namely the somitic paraxial mesoderm. In mice, lack of SCL affects blood and vascular development as SCL mutants are bloodless and show angiogenesis defects in the yolk sac (Grigorian, 2011).

Vertebrate homologs of the repressive Drosophila Hairy/E(spl) family of bHLH genes are the Hes and Hey (hairy/Enhancer-of-split related with YRPW motif) genes. A well studied member of the Hey family in zebrafish is gridlock, which is required for the specification of hematopoietic progenitors from the ICM. Hey 2 mutations in mice lead to severe congenital heart defects. In addition, the Hes protein plays a role in hematopoiesis as it is a positive regulator of Hematopoietic Stem Cell (HSC) expansion (Grigorian, 2011).

Genetic studies of the Notch receptors and their ligands in vertebrates support the idea that this pathway does indeed play a crucial role in the initial determination of hematopoietic stem cells. The yolk sac and the para-aortic splanchnopleura (P-Sp)/AGM (aorta gonad mesonephros) of Notch null mouse embryos lack HSCs. A similar phenotype is observed in mutants of Jagged 1, one of the Notch ligands. Notch is thought to be the deciding factor between hematopoietic and endothelial cell fates when the two originate from a common precursor or hemangioblast. In murine mutants exhibiting lower Notch1 mRNA levels, a lack of hematopoietic precursors is seen and is accompanied by an increase in the number of cells expressing endothelial cell markers. Likewise, in Drosophila, loss of Notch is associated with an increase in cardioblast number and a loss of blood precursor cells (Grigorian, 2011).

Larval and Pupal Stages

rhomboid expression upregulates Egfr signaling at wing vein primordia in the wing imaginal disc (Sturdevant, 1995).

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

Use of a dominant-negative form of the Egfr in the eye reveals that Egfr is required for differentiation of all photoreceptor cell types (R1-R8), including R7 which is also subject to the Sevenless signal. DN-Egfr 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-Egfr 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. Expression of DN-Egfr in the presumptive cone or pigment cells leads to them not differentiating. Overexpression of secreted Spitz, the ligand of Egfr 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 Egfr, 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 Egfr 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 Egfr 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 ligand, 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 photoreceptors 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 temporal switch in DER signaling controls the specification and differentiation of veins and interveins in the Drosophila wing

The Drosophila EGF receptor (Egfr) is required for the specification of diverse cell fates throughout development. How the activation of Egfr controls the development of vein and intervein cells in the Drosophila wing has been examined. Two distinct events are involved in the determination and differentiation of wing vein cells: (1) the establishment of a positive feedback amplification loop, which drives Egfr signaling in larval stages (at this time, rhomboid, in combination with vein, initiates and amplifies the activity of Egfr in vein cells); (2) the late downregulation of Egfr activity [at this point, the inactivation of MAPK in vein cells is necessary for the maintenance of the expression of decapentaplegic (dpp) and becomes essential for vein differentiation. Subsequently, Egfr becomes activated in intervein territories. During the time that dpp is expressed in vein territories, MAPK activity builds up in intervein territories, probably due to the presence of Vn, a weak Egfr activator. As a consequence, aos expression relocates to intervein territories. Together, these temporal and spatial changes in the activity of Egfr constitute an autoregulatory network that controls the definition of vein and intervein cell types (Martin-Blanco, 1999).

The reiterated use of Egfr is a common effector of differentiation. In the Drosophila eye, Egfr is required for the determination of all cell types. In this system, cell fate depends on the developmental stage at which the receptor is activated. By interfering with Egfr signaling activity, the specification of veins respond to the activation of receptor tyrosine kinase (RTK) signaling during larval stages, but continued activation of RTK signaling results in a failure of vein cells to differentiate. One explanation for these opposite effects could be that early activation of RTK signaling would specify vein cells, while late RTK signaling would implement intervein cell fates. Several observations provide support for this model. In pupae, MAPK is repressed in veins and activated in intervein cells. This activation of MAPK (and the expression of downstream genes, such as argos) responds to Ras signaling activity, and appears to be involved in the suppression of vein cell fates. Indeed, after ectopical activation of D-Raf during the pupal period, promoting intervein cell fates, the MAPK activity remains stimulated all over the wing blade (Martin-Blanco, 1999).

It seems that Egfr is the only receptor tyrosine kinase at work in the wing, able to activate Ras and Raf. While Egfr is ubiquitously expressed during larval imaginal disc development, EGFR mRNA levels are downregulated in the pupal period in presumptive vein cells. This downregulation of Egfr could be involved in the suppression of MAPK activity in vein territories. Furthermore, when a dominant negative-Egfr (DN-Egfr) molecule is overexpressed, titrating the endogenous Egfr, in pupae, extra vein tissue is induced. MAPK dephosphorylation in veins could also be induced by other mechanisms; for instance, the early expression of the inhibitor ligand Argos in veins up to 24 hours APF could cooperate in the inactivation of MAPK in these territories (Martin-Blanco, 1999).

What is the function of this change of expression? The first effect of this developmental switch is a modification in the expression of downstream targets. As a consequence of the reduction in MAPK activity from vein cells, aos is eliminated from veins between 24 and 30 hours APF. Conversely, it is upregulated in intervein territories. This scenario is reminiscent of the induction of Egfr ligands in the ventral ectoderm. Here, the primary signal, Spitz induces a relay mechanism by triggering the expression of Vn (and Aos) in adjacent cells. Aos reduces the overall level of Egfr signaling, whereas Vn provides a lower level of activation, capable of inducing only the lateral cell fates. In the larval wing, high levels of Egfr signaling are achieved in veins through a positive feedback loop. Here, Egfr activity promotes the expression of Aos. It is suggested that Aos diffusion from veins could prevent adjacent cells from responding to the vein inductive signals and producing high levels of Egfr activity ('remote inhibition'). Consistently, aos mutant flies display small deltas and extra veins clustered around vein territories. On the contrary, Aos overexpression in larval stages induces the suppression of veins. It is also proposed that, in pupae, while Egfr activity (and Aos) in veins are lost, Vn and Aos expression in intervein cells will reach a competitive balance leading to the activation of Egfr and MAPK, and intervein cell specification (Martin-Blanco, 1999 and references therein).

Several types of cell-cell communication have been proposed to be required during the latter stages of pupal wing development. The dpp gene encodes a member of the TGFbeta superfamily and is expressed during early pupal development in vein primordia. A class of loss-of-function dpp alleles and certain combinations of Dpp receptor mutants lead to vein-loss phenotypes. Mosaic analysis of dpps allele show that mitotic clones affect the differentiation of veins. Meanwhile, the effects of overexpression of dpp or an active form of its receptor thick veins (tkv) indicate that Dpp directs vein differentiation through activation of Tkv in pupal stages. The initiation of dpp expression in pupal stages depends on the activity of early acting genes, and in particular Egfr activity. However, although Egfr signaling is downregulated in vein territories during pupariation, dpp expression is maintained through an autoregulatory loop and remains high in vein cells until their final differentiation. Interestingly, in intervein cells, dpp expression is not activated in response to the Egfr activity described above. On the contrary, these cells express short gastrulation (sog), a gene that exerts an opposing effect to dpp. sog plays a role restricting vein formation to the center of the provein regions. dpp and sog interact antagonistically during vein differentiation. Ectopic activation of Egfr signaling in pupal stages abolishes dpp expression from veins. This suppression of dpp correlates with the loss of veins observed in this condition; it is reminiscent of the effect of Sog overexpression in pupal wings. Moreover, vein plexates induced by compromising Egfr activity in pupal wings, associate with a broadening of dpp-expressing areas (Martin-Blanco, 1999).

It is suggested that Egfr signaling downregulation from vein territories allows dpp to autoregulate dpp expression. It remains to be determined whether sog expression depends on Egfr in intervein territories, or is a consequence of the activity of intervein-specific genes such as blistered. The model presented here on how a single receptor (Egfr), triggering a conserved signal transduction pathway, is used reiteratively to implement two different cell fates in the development of the fly wing serves to reconcile many observations that have been made regarding cell fate specification in the wing. This may well provide a paradigm for the regulation of Egfr signal transduction in other developmental events (Martin-Blanco, 1999).

EGF receptor and Notch signaling act upstream of Eyeless/Pax6 to control eye specification

The Drosophila compound eye is specified by the concerted action of seven nuclear factors: Twin of eyeless (Toy), Eyeless (Ey), Eyes absent (Eya), Sine oculis (So), Dachshund (Dac), Eye gone (Eyg), and Optix (Opt). These factors have been called 'master control' proteins because loss-of-function mutants lack eyes and ectopic expression can direct ectopic eye development. However, inactivation of these genes does not cause the presumptive eye to change identity. Surprisingly, several of these eye specification genes are not coexpressed in the same embryonic cells -- or even in the presumptive eye. Surprisingly, the EGF Receptor and Notch signaling pathways have homeotic functions that are genetically upstream of the eye specification genes; specification occurs much later than previously thought -- not during embryonic development but in the second larval stage (Kumar, 2001).

The Egfr and Notch pathways function in the specification or determination of the eye. An ey-GAL4 driver was used to express target proteins; this element drives expression first in the eye and antenna anlagen in the embryo (by stage 11) and then in regions ahead of the furrow in just the eye imaginal disc. Egfr function was removed in this domain by expressing a dominant negative form of the receptor. Both the eye and antenna were deleted from eclosed adults indicating that both structures require Egfr signaling for their specification, determination, or survival. Under these conditions, the larval discs do not form, making analysis of later developmental phenotypes impossible. The same phenotype was obtained with dominant negative Ras indicating that this activity is Ras dependent. Wild-type and activated forms of several components of the Ras pathway were expressed in the eye anlagen using the same driver and it was found that hyperactivation of many elements leads to the homeotic transformation of the eye into a morphologically complete antenna. Homeotic transformation of the eye to antenna can also be induced by the Egfr ligand Spitz but not by two other known activators. The membrane bound version of Spitz does not induce homeotic transformations, suggesting a requirement for paracrine signaling. Wild-type and constitutively active forms of the Egfr and two other Drosophila RTKs (Breathless [Btl] and Heartless [Htl]) were expressed but only Egfr is able to induce the transformation. Expression of the constitutively active version of Egfr gives a significantly stronger phenotype than the wild-type version of Egfr, suggesting that the level of Egfr signaling is important for maintaining the balance between eye and antennal identities. The downstream elements of the pathway that can induce this transformation include Ras, Raf, and PntP1, while neither MEK, MAPK, nor PntP2 induced this effect in this assay. Aop, Tramtrack (Ttk), and BarH1/H2, each of which mediates negative feedback inhibition of Egfr signaling, delete the eye. The failure of Mek, Mapk, and PntP2 to induce this transformation reflects the existence of actual branch points in the pathway. However, it is also possible that the quantitative levels of expression of these three elements are not limiting for this signal at this time and place; indeed, their phosphorylation states may be more relevant (Kumar, 2001).

Notch and Egfr have been shown to often antagonize each other during cell fate decisions in the fly eye. Notch function was removed with a dominant negative form and results similar to the effects of Egfr signal hyperactivation were obtained. Consistent with this, when an activated form of Notch was expressed, the size of the eye was reduced and there were severe dysmorphies. Expression of dominant negative transgenes of the ligands Delta (Dl) or Serrate (Ser) also results in the eye to antenna transformation. Elevated expression levels of both Su(H) and many of the proteins of the E(spl) complex (m4, m7, m8, m8DN, malpha, mß, mgamma, and mdelta) were also expressed but no effect on either eye or antenna disc development was observed. However, homeotic eye to antenna transformations occurred when Mastermind (Mam) was expressed using a dominant negative construct. Mam is a member of the neurogenic gene group that encodes a nuclear protein of unknown function. These results suggest a Su(H) and E(spl)C independent pathway for eye and antenna disc development that involves Mam (Kumar, 2001).

Do Egfr and Notch Act upstream of the eye specification genes? A molecular epistasy study was undertaken, examining the expression of some of the eye and antennal specification genes in the transforming conditions during the third larval stage (before cell types differentiate). In eye specification gene mutants (such as ey), ommatidial development is blocked, but the eye disc remains in a reduced form. Conditions that produce eye to antenna transformations, whether through hyperactivation of Egfr or downregulation of Notch signaling, show a complete replacement of the eye disc with an antenna disc. Distal-less (Dll) and Spalt-Major are normally expressed within subdomains of the antenna disc and are required for antenna development. Dll and SalM are expressed in the correct locations in the transformed antenna disc suggesting that both endogenous and transformed antenna are also both morphologically and molecularly equivalent (Kumar, 2001).

The transcription of five of the seven known eye specification genes (toy, ey, eya, so, and eyg) was examined. In transforming conditions, transcription levels of all five of the seven genes are below the levels of detection. This is consistent with both Egfr and Notch signaling acting genetically upstream to both the eye and antennal specification genes. The downregulation of ey suggests that the ey-GAL4 driver may also be downregulated via an autoregulatory mechanism. That the transformation occurs despite this may reflect a phenocritical period for the eye-antenna transformation; once the transformation has occurred the system is refractory to the loss of Egfr signaling (Kumar, 2001).

The notch pathway signals differentially in the eye versus the antenna primordia in the second larval stage. Loss of Notch activity during the second larval stage results in the transformation of the eye into an antenna. Thus, it is predicted that Notch signaling should be elevated in the presumptive eye versus the antenna at the critical time. Cells that are actively receiving a Notch signal upregulate Notch protein expression. Thus elevated Notch antigen expression can be used as a reporter of elevated Notch signaling. Notch and ey expression were examined in imaginal discs from first, second, and third stage larvae. Both Notch and ey are expressed throughout the entire eye-antennal disc anlagen during the first larval stage. By the second larval stage, Notch is differentially upregulated within the presumptive eye. Interestingly, Notch appears especially active along the eye margins and midline, where it is thought to regulate retinal polarity. In contrast, ey appears to be exclusively within the eye field. In the third larval stage, Notch expression is upregulated in the morphogenetic furrow, where it acts to control ommatidial spacing while ey remains upregulated ahead of the furrow (Kumar, 2001).

Therefore, Egfr signaling promotes an antennal fate while Notch signaling promotes an eye fate. This role for Notch is consistent with the observation that removal of Notch signaling can partially inhibit compound eye development. Furthermore, several of the eye and antennal specification genes (ey, toy, eya, so, eyg, salM, and Dll) are downstream of the Egfr and Notch inputs. Wg and Hh pathway signaling affect this specification. The eye specification genes form a regulatory network and the direct control of any one of these genes may affect the others. Thus, which (if any) of the known eye specification genes is a direct target of Notch or Egfr signals may require direct biochemical assays (Kumar, 2001).

While activating Egfr or blocking Notch signals transforms the eye cleanly into an antenna, the reciprocal transformation is not complete, suggesting that there may be additional positive regulators of eye fate. The reciprocal transformation experiment could not be conducted (i.e., antenna to eye switch via hyperactivation of Notch or downregulation of Egfr signaling solely within the antennal anlagen). Unlike the ey-GAL4 driver, there is not an equivalent known driver that is expressed solely with the antennal anlagen. All known antennal-determining genes are also expressed in other imaginal discs. For instance, the Dll-GAL4 driver is expressed in several places within the embryonic head and leg imaginal disc. Expression of Egfr or Notch constructs with this driver results only in embryonic lethality. It may be that the antenna can be changed to an eye via alterations of Egfr or Notch signaling provided that the appropriate tools for their missexpression are available (Kumar, 2001).

Why do homozygous mutants for eye specification genes not transform the eye into an antenna? While it may be that some alleles are not nulls (e.g., ey1), a more interesting possibility is that there may be functional redundancy in some cases -- particularly that of ey and toy. Thus, only when both genetic functions are eliminated will a true null condition exist. Just such a situation confused the phenotypic analysis of two other twin homeodomain proteins, engrailed and invected. Unfortunately, mutations of the toy gene do not yet exist (Kumar, 2001).

How do the eye specification genes function? Published genetic epistasy and biochemical interaction data suggest that the seven known eye specification genes' products interact at the transcriptional and protein levels to direct cells toward eye fate. This requires that they are expressed in the same cells. Furthermore, it has been suggested that many, if not all, of these genes are 'master regulators' of eye fate -- that is, they are both necessary and sufficient for eye specification. Many very compelling experiments have been described showing the induction of ectopic eyes through the ectopic expression of these genes alone or in synergistic combinations. It is suggested that these genes come under separate regulation by different patterning signals in early development and that there are overlapping domains. Only when all of the domains coincide (during the second larval stage) do eye specification genes specify the eye. This seems to be the simplest explanation since the eye specification genes form a very tight genetic, biochemical, and transcriptional regulatory network suggesting that they are together required for eye specification. It may be that the final coexpression of the eye specification genes' products (and the exclusion of the antennal specification factors) is the last step required to allow the morphogenetic furrow to initiate in response to the next local expression of hh and for the final specification of retinal cell types and pattern (Kumar, 2001).

Regulation of Hh signal transduction as Drosophila eye differentiation progresses

Differentiation of the Drosophila retina occurs as a morphogenetic furrow sweeps anteriorly across the eye imaginal disc, driven by Hedgehog secretion from photoreceptor precursors differentiating behind the furrow. A BTB protein, Roadkill, is expressed posterior to the furrow and targets the Hedgehog signal transduction component Cubitus interruptus for degradation by Cullin-3 and the proteosome. Clonal analysis and conditional mutant studies establish that roadkill transcription is activated by the EGF receptor and Ras pathway in most differentiating retinal cells, and by both EGF receptor/Ras and by Hedgehog signaling in cells that remain unspecified. These findings outline a circuit by which Hedgehog signal transduction is modified as Hedgehog signaling initiates retinal differentiation. A model is presented for regulation of the Cullin-3 and Cullin-1 pathways that modifies Hedgehog signaling as the morphogenetic furrow moves and the responses of retinal cells change (Baker, 2009).

As the morphogenetic furrow crosses the eye disc, Ci155 accumulates most highly just anterior to the morphogenetic furrow, even though Hh is secreted posterior to the morphogenetic furrow. The sharp reduction in Ci155 as the furrow passes is associated with a switch from Cul1-dependent processing to Cul3-dependent degradation (Ou, 2002). The posterior eye expresses rdx, encoding a BTB protein that couples Ci 155 to the Cul3 pathway (Kent, 2006; Zhang, 2006). This study identified the signals that induce rdx and that process Ci155 in the posterior eye (Baker, 2009).

The induction of rdx transcription couples Ci155 processing to Cul3 (Kent, 2006; Zhang, 2006). rdx transcription is regulated by both Hh signaling and Ras signaling, and there were distinctions between cell types. The smo mosaic and hhts2 experiments show that Hh signaling is continuously required for rdx transcription in unspecified cells with basal nuclei. In the absence of smo, EGFR-dependent rdx transcription occurs in differentiating photoreceptor cells only, not in unspecified cells. The egfr mosaics show that EGFR is essential for rdx transcription in all cells except the R8 photoreceptor class. Thus, EGFR-dependent differentiation was sufficient to induce rdx in photoreceptors even without Hh signaling, but Hh was not sufficient to induce rdx anywhere without EGFR signaling, except for the R8 cells. Undifferentiated cells might require both the Ras and Hh signaling pathways to induce rdx because the level of Ras signaling is lower in unspecified cells than in differentiating cells of the ommatidia. Alternatively, there may be a combinatorial requirement for both pathways in unspecified cells (Baker, 2009).

There has been some discussion of whether proteolysis of Ci155 by Cul-3 is regulated directly by Hh, as is Cul-1 dependent Ci processing. The current studies provide no support for this idea. In all the genotypes examined, Ci proteolysis correlates with the expression of rdx, and the simplest explanation is that the only effect of Hh on the Cul-3 pathway is through rdx transcription, directly in unspecified cells, and indirectly via EGFR-mediated differentiation in most specified cells (Baker, 2009).

Two mechanisms, acting in different cells, appear to reduce Hh responses through Ci155 after the furrow passes. One also occurs in wing development, where rdx is transcribed only by cells experiencing high Hh signaling levels close to the source of Hh. In wing development, rdx and the Cul3-pathway modulate the amount of Ci155 available for Cul1-dependent processing, lowering the maximum level of Ci155 activity at high Hh levels. Rdx could lower Ci155 levels in unspecified eye cells posterior to the furrow by this mechanism, in which an equilibrium between Hh-dependent induction of rdx, and rdx- and Cul3-dependent degradation of Ci155, leads to a lower level of Ci155 protein than anterior to the furrow. By contrast, in the specified, differentiating eye cells, rdx transcription becomes independent of Hh signaling, and Ci155 is degraded more completely (Baker, 2009).

If there is Hh signaling posterior to the furrow, as these studies find maintains rdx transcription in unspecified retinal cells, why are genes such as atonal that are induced by Hh signaling ahead of the furrow not also expressed posterior to the furrow? There are at least three possible explanations. First, rdx may dampen Ci155 accumulation in unspecified cells such that the threshold necessary for ato expression is not achieved posterior to the furrow. This is unlikely to be the sole explanation, since mutating rdx or cul3 permits Ci155 accumulation but does not lead to ectopic R8 specification, but it could contribute in conjunction with other mechanisms. Secondly, other genes may interfere posterior to the furrow. This could include egfr induction of Bar gene expression, since Bar genes antagonize ato expression. There seem to be multiple respects in which EGFR-dependent differentiation renders cells unable to continue anterior responses to Hh, and it is also envisaged that egfr might play a role in further mechanisms that modulate the response to Dpp signaling posterior to the furrow, should such mechanisms exist. Finally, recent evidence suggests that induction of ato by Hh is not so simple as the induction of a target gene above a threshold in a morphogen gradient, but depends indirectly on Hh repressing Eyeless and activating Sine Oculis, so that these transcription factors are coexpressed and turn on ato only in a domain ahead of the furrow. In this case, persistent Hh signaling would not be expected to activate ato expression once Ey had been repressed (Baker, 2009).

Recently, Hh has been discovered to induce compensatory proliferation in response to eye disc cell death, a further example of post-furrow Hh function. The current results now suggest the model that loss of EGFR-dependent rdx expression elevates Ci155 locally to permit Hh responses when photoreceptor cells that secrete EGFR ligands are lost. Consistent with this idea, loss of rdx or cul3 also result in proliferation of eye disc cells (Baker, 2009).

The regulation of rdx expression and thus degradation of Ci by Cullin-3 may not be sufficient to explain Ci regulation posterior to the furrow. In order for Ci155 to be stable, as observed in cul3 mutant clones and egfr mutant clones, Ci155 must escape processing to Ci75 by Cul-1. Ahead of the furrow, and in most other tissues, rdx is not expressed, Ci is not coupled to Cul3, and Ci155 is stabilized wherever Hh inhibits Smo and the Cul1 pathway. The observation that Ci155 is stable in cul3 clones, or in the genotypes where rdx is not expressed, shows that Ci155 escapes processing by the Cul1 pathway in the posterior eye as well, but this is not due to Hh. Ci155 accumulates in smo egfr mutant clones that do not express rdx and cannot respond to Hh (Baker, 2009).

One model would be that once rdx is induced, Ci155 is sequestered and not available to be processed by Cul1. This model cannot explain why Ci155 accumulates in egfr clones that lack rdx expression, where Ci155 should be available for Cul1. Therefore Ci155 must escape Cul1-mediated processing in the posterior eye by a distinct mechanism. This could be explained by the induction of a component distinct from Rdx that inhibits the processing of Ci155 by Cul1, or sequesters Ci155. It is equally possible that a component essential for processing of Ci155 by Cul1 is repressed posterior to the morphogenetic furrow (Baker, 2009).

Previous studies show that Ci155 never accumulates in smo tkv clones that are unable to respond to either Hh or Dpp signaling. Clones of cells unable to respond to Dpp, but able to respond to Hh and Ras, show only a subtle change in Ci155 labeling. These previously published observations suggest that Ci155 remains a target of Cul1 in the absence of both Dpp and Hh signaling, perhaps through failure to transcribe or repress transcription of a gene that modulates Ci155 proteolysis by Cul1 posterior to the furrow (Baker, 2009).

It is now possible to account for why smo clones affect Ci155 levels differently from cul3 clones, a previously puzzling observation. In cul3 clones, or egfr clones that do not express rdx, the Cul3 pathway cannot degrade Ci155 and the Cul1 pathway is inactivated posterior to the furrow exactly as in wild type discs, so Ci155 accumulates. In smo clones, Ci155 transiently accumulates in those cells in which processing by Cul1 has been lost but rdx not yet induced. In such cells, Ci155 is not coupled to any cullin, and is stable. Eventually, differentiation spreads into the posterior of smo clones, leading to rdx expression, and Cul3-dependent Ci degradation. If differentiation and rdx expression are prevented, as in smo egfr clones, then Ci155 remains stable. Because there is a delay in expressing rdx in smo clones compared to wildtype, Ci155 is not subject to Cul3-mediated processing as soon as in wild type, and there is a period when Ci155 has been uncoupled from Cul1-processing but not yet coupled to the Cul3 pathway. It is during this period that Ci155 accumulates in smo mutant cells (Baker, 2009).

These findings help explain how a wave of differentiation moves across the eye disc uni-directionally. Hh, secreted from differentiating photoreceptor cells, must be present at highest concentrations posterior to the furrow. Indeed, ahead of the furrow Ci155 is stabilized in a decreasing posterior-to-anterior gradient, consistent with a gradient of Hh protein coming from a source posterior to the furrow. Yet, the cell-autonomous responses to Hh signaling that are seen ahead of the furrow, such as cell cycle arrest and atonal expression, do not occur posterior to the furrow, where Ci is rendered unstable by Rdx and Cul3, induced both directly by Hh itself, and indirectly by the photoreceptor differentiation that is largely induced by EGFR posterior to the furrow (Baker, 2009).

There are other examples where Hh-secreting tissues are not the targets of Hh signaling. For, example, in Drosophila wing development, anterior compartments respond to Hh secreted by posterior compartments, but posterior compartment cells do not respond because ci transcription is repressed by the posterior-specific protein Engrailed. In vertebrate development, notochord cells express Shh but the responses seen in the nearby spinal cord are not seen in notochord. Such segregation of Hh-producing cells from fields competent to respond to Hh makes sense, if the purpose of Hh signaling in development is to pattern new body regions. Hh signaling is also deregulated in many tumors. Whether any of these tumors activate Hh signaling by affecting GLI protein stability, or other normal down-regulatory mechanisms, remains to be seen. In any case, mechanisms that render cells unresponsive to Hh by coupling Ci155 to the proteosome might prove useful in the treatment of cancers that depend on Hh signaling (Baker, 2009).

Programmed cell death and context dependent activation of the EGF pathway regulate gliogenesis in the Drosophila olfactory system

In the Drosophila antenna, sensory lineages selected by the basic helix-loop-helix transcription factor Atonal are gliogenic while those specified by the related protein Amos are not. What are the mechanisms that cause the two lineages to act differentially? Ectopic expression of the Baculovirus inhibitor of apoptosis protein (p35) rescues glial cells from the Amos-derived lineages, suggesting that precursors are removed by programmed cell death. In the wildtype, glial precursors express the extracellular-signal regulated kinase (phosphoERK) transiently, and antagonism of Epidermal growth factor pathway signaling compromises their development. It is suggested that all sensory lineages on the antenna are competent to produce glia but only those specified by Atonal respond to EGF signaling and survive. These results underscore the importance of developmental context of cell lineages in their responses to non-autonomous signaling in the choice between survival and death (Sen, 2004).

Several lines of investigation have ascertained that the first cells to divide in the sensory lineages are the secondary progenitors: PIIa, PIIb and PIIc. The numbers of sensory cells undergoing division at different times in the developing antenna were estimated by staining mitotic nuclei with antibodies against phosphorylated histone. A peak of cell division was observed between 16 and 24 h after puparium formation (APF). It has been considered that only in those sensory lineages specified by Ato, PIIb produces a glial cell and a tertiary progenitor, PIIIb, which in turn divides to form the sheath cell and a neuron. In Amos dependent lineages, PIIb is believed to directly give rise to a neuron and a sheath cell. The difference between the two lineages could be entirely dependent on the nature of the proneural genes activated; Amos, for example, could direct a non-gliogenic lineage. Alternatively, the two proneural genes could specify similar division patterns but the glial cell precursor in Amos-lineages could be removed by PCD, resulting in non-gliogenic lineages (Sen, 2004).

To test the latter possibility, cell death profiles were examined in developing pupal antennae using the terminal transferase assay (TUNEL) and attempts were made to correlate the timing of PCD with cell division profiles discussed above. The appearance of TUNEL-positive cells peaked between 22 and 24 h APF consistent with the occurrence of PCD immediately after division of secondary progenitors (Sen, 2004).

TUNEL reactions were performed on 22-24 h APF antennae from lz-Gal4; UAS-lacZnls and ato-Gal4; UAS-lacZnls animals. Double labeling with antibodies against ß-galactosidase marked sensory cells arising from the Lz and Ato lineages. Lz::lacZ overlaps the regions of the antennal disc where amos expression occurs and labels all the basiconic and trichoid sensilla in the mature (36 h APF) antenna. Hence for the purpose of this study, all cells in which lz-Gal4 expresses will be considered to belong to the Amos-dependent lineages. ato-Gal4 drives reporter activity in proneural domains of the disc and subsequently in all cells of the coeloconic sense organs (Sen, 2004).

Most of the apoptotic nuclei observed during olfactory sense organ development co-localized with Lz::LacZ suggesting that death occurred mainly within the 'Amos-dependent' sensory clusters. Only very few TUNEL-positive cells were detected in regions where ato-lacZ expressed and these did not co-localize with the reporter expression. If PCD is the mechanism used to remove glial precursors from Amos lineages, then their rescue would be expected to result in additional peripheral glia in the antenna (Sen, 2004).

The GAL4/UAS system was used to target ectopic expression of baculovirus inhibitor of apoptosis protein (p35) to different cell types within the developing antennal disc. distalless981-Gal4 (henceforth called dll-Gal4), which drives expression in all cells of the antennal disc, resulted in the formation of >300 glial cells as compared to ~100 in the wildtype. Other sensory cells--neurons, sheath, socket and shaft cells--within sense organs were unaffected. Ectopic expression of p35 specifically in Ato lineages (ato::p35) did not alter glial number. This means that the `additional' glial cells rescued in dll::p35 must arise from lineages other than Ato. Mis-expression of p35 in Amos-dependent lineages using lz-Gal4, on the other hand, resulted in a significant increase in glial number. While other explanations are possible, it is believed that the somewhat lower number of glia obtained in lz::p35 as compared to dll::p35 could be accounted for by the strength of the P(Gal4) driver (Sen, 2004).

In order to identify the cell within the Amos lineage that is fated to die, the cellular events during development of sense organs were re-examined. At approximately 12-14 h APF, most sensory cells are associated in clusters of secondary progenitors. Two cells in each cluster -- PIIb and PIIc -- express the homeodomain protein Prospero (Pros). pros-Gal4;UAS-GFP recapitulates Pros expression at this stage and marks PIIb and PIIc and their progeny in all olfactory lineages. In the wildtype, a Repo-positive cell was associated with only a few of the total sensory clusters, these were all located within the coeloconic domain of the antenna. Targeted expression of p35 using pros-Gal4 increased glial number indicating that cells which are the progeny of either PIIb or PIIc could be rescued from apoptosis. In the pros-Gal UAS-2XEGFP/UAS-p35 genotype, a glial cell was associated with most clusters at 18 h APF rather than in Ato lineages alone (Sen, 2004).

In order to directly visualize the cell undergoing apoptosis, 22-24 h APF antenna from the neuA101 strain were stained with antibodies against ß-galactosidase to mark the sensory cells and with TUNEL. Sensory clusters located in basiconic and trichoid domains of the pupal antenna each had a single associated TUNEL positive cell. Since TUNEL reactivity data does not reflect the initiation of the death program, developing antennae were also stained at different time points with an antibody that recognized the activated caspase -- Drice. At 20 h APF, a single Drice-positive cell was found within each sensory cluster within the basiconic and trichoid domains of the pupal antenna. This cell also expressed low levels of Pros suggesting that it could arise from either PIIb or PIIc. This means that the PIIb/c in Amos lineages, like that in Ato, divides to give rise to a PIIIb and its sibling. The sibling in the former lineage was not previously detected because it expresses only low levels of Pros and soon dies. Since this cell is capable of expressing the glial-identity gene repo when rescued from death, it is denoted as a glial precursor (Sen, 2004).

How is apoptosis of a specific cell within the lineage regulated? In Drosophila three genes [reaper (rpr), grim and head involution defective (hid)] which all map under the Df(3L)H99 are necessary for the initiation of the death program. Heterozygotes of Df(3L)H99 show a small but significant increase in glial number over that of normal controls. hid-lacZ was used to follow expression during antennal development; reporter activity occurs at low levels ubiquitously including in glial cells. Levels of reporter expression indicate somewhat higher hid transcription in glia rescued by p35 mis-expression. The presence of Hid in the 'normal' glial precursors suggests a mechanism dependent on possible trophic factors to keep cells alive. In several other systems signaling, mainly through the EGFR pathway, results in an antagonism of Hid action and transcription. The sustained levels of hid transcription in the rescued glia, is not unexpected since inhibitors of apoptosis act by antagonizing a downstream event of caspase activation, rather than on Hid itself (Sen, 2004).

The peripheral olfactory glia ensheath the axonal fascicles as they project towards the brain. Antennae from animals deficient for ato (ato1/Df(3R)p13), lack a large fraction of glia; the ~30 which remain, appear to arise elsewhere and migrate into the third segment of the antenna somewhat later during development (Sen, 2004).

Is death the default fate for all glial precursors and are there signals that keep Ato-glia alive? Several studies have provided compelling evidence for the role of receptor tyrosine kinase signaling in glial survival. In order to test this in the olfactory glia, 14-16 h APF pupal antennae were stained with antibodies against Pros and the phosphorylated form of ERK (pERK). The PIIb and PIIc cells are recognized by expression of Pros, which becomes asymmetrically localized in PIIb during mitosis. At this stage sensory cells do not express pERK. pERK was detected in the daughter of the first secondary progenitor to divide (probably PIIb). Staining with anti-Repo antibodies shows that this cell is glial. Clusters showing pERK expressing cells were observed only in regions of the pupal antenna from where the coeloconic sensilla originate and not in the Amos-lineage sensory clusters. pERK expression decays rapidly as Repo rises; only occasional cells can be detected that show immunoreactivity to both (Sen, 2004).

The presence of pERK in nascent glial precursors is evidence for receptor tyrosine kinase pathway signaling in these cells. Since EGFR activation is a well recognized signal for glial survival, the role of this pathway in glial cell development was tested. In order to demonstrate a role for EGF signaling during glial formation in the antenna, a hs-Gal4 strain and carefully timed heat-pulses were used to drive transient expression of various antagonists of the pathway. Ectopic expression of a dominant negative form of human Ras (DN-RasN17) between 14 and 15 h APF, severely reduced the numbers of antennal glia. Similarly, expression of the inhibitory ligand Argos or a dominant negative construct of Drosophila EGFR (DN-EGFR) compromises glial development. Since abrogation of signaling could, in principle, affect other developmental events, antenna from 36 h APF pupae of relevant genotypes were stained with support cell and neuronal markers to ascertain that these treatments did not affect sense organ development generally (Sen, 2004) (Sen, 2004).

Interference with EGFR activity affects glial number, suggesting that signaling is required for glial development and/or survival. Cells in the Amos-lineage also produce glial precursors, which do not express pERK. This implies that Amos-lineage glial precursors fail to experience EGF signaling and are fated to die. This hypothesis could, in principle, be tested by constitutively activating the pathway in Amos lineages. These experiments were not possible to carry out since activation of EGFR at the time when Amos-dependent secondary progenitors were undergoing division resulted in pupal lethality. The spatial and temporal expression of Vein-lacZ and Sprouty-lacZ (Sty-LacZ) supports a role in development of glia and requires further genetic analysis. Vein-LacZ was first detected at 18 h APF and expression was elevated at 20 h APF in a domain of the pupal antenna occupied mainly by coeloconic sensilla. The fact that this is the region of the antenna from where most glia originate, is interesting in the light of data from other systems that demonstrate that Vein acts as gliotrophin (Sen, 2004).

The spatial and temporal pattern of Vein and Sty expression is consistent with a role for these ligands in glial development. The region where Vein is expressed shows high pERK activity suggesting activation of the EFGR pathway in the coeloconic (Ato) domains of the developing antenna. Sty, however, is present in the basiconic and trichoid (Amos) domains of the antenna. At 10-14 h, when the secondary progenitors have not yet divided, Sty expressing cells lie adjacent to the PIIb and PIIc; these cells are labeled by Pros. This localization is consistent with a role for Sty in antagonizing EGF activity in the secondary progenitors (Sen, 2004) (Sen, 2004).

This work provides one more instance where PCD plays a crucial role in the selection of a specific population of cell types during development although the mechanisms employed still need to be elucidated. How does the development context of lineages of cells within a single epidermal field together with non-autonomous cues result in distinct consequences? The Drosophila antenna is a valuable system to address these issues because of the diversity of morphologically and molecularly distinct cell types located in a highly stereotyped architecture and the wealth of reagents available for study (Sen, 2004).

Interaction between EGFR signaling and DE-cadherin during nervous system morphogenesis

Dynamically regulated cell adhesion plays an important role during animal morphogenesis. The formation of the visual system in Drosophila embryos has been used as a model system to investigate the function of the Drosophila classic cadherin, DE-cadherin, which is encoded by the shotgun (shg) gene. The visual system is derived from the optic placode which normally invaginates from the surface ectoderm of the embryo and gives rise to two separate structures, the larval eye (Bolwig's organ) and the optic lobe. The optic placode dissociates and undergoes apoptotic cell death in the absence of Shotgun, whereas overexpression of Shotgun results in the failure of optic placode cells to invaginate and of Bolwig's organ precursors to separate from the placode. These findings indicate that dynamically regulated levels of Shotgun are essential for normal optic placode development. Overexpression of Shotgun can disrupt Wingless signaling through titration of Armadillo out of the cytoplasm to the membrane. However, the observed defects are likely the consequence of altered Shotgun mediated adhesion rather than a result of compromising Wingless signaling, since overexpression of a Shotgun-alpha-catenin fusion protein, which lacks Armadillo binding sites, causes similar defects as Shotgun overexpression. The genetic interaction between Shotgun and the Drosophila EGF receptor homolog, Egfr, was studied. If Egfr function is eliminated, optic placode defects resemble those following Shotgun overexpression, which suggests that loss of Egfr results in an increased adhesion of optic placode cells. An interaction between Egfr and Shotgun is further supported by the finding that expression of a constitutively active Egfr enhances the phenotype of a weak shg mutation, whereas a mutation in rhomboid (rho) (an activator of the Egfr ligand Spitz) partially suppresses the shg mutant phenotype. Finally, Egfr can be co-immunoprecipitated with anti-Shotgun and anti-Armadillo antibodies from embryonic protein extracts. It is proposed that Egfr signaling plays a role in morphogenesis by modulating cell adhesion (Dumstrei, 2002).

The head ectoderm of early Drosophila embryos is subdivided into several domains that realize different morphogenetic programs. The embryonic eye field is the posterior-medial region of the procephalic neurectoderm that gives rise to the visual system, which includes the larval eye (Bolwig's organ) and adult eye, as well as the optic lobe. Around gastrulation, cells of the eye field undergo a convergent extension directed laterally. Shortly afterwards these cells form two morphologically visible placodes, one on either side of the embryo. These optic placodes sink inside and become the optic lobe primordia, epithelial double layers attached to the posterior surface of the brain. The optic placode of a stage 12-13 embryo is V-shaped, with the anterior leg of the V representing the anterior lip, which later forms the inner anlage of the optic lobe, and the posterior leg forming the posterior lip, later forming the outer anlage. As the invagination deepens and the two lips 'sink' inside the embryo, ectodermal cells that earlier surrounded the perimeter of the optic placode approach each other and eventually form a closed epidermal cover. Abundant cell death accompanies the closing of the head epidermis over the optic lobe anlage, and the subsequent separation of this anlage from the epidermis. A small number of cells that originally formed part of the posterior lip of the optic placode remain integrated in the head epidermis and form the larval eye or Bolwig's organ. As these cells move away from the optic lobe anlage their basal ends become drawn out and form axons that constitute Bolwig's nerve (Dumstrei, 2002).

Shotgun is expressed throughout the ectoderm including the eye field and its epithelial derivatives. One would expect that normal optic lobe development requires modulation of Shotgun activity to allow, for example, the segregation of the invaginating optic placodes from the surrounding ectoderm. Since cell culture studies have indicated that the mammalian EGF receptor can disrupt cadherin-based adhesion, it was of interest to see whether Drosophila Egfr is expressed in the visual system to allow for such a possibility in Drosophila as well. Egfr is expressed in a complex and dynamic pattern that closely parallels the pattern of double-phosphorylated ERK (dpERK) expression, indicating activation of the MAP kinase signaling pathway. During stage 7 both rho (an activator of Egfr signaling) and dpERK are expressed in two stripes in the head ectoderm. The expression of dpERK in these two stripes is the result of Egfr activity. The anterior stripe corresponds to part of the head midline, while the posterior stripe reaches into the eye field. Distribution of dpERK in the two stripes becomes patchy during stage 10. At the same time, the posterior stripe widens dorsally to overlap with part of the optic lobe placode. Finally, at the late extended germ band stage and during germ band retraction, dpERK becomes restricted to the optic lobe placodes and cells of the dorsal head midline. This expression pattern demonstrates that Egfr activation accompanies the determination, morphogenesis and differentiation of the embryonic visual system (Dumstrei, 2002).

On the subcellular level, Egfr is expressed diffusely on the membrane of epithelial cells and neuroblasts. Egfr overlaps with Armadillo, the Drosophila ß-catenin homolog, which is an integral component of the cadherin-catenin complex. Like Shotgun, Armadillo is concentrated strongly in the apically located zonula adherens but is also found at lower levels in the entire lateral membranes (Dumstrei, 2002).

A second type of junction, called a septate junction, develops in Drosophila epithelial cells at a slightly later stage than the zonula adherens. Septate junctions have been implicated in maintaining epithelial stability. The Coracle protein forms part of the septate junctional complex, and an antibody against Coracle serves as a sensitive marker for this junction. Applying this marker to embryos of different stages it was found that all ectodermally derived epithelia express Coracle, except for the optic lobe and the invaginations that form the stomatogastric nervous system. Accordingly, no septate junctions have been reported in previous electron microscopic investigations of these tissues. The reliance on adherens junctions alone may make the optic lobe (and stomatogastric nervous system) susceptible to changes in the stability of these junctions; such changes occur resulting from manipulations of Shotgun and Egfr (Dumstrei, 2002).

A finely adjusted level of Shotgun is required for optic placode morphogenesis, and ß-catenin, as well as EGFR signaling, is involved in this process. Reduction in Shotgun results in dissociation of the placode around the time when it normally invaginates, suggesting that the forces exerted on the epithelial sheet while folding may disrupt cell contacts. A similar phenotype was described for other epithelial invaginations, including the Malpighian tubules and stomatogastric nervous system. Abolishing Armadillo/ß-catenin function results in a similar, if somewhat weaker phenotype. If Shotgun is overexpressed, invagination is also impaired. Cells stay together in a placode-like formation (as would be expected from 'hyperadhesive' epithelial cells), but do not noticeably constrict apically. It should be noted that the interpretation of this failure of optic placode cells to constrict is complicated by the accompanying increase in cell death in surrounding head epidermal cells. This phenomenon, in addition to a direct effect of an increased amount of Shotgun in the optic placode cells, could be part of the pathology responsible for the non-invagination phenotype. By contrast, the non-disjunction of optic lobe and larval eye is likely to be a rather direct consequence of an increased amount of Shotgun expression. Interestingly, other adhesion systems, notably the Drosophila N-CAM homolog FasII, are also involved in optic lobe-larval eye separation. Thus, the down regulation of FasII by the 'anti-adhesion' molecule Beaten path is also required for normal larval eye morphogenesis (Dumstrei, 2002).

The findings suggest the Egfr pathway is involved in modulating cadherin-mediated adhesion and thereby controls morphogenesis. Egfr, similar to its function in the developing compound eye, is activated in the precursors of the larval eye and adjacent optic lobe at a stage preceding optic lobe invagination and larval eye separation. The ligand for Egfr is Spitz, which is activated by Rhomboid (Dumstrei, 2002).

In a small subset of larval eye precursors (the 'Bolwig's organ founders') loss of Egfr signaling results primarily in cell death, lending further support to the view that Egfr signaling functions generally in the ectoderm and its derivatives to maintain cell viability. Recent studies in Drosophila indicate that MAPK directly controls the expression and protein stability of the cell death regulator, Hid (W; Wrinkled). If cell death is prohibited by a deficiency of the reaper-complex, cells of the optic placode and most other embryonic cells that undergo apoptosis in Egfr loss-of-function mutants survive. Both optic lobe and Bolwig's organ express several of their normal differentiation markers, but show a characteristic 'hyperadhesive phenotype', consisting in the failure of optic Iobe invagination and Bolwig's organ separation. Based on the similarity of this phenotype to the one resulting from Shotgun overexpression, and the genetic interaction between Egfr and Shotgun mutants in the ventral ectoderm, it is proposed that Egfr activation is required in normal development to phosphorylate the cadherin-catenin complex and thereby allows optic lobe invagination and Bolwig's organ separation to occur. This would be in line with experimental results obtained in vertebrate cell culture studies, which have demonstrated that drug- or Egfr-induced phosphorylation of the cadherin-catenin complex leads to dissociation between cadherin-catenin complex and cytoskeleton. Recent findings have shown that another phosphorylation event, mediated by the rho/rac GTPases, also affects adhesion by dissociating alpha-catenin from the cadherin-catenin complex (Dumstrei, 2002).

Co-IP data indicates that Egfr is linked to the cadherin-catenin complex in Drosophila as well. This implies that the effect of Egfr on Shotgun mediated adhesion could be a direct one that occurs at the cell membrane and does not involve MAPK signal transduction to the nucleus. It has been shown in a number of vertebrate cell culture systems that tyrosine phosphorylation of ß-catenin results in a disassembly of the cadherin-catenin complex complex and a consecutive loss in cadherin-mediated adhesion. Phenotypically, this results in increased invasiveness of tumor cell lines, neuronal and growth cone motility. Several tyrosine kinases and phosphatases have been identified that can increase or decrease the degree of phosphorylation of the cadherin-catenin complex. For example, v-src transfected into cultured cells phosphorylates ß-catenin and causes cells to dissociate, round up, and become more motile. Egfr also phosphorylates the cadherin-catenin complex and forms an integral part of this complex. This opens up the possibility that growth factors, with their widespread expression and biological activity in the developing embryo, may exert part of their effect on cell behavior by modulating, in a rather direct way, cell adhesion at the membrane. Such a mechanism would account for the 'rapid mode' of control of adhesion molecules. Systems such as the optic placode of the Drosophila embryo, where matters of different cell fates are decided at the same time when morphogenetic movements change the arrangement and shape of the cells involved, constitute favorable paradigms to address how signaling systems control both processes (Dumstrei, 2002).

Fat/Hippo pathway regulates the progress of neural differentiation signaling in the Drosophila optic lobe

A large number of neural and glial cell cell types differentiate from neuronal precursor cells during nervous system development. Two types of Drosophila optic lobe neurons, lamina and medulla neurons, are derived from the neuroepithelial (NE) cells of the outer optic anlagen. During larval development, epidermal growth factor receptor (EGFR)/Ras signaling sweeps the NE field from the medial edge and drives medulla neuroblast (NB) formation. This signal drives the transient expression of a proneural gene, lethal of scute, and its signal array is referred to as the 'proneural wave', since it is the marker of the EGFR/Ras signaling front. This study shows that the atypical cadherin Fat and the downstream Hippo pathways regulate the transduction of EGFR/Ras signaling along the NE field and, thus, ensure the progress of NB differentiation. Fat/Hippo pathway mutation also disrupts the pattern formation of the medulla structure, which is associated with the regulation of neurogenesis. A candidate for the Fat ligand, Dachsous is expressed in the posterior optic lobe, and its mutation was observed to cause a similar phenotype as fat mutation, although in a regionally restricted manner. It was also shown that Dachsous functions as the ligand in this pathway and genetically interacts with Fat in the optic lobe. These findings provide new insights into the function of the Fat/Hippo pathway, which regulates the ordered progression of neurogenesis in the complex nervous system (Kawamori, 2011).

The Fat/Hippo pathway has been known as a tumor suppressor pathway. This study and in the report of Reddy (2010), it was shown that the loss of Fat/Hippo signaling causes a delay of NB differentiation in the optic lobe. In contrast, dachs;ft double mutation, which is expected to stabilize the Fat/Hippo pathway, causes an advance of NB differentiation. This led to the question of how the Fat/Hippo pathway controls NB differentiation (Kawamori, 2011).

It has been reported that EGFR/Ras signaling is necessary and sufficient for NB induction, and its transduction is the driving force of the progress of the proneural wave. EGFR/Ras signaling sweeps the NE field through the gradual activation of Ras and its downstream EGF secretion by Rho. It was reasoned that this signal transduction is the target of the Fat/Hippo pathway in the control of NB differentiation. Indeed, ectopic expression of the EGFR/Ras signaling components RasV12 and rho was sufficient to induce NBs in the ft mutant background (Kawamori, 2011).

Which step of this cycling process does Fat/Hippo pathway mutation affect? Based on ectopic expression experiments, the Fat/Hippo pathway lies upstream of Ras and Rho, and it is expected to control the process from EGF transmission to Ras activation. The phenotypic difference produced by RasV12 and rho overexpression should be noted. When rho was expressed in the ft mutant background, several NE clones with abnormal morphology remained. This phenotype was not observed when RasV12 was expressed. In this model, RasV12 drives NB differentiation in RasV12-expressing cells in a cell-autonomous manner. In contrast, Rho activates Ras signaling in neighboring cells through the secretion of an EGF ligand. Based on these phenotypic differences, the Fat/Hippo pathway is expected to control the cell-to-cell EGF transmission, including its secretion, distribution or reception at the cell surface. This hypothesis is supported by the fact that several signaling components of the EGFR/Ras pathway, including Rho and EGFR, are localized to the apical side of epithelial tissues, and it is thought that this signal is transmitted along the apical side in epithelial tissues. It has also been reported that Fat/Hippo pathway mutations enhance the expression level of several apically localized molecules, such as aPKC, PatJ, Crumbs and E-cadherin. Thus, Fat/Hippo signaling targets could include unknown apical components that are involved in EGF transmission and this could account for the incomplete NB induction by rho overexpression. rho-expressing cells secret the EGF ligand, which diffuses in the NE surface, but Fat/Hippo pathway mutation would prevent its cell-to-cell transmission and subsequent EGFR/Ras pathway activation in the receiving cells. In this hypothesis, EGF transmission would be disturbed in the NE mutant for the Fat/Hippo pathway, causing the delay of proneural wave progress (Kawamori, 2011).

As an alternative hypothesis, the Fat/Hippo pathway could regulate signal transduction from the EGFR to Ras activation. If this is the case, the Fat/Hippo pathway regulates the intracellular signal transduction of the EGFR pathway. Many of the known targets of the Fat/Hippo pathway are components of growth regulatory, cell survival and cell adhesion molecules. There could be unknown targets that modulate other signaling pathways, including the EGFR pathway, and the NB differentiation defect would thus be caused by a failure in the activation of differentiation signals in the absence of Fat/Hippo signaling (Kawamori, 2011).

This study shows that the Fat/Hippo pathway mutation also affected the morphological character of the NE. Fat/Hippo pathway mutant clones were induced, and they often included NE tissue with a folded morphology and disrupted the medulla structure. The results showed that the Fat/Hippo pathway functions in the regulation of NB differentiation and in NE morphology are distinct, but the two functions could affect each other. The morphological defect of the NE could affect EGFR/Ras signal transduction. The possibility is discussed that the EGF ligand could be distributed along the apical membrane of the NE. The invagination of the apical membrane of the folded NE into the inner region could prevent EGF ligand signaling. There were clones with a normal NE morphology in which NB differentiation was delayed and, thus, morphological defects are not determinate, but they could promote the delay of NB induction (Kawamori, 2011).

How is the activity of the Fat/Hippo pathway regulated throughout the development of the optic lobe? Ft is a member of the cadherin family, and an extracellular molecule is expected to regulate its activity. Ds is a candidate for the Ft ligand that regulates planar cell polarity and Fat/Hippo signaling activity in other epithelial tissues. The expression of ds with a posterior-specific pattern in the developing optic lobe (Reddy, 2010) was confirmed. In the rescue experiments for the ds mutation, the expression of either ds lacking its intracellular domain (dsΔICD) or ft lacking its extracellular domain (ftΔECD) was sufficient to compensate for ds function, suggesting that Ds functions as a ligand and that Ft lies downstream of Ds in this context (Kawamori, 2011).

The phenotypes of ds and ft mutants were compared to assess whether the mutation of ds by itself accounts for the phenotype of the ft mutants. In contrast to the ft mutants that exhibited altered NB differentiation in the entire outer optic anlagen, the ds mutant phenotype was regionally specific; NB differentiation was severely delayed in the posterior region, and the development of the anterior region was not significantly affected. These differences suggest that there might be some regulatory mechanisms that control Ft activity independently of Ds in the anterior region of the optic lobe (Kawamori, 2011).

The Fat/Hippo pathway is known as a tumor suppressor pathway, and many studies related to this pathway have focused on tissue growth or cell survival. This study has reported a new function of the Fat/Hippo pathway in the regulation of neural differentiation. The Fat/Hippo pathway regulates the progress of neural differentiation signaling, and the EGFR/Ras pathway is a candidate target of this pathway. The data suggest that the Fat/Hippo pathway includes unknown targets involved in EGFR/Ras signal transduction. Further studies are required to identify the targets of the Fat/Hippo pathway and determine the interplay between Fat/Hippo and EGFR/Ras pathways, specifically in NB differentiation (Kawamori, 2011).

Distalization of the Drosophila leg by graded EGF-receptor activity

Arthropods and higher vertebrates all possess appendages, but these are morphologically distinct and the molecular mechanisms regulating patterning along their proximodistal axis (base to tip) are thought to be quite different. In Drosophila, gene expression along this axis is thought to be controlled primarily by a combination of transforming growth factor-ß and Wnt signalling from sources of the ligands, Decapentaplegic (Dpp) and Wingless (Wg) in dorsal and ventral stripes respectively. In vertebrates, however, proximodistal patterning is regulated by receptor tyrosine kinase (RTK) activity from a source of the ligands, fibroblast growth factors (FGFs), at the tip of the limb bud. This study revises understanding of limb development in flies and shows that the distal region is actually patterned by a distal-to-proximal gradient of RTK activity, established by a source of epidermal growth factor (EGF)-related ligands at the presumptive tip. This similarity between proximodistal patterning in vertebrates and flies supports previous suggestions of an evolutionary relationship between appendages/body-wall outgrowths in animals (Campbell, 2002).

Wingless (Wg) and Decapentaplegic (Dpp) regulate proximodistal patterning in the Drosophila leg imaginal disc, and it was proposed originally that they do this indirectly by defining an organizer in the center: the presumptive tip of the leg, which would be the source of additional secreted signal(s). However, more recent studies suggest that Wg and Dpp act more directly in proximodistal patterning and may function as morphogens to activate genes such as Distal-less (Dll) and dachshund (dac): the more Wg and Dpp a cell receives, the more distal it becomes. At present, however, it is unclear how much of the proximodistal axis is actually specified directly by Wg and Dpp. This is important when considering appendage evolution because the legs of arthropod ancestors may largely correspond only to the distal region of Drosophila legs, the tarsus, which constitutes most of the Dll domain. This was revealed by removing Hox gene influence in legs and antennae, the supposition being that this would produce a 'ground state' appendage that would reflect the nature of the leg in an arthropod ancestor before leg patterning was modified by Hox gene activity. These ground-state appendages consist of a proximal region with an irregular morphology unlike that of an appendage, and a distal leg-like region consisting of an almost normally patterned tarsus (Campbell, 2002).

Initially, tests were performed to see whether Wg and Dpp directly pattern the proximodistal axis of the tarsus by determining their role in activation of the aristaless (al) gene in the center of the disc. al encodes for a homoeodomain protein required for development of structures found at the tip of the leg, including the claws. Previous studies indicated that al expression is activated by Wg and Dpp and this was confirmed with loss of function studies: al expression is absent from the center of wg and dpp mutant discs. However, this does not rule out al being activated by a secondary signal, which in turn is activated by Wg and Dpp. To test this, al expression was monitored in discs containing clones of cells mutant for genes required for transduction of Wg or Dpp signals, including arrow (arr), which encodes for a Wg co-receptor, and thickveins (tkv), which encodes a Dpp receptor (clone founder cells were generated before the onset of al expression). Central al expression is absent in discs consisting largely of arr mutant clones, but, as in wgts discs, such large clones would remove any putative secondary signal, and, in fact, further analysis revealed that al is still expressed in arr mutant cells located outside of the very center. Similarly, al can still be detected in tkv mutant cells. Thus, Wg and Dpp signalling are required, but not directly, to induce al, suggesting that it is activated by a secondary signal, which in turn is activated by Wg and Dpp (Campbell, 2002).

The EGF-receptor (EGFR) signalling pathway was tested as a potential activator of al because the claws are lost in Egfr hypomorphic mutants (the claws are also lost in al mutants. Use of a temperature-sensitive (ts) allele showed that Egfr is required for development of almost all of the tarsus. After a 24-h shift to the restrictive temperature during the first half of the third instar, Egfrts adults have leg truncations with the size of the truncation increasing with temperature. At lower temperatures only the most distal structures, the claws, are lost, but at high temperatures tarsal segments II–V are absent. Intermediate truncations result at temperatures between these extremes. These shifts affect only patterning of the tarsus, apart from at the highest temperature, 33°C, when development of more proximal regions is occasionally disrupted; this may reflect a low level EGFR signalling requirement for proliferation as has been shown in the eye disc. The temperature-dependent truncations of the tarsus suggest that development of the most distal region requires the highest level of EGFR activity, whereas more proximal regions require progressively less; that is, the tarsus may be patterned by a distal-to-proximal gradient of EGFR activity (Campbell, 2002).

Similar results were obtained analyzing marker gene expression in Egfrts discs, including al, Bar (B, expressed in segments IV and V) and rotund (rn, expressed in segments II–IV). Loss of EGFR activity results in loss of al, B and rn expression, but al is lost at a lower temperature than B, which in turn is lost at a lower temperature than rn, indicating that the more distal the marker, the higher the EGFR activity level required for expression. Clonal analysis with Egfrts showed that this response to EGFR is cell autonomous, and that again, al requires higher EGFR activity than B. It was not possible to do similar tests for rn because at temperatures above 31°C Egfrts clones do not survive in distal regions, raising the possibility that rn expression may be lost in Egfrts discs simply because of reduced growth or cell survival in this region. However, expression of other genes, including wg and dpp, appears normal in Egfrts discs. This is also true for the Wg and Dpp target Dll, clearly demonstrating that Wg, Dpp and Dll are not sufficient to distalize the leg (Campbell, 2002).

Ectopic activation of EGFR signalling results in autonomous, ectopic expression of al and B in mid-third-instar discs. Curiously, not all regions of the disc respond identically, with ventral regions being the most responsive and lateral regions the least. The reason for this is unclear but it indicates that factors in addition to EGFR may be regulating expression of tarsal genes such as al, at least outside of their normal domains. Only the presumptive tarsus is responsive to ectopic EGFR activity; this is most evident in adult appendages where no defects in patterning can be observed outside of here. Other regions may be refractive to ectopic EGFR activity because expression of tarsal genes requires Dll, which is expressed only in distal regions under Wg and Dpp control (Campbell, 2002).

In addition to activating genes, EGFR signalling is required to repress genes in distal regions, and again different genes appear to be differentially sensitive, with some, such as B and rn, possibly being both activated and repressed above different thresholds. B, rn and dac are repressed in the center of wild-type discs, with dac being repressed over a wider region than B and rn. Lowering EGFR activity in Egfrts discs to a level sufficient only for loss of al, results in expression of B and rn in the center, but not dac. Raising the temperature still further results in extension of the dac domain to fill the center. Clonal analysis shows that Egfr acts autonomously to repress dac. Ectopic EGFR activity can also repress B, dac and rn but again predominantly in ventral regions (B is repressed mainly at later stages). Previous studies have shown that repression of dac in distal regions requires high levels of Wg and Dpp signalling, so all three pathways appear to be required to achieve this (Campbell, 2002).

A distal-to-proximal gradient of EGFR activity predicts a source of ligand(s) at the presumptive tip. Potential ligands are the TGF-alpha family members Spitz and Keren, and the neuregulin, Vn; the former require activation by the membrane protein Rhomboid (Rho), or the homolog Roughoid (Ru). Both vn and rho are expressed in the center of the leg disc in early third instars. Genetic studies show that they are redundant so that loss of either gene alone has no effect on tarsus development, but loss of both together along with ru, which shows partial redundancy with rho even though no expression can be detected, has marked effects on leg patterning and growth. Large ru rho vn triple mutant clones can result in truncations of the tarsus, although these are never as extreme as in Egfrts mutants, possibly because of the difficulty of removing all ligand-expressing cells at the center of early leg discs using this technique, or because the ru mutant used is not null. Wild-type tissue located at the tip of adult legs always correlates with rescue of tarsal development. In addition, misexpression of a secreted form of Spitz results in non-autonomous activation of al. Verification of high levels of EGFR signalling in the distal leg is revealed by expression of sprouty in this location; this is upregulated in many tissues by EGFR signalling (Campbell, 2002).

Although a quantitative response to EGFR activity is important, an additional factor that can influence patterning of the tarsus is timing, revealed by shorter temperature shifts with the Egfrts mutant. A 12-h shift to 33°C in the first quarter of the third instar also results in leg truncations, although these are less severe than those produced by the 24-h shifts described above. However, a similar 12-h shift later during the second quarter of the third instar results in legs with normal distal pattern elements (claw, tarsal segment V), but with fusions or deletions of intermediate segments II–IV, suggesting that distal regions are specified before more proximal ones. This phenomenon mirrors the temporal pattern of gene expression in these regions in wild-type discs: the distal markers al and B are expressed in very early third instars whereas the intermediate tarsal marker, rn, cannot be detected until several hours later. The early 12-h shift can prevent all al and B expression, correlating with the pattern defect. However, the later shift does not result in loss of rn expression (although the domain is smaller than in wild-type discs. It is possible that the patterning defect resulting from the later shift may be caused by the loss of other, as yet unidentified, EGFR targets at this time. Alternatively, growth and patterning of this region requires segmentation of the tarsus, which is controlled by Notch signalling, so it is possible that EGFR signalling may be involved in regulating this process in the tarsus. In addition, these shifts are associated with cell death, which may also contribute to the defective patterning (Campbell, 2002).

The present results are consistent with the proposal that the tip of the leg is the source of EGF-related ligands and acts as an organizer to regulate proximodistal patterning of the tarsus, the evolutionarily 'ancient' portion of the Drosophila leg. This does not rule out a role for Wg and Dpp, but these proteins appear to be required only indirectly for regionalization of the tarsus, first to activate Dll during the first/second instar and then probably to activate rho and vn in the center of the disc. The resulting EGFR gradient is then used to establish the patterns of tarsal gene expression during the third instar. This can be compared to proximodistal patterning in vertebrates where the apical ectodermal ridge at the tip of the limb bud is the source of FGFs that are required for outgrowth and formation of the proximodistal axis -- both EGF and FGF receptors are RTKs. This similarity could indicate an evolutionary link between outgrowths from the body wall in animals, a possibility already revealed by the expression of Dll homologs in most of these outgrowths. This is not to imply that vertebrate and insect limbs evolved directly from a putative body-wall outgrowth in their last common ancestor, but that this putative outgrowth probably used a developmental genetic pathway involving Dll and distally expressed RTK ligands, which has subsequently been used in new ways in these two phyla and possibly elsewhere in the animal kingdom (Campbell, 2002).

Egfr/Ras pathway mediates interactions between peripodial and disc proper cells in Drosophila wing discs

All imaginal discs in Drosophila are made up of a layer of columnar epithelium or the disc proper and a layer of squamous epithelium called the peripodial membrane. Although the developmental and molecular events in columnar epithelium or the disc proper are well understood, the peripodial membrane has gained attention only recently. Using the technique of lineage tracing, it has been shown that peripodial and disc proper cells arise from a common set of precursors cells in the embryo, and that these cells diverge in the early larval stages. However, peripodial and disc proper cells maintain a spatial relationship even after the separation of their lineages. The peripodial membrane plays a significant role during the regional subdivision of the wing disc into presumptive wing, notum and hinge. The Egfr/Ras pathway mediates this function of the peripodial membrane. These results on signaling between squamous and columnar epithelia are particularly significant in the context of in vitro studies using human cell lines that suggest a role for the Egfr/Ras pathway in metastasis and tumor progression (Pallavi, 2003).

A significant finding of this study is the role of the peripodal membrane (PM) in wing/notum/hinge decision. The wing disc is initially divided into anterior and posterior compartments by virtue of En expression only in a subset of disc cells. Later, it is subdivided into three distinct groups of cells: wing, notum and the hinge. This is marked by the expression of Wg in the presumptive wing region -- Pnr in the presumptive notum and Tsh in the presumptive hinge. PM cells over the notum and the pouch may provide positional cues for notum/hinge-wing decision. The Egfr pathway functions in the PM to specify notum-specific genes and/or to inhibit wing-specific genes. Mis-expression of dominant negative Egfr (DNDER), DN-Ras, DN-Raf or Argos (Aos; a negative regulator of the Egfr pathway) in the PM is enough to induce notum/hinge-to-wing transformations. The dynamic expression pattern of Aos marks the spatial and temporal pattern of Egfr activation. In the second instar larval stage, when wing-notum decision is made, Aos is expressed specifically in those PM cells that overlay the posterior notum. Once the wing-notum decision is made, Aos expression recedes from PM cells and it starts expressing in the notum-associated myoblasts and in the pouch. Although, at this stage. the possibility cannot be ruled out that the Egfr pathway is required in both PM and DP cells to specify notum fate, the results suggest that the Egfr pathway mediates interactions between PM and disc proper (DP) cells during the notum/hinge specification (Pallavi, 2003).

Observations on notum/hinge-to-wing transformations in this report and elsewhere are restricted to the posterior compartment. However, ectopic expression of Wg can cause notum-to-wing transformation in both the anterior and posterior compartments. En is expressed in large numbers of PM cells that overlay part of the anterior compartment. Ubx, which is expressed only in the posterior compartment of T2 (parasegment 5), is expressed in all PM cells. In addition, overexpression of Hh in PM cells does not induce ectopic dpp-lacZ expression in those cells. These observations suggest posterior identity of all PM cells. Is this the reason for observed notum/hinge-to-wing transformations only in the posterior compartment? If the answer is yes, how is the notum specified in the anterior compartment? Further investigation is needed to determine the compartmentalization within the PM and compartment-specific interactions between PM and DP (Pallavi, 2003).

Although these results suggest a role for the PM in specifying notum development, further investigation is required to determine how the Egfr pathway is activated in PM cells. Does DP play a role in activating the Egfr pathway in PM cells, which in turn specifies notum development in the DP? This would be analogous to oocyte-follicle cell interactions, wherein Gurken expressed in the oocyte is responsible for the activation of Egfr pathway in the follicle cells. Subsequently, follicle cells signal to the oocyte, which results in the re-organization of the cytoskeleton of the latter. Identification of the ligand for Egfr in PM cells and the signal that goes from PM to DP may lead to the mechanism by which it is activated in PM cells. Because only PM cells over the notum send out microtubule extensions, any molecule that signals to DP to specify notum development may depend on these processes. Such microtubule extensions have also been observed in the eye disc and are shown to be required for proper signaling from PM to DP. Overexpression of a dominant-negative form of glued (DN-Glu), a component of microtubule-binding motor complex proteins in the eye disc PM causes delay in the progression of the morphogenetic furrow. However, no effect was seen of expressing DN-Glu in the wing disc PM using Ubx-GAL4 driver. The role, if any, of microtubule extensions in the wing disc would probably be independent of the motor complex. Further investigation is needed to identify signal molecules involved in PM to DP interactions (Pallavi, 2003).

Drosophila adult muscle precursors form a network of interconnected cells and are specified by the rhomboid-triggered EGF pathway

In Drosophila, a population of muscle-committed stem-like cells called adult muscle precursors (AMPs) keeps an undifferentiated and quiescent state during embryonic life. The embryonic AMPs are at the origin of all adult fly muscles and, as is demonstrated in this study, they express repressors of myogenic differentiation and targets of the Notch pathway known to be involved in muscle cell stemness. By targeting GFP to the AMP cell membranes, it was shown that AMPs are tightly associated with the peripheral nervous system and with a subset of differentiated muscles. They send long cellular processes running along the peripheral nerves and, by the end of embryogenesis, form a network of interconnected cells. Based on evidence from laser ablation experiments, the main role of these cellular extensions is to maintain correct spatial positioning of AMPs. To gain insights into mechanisms that lead to AMP cell specification, a gain-of-function screen was performed with a special focus on lateral AMPs expressing the homeobox gene ladybird. The data show that the rhomboid-triggered EGF signalling pathway controls both the specification and the subsequent maintenance of AMP cells. This finding is supported by the identification of EGF-secreting cells in the lateral domain and the EGF-dependent regulatory modules that drive expression of the ladybird gene in lateral AMPs. Taken together, these results reveal an unsuspected capacity of embryonic AMPs to form a cell network, and shed light on the mechanisms governing their specification and maintenance (Figeac, 2010).

In late Drosophila embryos, each abdominal hemisegment features six AMPs at stereotypical positions associated with differentiating muscle fibres. To better characterize these cells, tests were performed to see whether the Notch pathway, which is known to be required for generation of satellite cells from muscle progenitors and for keeping them ready to engage in muscle regeneration, is also active in AMPs. Analysis of a GFP reporter line, E(spl)M6-GFP, described as a read-out of the Notch pathway in Drosophila, revealed that it is co-expressed with Twist in AMPs. Also, transcripts of another Notch target, Him, specifically accumulated in AMPs. By testing several mesodermal cell markers, it was found that, in addition to Twist, two other transcription factors, Zfh1 and Cut, are expressed in all AMPs. Zfh1 expression in embryonic AMPs has been reported previously, whereas cut has been used to reveal a subset of AMPs associated with larval wing and leg imaginal discs. Despite expressing common markers, the AMPs are heterogenous and differ by the expression of muscle identity genes. For example, slouch (S59) and Pox meso are specifically expressed in ventral (V) AMPs whereas ladybird (lb) and Kruppel (Kr) display lateral (L) AMP-specific expression (Figeac, 2010).

To gain insights into AMP cell shapes and their behaviour, an E(spl) M6-GAL4 line was generated that recapitulates M6-GFP expression, and it was used it to drive a membrane-targeted GFP. It has been previously reported that AMPs are associated with the larval peripheral nervous system (PNS) and that in daughterless mutant embryos lacking all the larval sensory system, the final pattern of AMPs is deranged. This study showed that all embryonic AMPs are closely associated with both the PNS and the differentiated muscles, sitting either at the top of muscle fibres [LAMPs and dorsal (D) AMPs] or on their internal face [dorsolateral (DL) AMPs and VAMPs)]. In late embryos, the AMPs form a network of cells displaying irregular shapes and are interconnected by long cellular processes aligning PNS nerves. Connections between the AMPs initially form within the parasegments, but the AMPs very quickly send filopodia posteriorly and make contact with DLAMPs of the adjacent segment, thus interlinking all AMPs. In addition to the interconnected M6+/twi+ AMPs, a population of morphologically distinct M6+/twi- cells of unknown fate, located more internally in central and posterior regions of the abdomen, was identified (Figeac, 2010).

It has been reported that a subset of muscle progenitors divides asymmetrically and gives rise to numb-positive founder cells that undergo differentiation and to Notch-expressing AMPs. Through this pathway, six AMPs are born in each abdominal hemisegment. In contrast to founders, AMPs express the Notch target Holes in muscle (Him) and Zfh1, the Drosophila homolog of ZEB, both of which are able to counteract Mef2-driven myogenic differentiation. Interestingly, another general AMP marker, E(spl)M6, also corresponds to a Notch target, suggesting that Notch signalling could play an evolutionarily conserved role in muscle cell stemness. It operates not only in vertebrate satellite cells but also, as shown in this study, in Drosophila AMPs. Finally, it is reported that, similar to muscle progenitors, the AMPs are heterogenous and express different muscle identity genes, such as lb or slou. This strongly suggests that AMPs acquire a positional identity that makes them competent to form a given type of muscles during adult myogenesis. For example, the lateral AMPs expressing lb are at the origin of all lateral body wall muscles of the adult fly. In support of the specific positional identities of AMPs comes also the analysis of lame duck (lmd) mutant embryos known to be devoid of fusion-competent myoblasts (FCMs). In this mutant context, the number of Twi-positive and Zfh1-positive AMP-like cells is highly increased, while the number of Lbe- and Twi-positive LAMPs committed to the lateral lineage remains unchanged. Thus in the absence of lmd, some presumptive FCMs can adopt the AMP-like fate but they do not carry positional information transmitted by the identity genes such as lb (Figeac, 2010).

Based on the premise that the AMPs correspond to a novel population of transient stem cells, their shapes and behaviour were analyzed in living embryos carrying M6-GAL4 and UAS-GAP-GFP transgenes. Surprisingly it was found that shortly after their specification, the AMPs start to send cellular processes that align along the nerves of the PNS, with the result that, by the end of embryogenesis, all AMPs become linked together. Interestingly, the intersegmental connections are made via an intermediary M6+ twi- cell of unknown fate. In addition to this particular cell, which ensures the intersegmental link between AMPs, the embryos also contained other M6+ twi- non-neural cells of rounded morphology located more internally that were unconnected to the AMP cell network. The origin and identity of these cells remain unknown (Figeac, 2010).

Exploiting the possibility of following AMPs in vivo, test were performed to see how AMPs would behave if their connections were broken. Since the AMPs separated from the network by laser ablation changed shape and lost their normal positions, it is concluded that one important reason for which AMPs form a cell network is to keep precise spatial positioning. Based on the observation that AMPs send long cellular processes along the peripheral nerves, it is probable that nerves serve as a support for extending AMP cell protrusion. This possibility is supported by the abnormal pattern of AMPs observed in daughterless mutant embryos lacking the PNS and in embryos in which the PNS was affected by the Elav-GAL4 driven expression of the inducer of apoptosis, Reaper. PNS nerves might also represent a source of signals for AMPs such as Delta in order to maintain Notch activity. However, analysis of the lateral domain revealed that Delta expression was associated with the segment border muscle (SBM) precursor but not with the PNS neurons, indicating that Notch activity in lateral AMPs is regulated by Delta produced in the SBM rather than in nerves (Figeac, 2010).

Taking advantage from the restricted number of embryonic AMPs and the genetic tools available in Drosophila, a large-scale gain-of-function screen was performed to identify the genes involved in AMP specification. rho and other components of the EGF signalling pathway were found to be crucially required for both specification and maintenance of AMPs. Importantly, as reported by Krejci (2009), several components of EGF signalling are direct targets of Notch in AMPs, thus creating a link between the two signalling pathways. The high number of AMPs in EGFRCA and RAS gain-of-function contexts provides evidence that RAS signalling not only promotes muscle founder specification, but is also crucial for specifying AMPs when induced by EGF signals. Further support for a key role of the EGFR pathway is the identification of cells sending EGF to lateral AMPs and the demonstration of their role in AMP cell maintenance. It also turns out that the anti-apoptotic role of the EGFR pathway in Drosophila AMPs described in this study is conserved across evolution, since EGF signalling also promotes survival of vertebrate satellite cells (Figeac, 2010).

The evidence for a major role of the EGFR pathway in the specification and maintenance of AMPs raises important questions about EGF targets operating in these muscle-committed stem-like cells in Drosophila. lb genes have been shown to be required for specification of LAMPs, making them candidate targets of EGF signalling in the lateral region. This study shows that lb regulatory modules contain binding sites for ETS factors that act as EGFR effectors and goes on to demonstrate their crucial role in AMP enhancer activity. The proximity of the ETS binding sites and homeodomain binding sites in the AMP element suggests that an adapted spatial conformation of interacting factors is important in allowing simultaneous binding and thus maintenance of the lineage-restricted activity of this enhancer. Interestingly, the main difference between regulatory modules driving expression in differentiated muscle lineages versus regulatory modules that act in non-differentiated AMPs is the responsiveness of the latter category to extrinsic EGF signals. In opposition to this, this study found that intrinsic Mef2 inputs are sufficient to drive expression in differentiated muscle lineage. The ETS and Mef2-driven expression of these two distinct regulatory modules is positively regulated by lb, which is known to play a pivotal role in the specification of muscle lineages in the lateral domain. The specific expression of lb in a subset of AMP cells and of its ortholog Lbx1 in activated satellite cells suggests that similarities in genetic control of Drosophila and vertebrate muscle stem cells might extend beyond those discussed here (Figeac, 2010).

Egfr in oogenesis

The Drosophila EGF receptor (Torpedo/DER) and its ligand (Gurken) play roles in the determination of anterioposterior and dorsoventral axes of the follicle cells and oocyte. The roles of DER in establishing the polarity of the follicle cells were examined further, by following the expression of Egfr-target genes. One class of genes (e.g. kekon) is induced by the Egfr pathway at all stages. Kekon is a novel member of both the leucine-rich repeat and immunoglobulin superfamilies and is a target for Epidermal growth factor signaling in the dorsal-anterior follicle cells and other domains of Egfr signaling. Broad expression of kekon at the stage in which the follicle cells migrate posteriorly over the oocyte, demonstrates the capacity of the pathway to pattern all follicle cells except the ventral-most rows. This may provide the spatial coordinates for the ventral-most follicle cell fates. A second group of target genes (e.g. rhomboid (rho)) is induced only at later stages of oogenesis, and may require additional inputs by signals emanating from the anterior, stretch follicle cells. The function of Rho was analyzed by ectopic expression in the stretch follicle cells, and shown to induce a non-autonomous dorsalizing activity that is independent of Gurken. Rho thus appears to be involved in processing an Egfr ligand in the follicle cells, to pattern the egg chamber and allow persistent activation of the Egfr pathway during formation of the dorsal appendages (Sapir, 1998).

The expression of Kekon provides a sensitive reporter for Egfr activation, and clarifies the spatial and temporal aspects of dorsoventral patterning by the pathway. At stages 8-9, when the follicle cells migrate posteriorly over the oocyte, expression of Kekon is very broad. This pattern is induced along the two axes. Initially, posterior migration of the follicle cells over the source of high Gurken induces activation of the Egfr pathway in all follicle cells passing over the oocyte nucleus. Next, the lateral diffusion of the Grk signal leads to a symmetrical lateral activation, which decreases toward the ventral follicle cells. Consequently, expression of the marker is induced in all follicle cells, except in the ventral-most rows. This pattern has the capacity to define, by default, the fate of the ventral-most rows of cells (Sapir, 1998).

The next cycle of Egfr activation takes place at stage 10, when the follicle cells have completed their posterior migration. The induction of rho expression is critical, triggered by Gurken-mediated Egfr activation. Rho expression is essential for dorsoventral patterning, since expression of antisense rho in all follicle cells can lead to the generation of ventralized egg chambers and embryos (Sapir, 1998).

However it is implausible that this dorsoanterior expression domain can define the dorsal and ventral regions of the egg chamber. What then is the function of this wave of Egfr activation with respect to dorsoventral patterning? One possibility is that this phase has an additive effect to the previous activation of Egfr, which took place during follicle cell migration. The combined effects of both phases would determine the capacity of the follicle cells to become dorsal. Normally, activation of the Egfr pathway in the Rho-expressing cells does not seem to extend beyond these cells, as monitored by expression of the Egfr-target gene kekon. It is thus possible that relay mechanisms extend a second, unknown dorsalizing signal from the Rho-expressing cells to the more lateral and posterior follicle cells. In conclusion,, Rho-dependent signaling appears to be important for patterning the dorsal appendages. This is supported by the persistent expression of kekon and rho in the precursors of the dorsal appendages until the final stages of oogenesis, and by the induction of multiple dorsal appendages following ectopic Rho expression. Patterning of the dorsal appendages may thus represent another distinct Egfr-dependent process (Sapir, 1998).

While the kekon gene is induced at each phase of Egfr activation, rho expression is triggered only from stage 10 onwards. A mechanism must exist to prevent rho, and possibly other genes (e.g. bunched), from being triggered by the same pathway at earlier stages. One option is that induction by the DER pathway is not sufficient to trigger rho, and an additional input must be provided by a different group of genes from the stretch follicle cells. Dpp is expressed in these cells and may prove to be a likely candidate. Multiple requirements for triggering rho expression may thus assure that it will normally be induced at a restricted point in space and time, only when the Gurken-induced signal emanating from the oocyte nucleus can be combined with a signal originating from the stretch follicle cells (Sapir, 1998).

Intercellular signaling through the EGF receptor (EGFR) patterns the Drosophila egg. The TGF alpha-like ligand Gurken signals from the oocyte to the receptor in the overlying somatic follicle cells. In the dorsal follicle cells, this initial paracrine signaling event triggers an autocrine amplification by two other EGFR ligands: Spitz and Vein. Spitz becomes an effective ligand only in the presence of the multitransmembrane domain protein Rhomboid. Consequent high-level EGFR activation leads to localized expression of the diffusible inhibitor Argos, which alters the profile of signaling. This sequential activation, amplification, and local inhibition of the EGFR forms an autoregulatory cascade that leads to the splitting in two of an initial single peak of signaling, thereby patterning the egg (Wasserman, 1998).

Although Gurken is the only ligand previously reported to activate the Egfr during oogenesis, the requirement for Rhomboid in the follicle cells of the egg chamber led to an examination of the necessity for Spitz. Complete loss of Spitz function causes embryonic lethality, but spitz hypomorphs are viable. Adult females with reduced spitz lay eggs with a significant loss of the most dorsal tissue, implying that Spitz is indeed required for normal development of the egg. This phenotype was quantified by measuring the gap between the dorsal appendages: Their mean separation was found to be reduced in spitz hypomorphs; furthermore, in 20% of the mutant eggs the dorsal appendages were fused at the point of attachment, a phenotype never seen in wild-type eggs. Spitz could be required in the oocyte or in the somatic follicle cells that surround it. To examine this, germline clones of spitz null mutations were generated; in these, the oocyte is mutant but the follicle cells are wild type. This causes no defects, either in patterning the egg or in the viability or patterning of the embryos derived from such eggs. There is no requirement for the Egfr in the oocyte, only in the follicle cells. There is therefore an essential function for the Egfr ligand Spitz in dorsal-anterior patterning of the egg, and it is required only in the somatic follicle cells, where the receptor is also needed (Wasserman, 1998).

In other tissues Rhomboid appears to activate Spitz/Egfr signaling, leading to the suspicion that Rhomboid might mediate autocrine Spitz signaling in the follicle cells. Consistent with this idea, the phenotype caused by loss of Spitz from the follicle cells is similar to that caused by loss of Rhomboid. Expression of antisense rhomboid causes loss of dorsal tissue and fusion of the appendages in eggs from heat-shocked females expressing HS-as-rho. Unmarked follicle cell clones of a rhomboid null mutation also give fused appendage phenotypes; as with spitz clones, these range from mild to severe fusions. Like Spitz and the Egfr, Rhomboid is not needed in the oocyte, implying that it, too, is only required in the follicle cells (Wasserman, 1998).

spitz is expressed uniformly in all follicle cells (region 2 of the germarium) from very early in oogenesis through stage 13, when egg patterning is complete. No transcript can be detected in the oocyte, consistent with a lack of Spitz requirement. This expression domain coincides exactly with that of the Egfr itself. spitz expression in follicle cells is unaffected by Egfr signaling; in egg chambers from gurken or Egfr mutant mothers, spitz expression is unaltered. The same is true of egg chambers from fs(1)K10 mothers, which have ectopic Egfr signaling. These expression data show that Gurken/Egfr signaling does not affect Spitz transcription, implying that the dependence of Spitz signaling on prior Gurken signaling must be posttranscriptional. In contrast, rhomboid is expressed in a dynamic pattern in follicle cells and is dependent on EGFR signaling. At stages 9-10a of oogenesis, rhomboid is expressed in a central patch of the dorsal-anterior follicle cells: this resolves to a stripe of cells on either side of the dorsal midline by stage 10b. In the absence of Egfr signaling, rhomboid expression is lost and, conversely, it is ectopically expressed in fs(1)K10 egg chambers. These expression profiles of spitz and rhomboid are consistent with Gurken signaling from the oocyte activating the expression of rhomboid in the follicle cells. This may in turn allow Spitz to become an autocrine ligand in the follicle cells and thus establish an autocrine amplification of the initial paracrine signal. The expression of the neuregulin-like Egfr ligand vein was also examined. It is also expressed in two stripes of follicle cells at stage 10b. Interestingly, vein expression is dependent on Egfr signaling: it is ectopically expressed in fs(1)K10 eggs and absent from gurken null eggs, establishing another potentially important feedback mechanism. This suggests that the autocrine amplification of Egfr signaling also involves Vein, although in this case the feedback occurs by direct transcriptional activation of the ligand (Wasserman, 1998). vein expression has also been found to be dependent on Egfr signaling during embryogenesis (T. Volk, personal communication to Wasserman, 1998).

The expression of the secreted Egfr inhibitor, Argos, is dependent on Egfr signaling in many tissues. Consistent with this, argos is expressed in the dorsal-anterior follicle cells at the time when Egfr signaling occurs. At stage 11 the RNA is detectable in a single, T-shaped group of cells centered on the dorsal midline, and by stage 13, argos, like rhomboid and vein, is found in two groups of cells: one on either side of the midline. As elsewhere, argos expression is dependent on Egfr activation: in gurken mutant egg chambers it is lost, and it is ectopically expressed in fs(1)K10 egg chambers. Is argos expression dependent on Spitz amplification of Egfr signaling? An examination was performed to see if Spitz contributes to a signaling threshold required to induce argos expression. argos expression is normal in eggs from mothers with reduced Ras1, but when Spitz is halved, dorsal-anterior argos expression is abolished in most egg chambers. Therefore, there is indeed a threshold of Egfr signaling required to switch on argos, and both Gurken and Spitz participate in reaching this threshold (Wasserman, 1998).

The initial expression of argos at the dorsal midline led to the speculation that it might cause a reduction of Egfr signaling near the midline, thereby splitting the single signaling peak in two. The resulting twin peaks of Egfr activation would then specify the location of the dorsal appendages. A prediction of this model is that loss of Argos should remove inhibition of the Egfr at the midline and produce a single peak of signaling, leading to the formation of a fused appendage phenotype. The eggs from females with hypomorphic argos mutations were examined. A significant proportion of these eggs have a partially or, in the most severe cases, fully fused phenotype. The same fused appendage phenotype is observed in follicle cell clones of an argos null mutation. These data imply that there is a requirement for Argos in eggshell patterning and that, as with Spitz, Rhomboid, and the Egfr, this requirement is confined to the follicle cells (Wasserman, 1998).

It is proposed that Argos modifies the initial Egfr activation profile in the follicle cells, producing twin peaks of activity displaced from the midline. These specify the position of the dorsal appendages. Direct evidence for a transition from one to two peaks of signaling was obtained with an antibody that recognizes only the activated, diphosphorylated form of MAP kinase, a key member of the signal transduction pathway downstream of the receptor. At stages 9-10, there is a single domain of activated MAP kinase in the follicle cells, centered on the dorsal midline. By stage 11, two domains, are observed: one on each side of the dorsal midline. From their position, these cells correspond to the cells that will form the dorsal appendages. In Egfr hypomorphs, which have a fused appendage phenotype, the single peak of activated MAP kinase does not split in two. These results clearly demonstrate that Egfr signaling does indeed evolve from a single peak into twin peaks of activation. This is supported by examining the expression pattern of known Egfr target genes in the follicle cells. By stage 11 these targets (pointed, rhomboid, argos, vein, and Broad) are expressed in two dorsal anterior domains, one on each side of the midline. This is taken as additional evidence for twin peaks of Egfr activation. Earlier, pointed, rhomboid, and argos are all also detectable in a single peak at the dorsal midline (Wasserman, 1998).

Egfr signaling specifies the dorsoventral axis and patterns the eggshell. It is suggested that these two functions are controlled by temporally separate phases of Egfr activation. When amplification and splitting of Egfr signaling do not occur, eggs have only a single, fused appendage. Surprisingly, larvae emerge from these eggs at the frequency predicted by Mendelian principles, and those that emerge have no apparent dorsoventral defects. When follicle cell clones of a spitz null are induced, the hatching rate of eggs with fused appendages of 82% of the predicted number. Similarly, all of the predicted number of eggs with a single fused appendage hatch from mutant females. The same is true of eggs with fused appendages caused by follicle cell clones of argos null mutations. Therefore, disruption of the amplifying and splitting process does not perturb dorsoventral axis specification, implying that the initial Gurken signal to the Egfr is sufficient to specify the axis. The subsequent cascade of amplification and splitting then patterns the eggshell (Wasserman, 1998).

Early Drosophila development requires two receptor tyrosine kinase (RTK) pathways: the Torso and the Epidermal growth factor receptor (EGFR) pathways, which regulate terminal and dorsal-ventral patterning, respectively. Previous studies have shown that these pathways, either directly or indirectly, lead to post-transcriptional downregulation of the Capicua repressor in the early embryo and in the ovary. This study shows that both regulatory effects are direct and depend on a MAPK docking site in Capicua that physically interacts with the MAPK Rolled. Capicua derivatives lacking this docking site cause dominant phenotypes similar to those resulting from loss of Torso and EGFR activities. Such phenotypes arise from inappropriate repression of genes normally expressed in response to Torso and EGFR signaling. These results are consistent with a model whereby Capicua is the main nuclear effector of the Torso pathway, but only one of different effectors responding to EGFR signaling. Finally, differences in the modes of Capicua downregulation by Torso and EGFR signaling are described, raising the possibility that such differences contribute to the tissue specificity of both signals (Astigarraga, 2007).

Distinct activation patterns of EGF receptor signaling in the homoplastic evolution of eggshell morphology in genus Drosophila

Homoplasy is a phenomenon in which organisms in different phylogenetic groups independently acquire similar traits. However, it is largely unknown how developmental mechanisms are altered to give rise to homoplasy. In the genus Drosophila, all species of the subgenus Sophophora, including D. melanogaster, have eggshells with two dorsal appendages (DAs); most species in the subgenus Drosophila, including D. virilis, and in the subgenus Dorsilopha (represented by a single species; Drosophila busckii), have four-DAs. D. melanica belongs to the Drosophila subgenus, but has two-DAs, and phylogenetic analyses suggest that it acquired this characteristic independently. The patterning of the DAs is tightly regulated by epidermal growth factor receptor (EGFR) signaling in D. melanogaster. Previous studies suggested that a change in the EGFR signal activation pattern could have led to the divergence in DA number between D. melanogaster and D. virilis. This study compared the patterns of EGFR signal activation across the Drosophila subgenera by immunostaining for anti-activated MAP kinase (MAPK). This analysis revealed distinct patterns of EGFR signal activation in each subgenus that were consistent with their phylogenetic relationship. In addition, the number of DAs always corresponded to the number of EGFR signaling activation domains in two, three, and four-DA species. Despite their common two-DA characteristic, the EGFR signaling activation pattern in D. melanica diverged significantly from that of species in the subgenus Sophophora. These results suggest that acquisition of the homoplastic two-DA characteristic could be explained by modifications of the EGFR signaling system in the genus Drosophila that occurred independently and at least twice during evolution (Kagesawa, 2008).

This study examined the patterns of MAPK activation in the follicle cells during oogenesis in five, eight, and one species belonging to subgenera Sophophora, Drosophila, and Dorsilopha, respectively. The comparative analyses revealed that MAPK was activated in a very similar pattern at the initial stages in all species examined, regardless of the DA numbers that eventually formed on their eggshells. MAPK activation was first detected at the dorsal midline of the follicle cells in all of the examined species of the Drosophila genus at stage 9/10. In D. melanogaster, Grk is specifically localized to the dorsoanterior midline of the oocyte and activates EGFR signaling in the overlying follicle cells at this stage. Therefore, the initial activating mechanism for EGFR signaling in the follicle cells is probably conserved among the species of the Drosophila genus. In addition, a mathematical study predicted that changes in the amount and distribution of Grk protein in the oocyte could account for the formation of zero to four-DAs. Therefore, it remains possible that the level of MAPK activation in the follicle cells at stage 9/10 differs quantitatively with the number of DAs, because this analysis was mostly qualitative and did not provide the relative values of MAPK activation level (Kagesawa, 2008).

Although the activation patterns of MAPK in follicle cells were evolutionarily conserved in the Drosophila genus through stage 10A, they diverged significantly between subgenus Sophophora and the rest of subgenera from stage 10B. That is, Sophophora showed two 'L-shaped' MAPK activation domains, but the domains were 'V-shaped' in the other subgenera. However, among the species of each subgenus, the MAPK activation pattern was very similar, with a few exceptions in subgenus Drosophila exemplified by D. melanica, D. phalerata, and D. guttifera (Kagesawa, 2008).

Molecular phylogenetic analysis revealed that the subgenus Dorsilopha, which has the V-shaped pattern of MAPK activation, diverged from a common ancestor of subgenus Sophophora. This finding suggests that the two-L-shaped-domains pattern of MAPK activation is a trait derived from the V-shaped MAPK activation pattern. Furthermore, the two-DA characteristic and the two-L-shaped-domains MAPK activation pattern probably co-segregated early in the divergence of subgenus Sophophora. Therefore, it is conceivable that the events responsible for the change from the V-shaped to the two-L-shaped pattern of MAPK activation played a crucial role in the morphological evolution from four to two-DAs. However, it remains unclear whether the two-L-shaped pattern of MAPK activation is prerequisite for the activation domain of EGFR signaling to remain unseparated, which consequently results in the formation of two MAPK activation domains. It is also possible that the crucial step for the development of two-DAs is attributable to the lack of a mechanism for separating the MAPK activation domain into two regions in each lateral half at stage 12, instead of the two-L-shaped pattern of MAPK activation at stage 10B (Kagesawa, 2008).

The present results suggest that modifications in EGFR signaling, which independently occurred in different phylogenetic groups, were responsible for the homoplastic evolution of the two-DA eggshell. All of the Sophophora species tested in this study showed two L-shaped MAPK activation domains. In contrast, the species of subgenus Drosophila, except for the three-DA species, showed a V-shaped MAPK activation domain. By stage 10B, the MAPK activation of D. melanica conformed to the typical V-shaped pattern of subgenus Drosophila, as predicted from its phylogenetic status. However, after this stage, the pattern of EGFR signaling activation became distinctive among the species of subgenus Drosophila. In D. melanica, MAPK was not activated in the anterolateral regions at stage 12, which could account for the two-DA phenotype. Based on the comparison of MAPK activation patterns among the species of subgenus Drosophila, it is proposed that at least two alterations of MAPK activation patterns account for the absence of MAPK activation in the anterolateral regions. First, in D. melanica, the two linear domains of MAPK activation were shorter than those of the other subgenus Drosophila species at stage 10B, which could result in the lack of MAPK activation in the anterolateral regions at stage 12. Second, the single large MAPK activation domain failed to be divided into two domains in each lateral set of follicle cells at stage 12. However, it is difficult to determine which changes in EGFR signaling activity played crucial roles in the evolution from the four- to two-DA phenotype in these species. Therefore, it remains unclear whether the modifications of EGFR signaling responsible for the acquisition of the two-DA characteristic are similar between D. melanica and the species of subgenus Sophophora, although phylogenetic analyses indicated that these two modifications occurred independently (Kagesawa, 2008).

It has been shown that aos, a negative regulator of EGFR signaling, is expressed in the regions that are posteriorly juxtaposed to the MAPK activation domains in D. virilis. Similarly, other putative negative regulator(s) of EGFR signaling might be expressed in the triangular region at the dorsal midline, where EGFR signaling is suppressed. Based on these results, it has been proposed that these negative regulators of EGFR signaling might suppress MAPK activation in the middle part of its linear activation domains, resulting in the division of the MAPK activation domain into two in each lateral set of follicle cells. Applying this assumption to the other species of subgenus Drosophila, an absence of these inhibitors could be the reason why the MAPK activation domain is not split into two domains, but remains as a single large domain at stage 12 in D. melanica. Alternatively, these negative regulators of EGFR signaling might suppress the MAPK activation in the anterior part of the linear MAPK activation domains, consequently shortening these domains, at stage 10B in this species. These two possible modifications could result in the acquisition of the two-DA characteristic by D. melanica, although there may be other explanations (Kagesawa, 2008).

Although the developmental basis of homoplastic evolution remains largely unknown, previous studies have provided some insight into the underlying genetic mechanisms. Extensive studies on evolutionary modifications in the pattern of dorsal trichomes in Drosophila larvae revealed that changes in the expression pattern of a single gene, shavenbaby, underlie the observed convergent evolution. Similarly, changes in the cis-regulatory elements of the yellow gene are responsible for independently occurring modifications of similar body-color traits in the subgenus Sophophora. Therefore, evolutionary changes occurring in the same gene could account for the homoplastic evolution of animal morphology. In contrast, it was also reported that the evolution of similar body-color traits can also be achieved by different alterations in the functions or regulation of different genes. Thus, it is important to determine whether the two-DA characteristic was acquired by modifications occurring in the same gene or different genes, or by the same or different modifications of a common gene in D. melanica and the species of subgenus Sophophora. Further comparative studies on the expression patterns of genes that control EGFR signaling activity might provide the information needed to clarify this issue (Kagesawa, 2008).

During the homoplastic evolution of the two-DA characteristic, the expression pattern of rho diverged. However, it has been found that the functions of the rho enhancers were not altered significantly in the divergence of D. virilis and D. melanogaster. In addition, the previous results suggested that the global activity of trans-acting factors that regulates the activation of rho genes did change during the evolution of the four- and two-DA characteristics between these two species. Therefore, it is conceivable that changes in the transcriptional landscape, including divergences in the distributions and/or activation patterns of trans-acting factor(s), could give rise to the independent evolution of similar morphological traits. However, as reported for other examples of homoplastic evolution, it is also possible that modifications of the cis-regulatory elements of a gene that regulates EGFR signaling activity played a crucial role in the evolution of the two-DA phenotype in D. melanica and the species of subgenus Sophophora (Kagesawa, 2008).

Among the Hawaiian species of Drosophila, one, which lays eggs deep within the breeding substrate (e.g., tree or soil fluxes) has very long and thick DAs; the other species, which simply drop their eggs on surfaces, such as leaves, have extremely short and thin DAs. Therefore, the morphologies and numbers of DAs could be adaptations to specific environments. To investigate this issue, it will be important to know whether the homoplastic evolution of the two-DA characteristic is the outcome of adaptation to a common environment. It is interesting to speculate that D. melanica and the species of subgenus Sophophora lay their eggs in similar environments, with the result that these species all acquired a common two-DA phenotype (Kagesawa, 2008).

Pattern formation by a moving morphogen source.

During Drosophila melanogaster oogenesis, the follicular epithelium that envelops the germline cyst gives rise to an elaborate eggshell, which houses the future embryo and mediates its interaction with the environment. A prominent feature of the eggshell is a pair of dorsal appendages, which are needed for embryo respiration. Morphogenesis of this structure depends on broad, a zinc-finger transcription factor, regulated by the EGFR pathway. While much has been learned about the mechanisms of broad regulation by EGFR, current understanding of processes that shape the spatial pattern of broad expression is incomplete. It is proposed that this pattern is defined by two different phases of EGFR activation: an early, posterior-to-anterior gradient of EGFR signaling sets the posterior boundary of broad expression, while the anterior boundary is set by a later phase of EGFR signaling, distributed in a dorsoventral gradient. This model can explain the wild-type pattern of broad in D. melanogaster, predicts how this pattern responds to genetic perturbations, and provides insight into the mechanisms driving diversification of eggshell patterning. The proposed model of the broad expression pattern can be used as a starting point for the quantitative analysis of a large number of gene expression patterns in Drosophila oogenesis (Zartman, 2011).

The EGFR-mediated patterning of the follicular epithelium provides a striking example of how complex gene expression domains can be specified parsimoniously by a single pathway. A simple model is presented that explains the two-dimensional pattern of BR, a transcription factor that marks the cells contributing to the roof part of the dorsal appendages in Drosophila oogenesis. This model can be summarized as follows: the two BR domains are limited to the anterior domain by the earlier phase of EGFR signaling, which defines an anterior band of cells competent to express high levels of BR necessary for the formation of dorsal appendages. At a later stage of oogenesis, the level sets, i.e. lines of constant concentration, of the dorsoventral EGFR signaling gradient intersect with this competence zone, splitting the BR expression domain into two patches (Zartman, 2011).

In addition to rationalizing the wild-type pattern of BR, the model can be used to generate hypotheses regarding the patterning of eggshell morphologies in other Drosophila species. The number and size of dorsal appendages vary greatly across the phylogenetic spectrum, providing a mechanism of adaptation to the nature of the oviposition substrate. Since BR is a key regulator of dorsal appendage morphogenesis, one can expect that changes in the expression pattern of BR can provide a mechanism for the diversification of dorsal appendages. As a first step toward testing this hypothesis, the expression of BR was analyzed in mid-oogenesis, when the domains of elevated BR are first established (Zartman, 2011).

It was found that the BR pattern is dynamic and shows significant transitions during development; however, several stereotypic patterns at stage 10B of oogenesis emerge for species that have 2, 3 or 4 dorsal appendages. The number of contiguous BR patches is not equal to the number of dorsal appendages. At the same time, a clear pattern emerges regarding changes in the concavity of the BR domain. For species with two appendages, the BR patches are split along the dorsal midline (along the DV axis) and each of the two patches has a dorsal anterior boundary that is concave relative to the DV/AP coordinate system. Species with three dorsal appendages show a continuous BR patch that is convex. For four appendages, the curvature of the boundary appears to switch between concave and convex and then back to concave (Zartman, 2011).

Thus, the shape of the boundary of the BR expression domain diverges across species and may alternate between convex and concave (D. virilis) or may be simply convex (D. phalerata). With the change in concavity, the number of appendages also changes from 2 for Dm to 3 for Dp and 4 for Dv. A mathematical model can recapitulate qualitatively some aspects of the transition in BR expression, simply by varying the strength of the negative feedback and thresholds. One mechanism for converting a Dm pattern into a Dp pattern involves reducing the strength of inhibition (or the shape of the GRK source) and shifting the anterior/posterior pre-pattern in the posterior direction. A comprehensive comparison of the shape of the GRK source and the function of the feedback inhibitors across species will provide a further test of this model. At the same time, investigation of BR patterning in other species can establish the limits of the model (Zartman, 2011).

One of the most important questions for future work is how the quantitative changes in the expression pattern of BR give rise to discrete changes in the number of dorsal appendages. It is speculated that the local concavity of the BR pattern drives the temporal order of cell intercalations and specifies where the floor cells form a hinge that closes the forming tube. As such, changes in concavity could lead to mechanical 'instabilities' that further subdivide the BR cells into smaller domains to form extra tubular appendages. Interestingly, recent work by Boyle (2010) provides possible support for this model: that study found that genetic perturbations or laser ablation of the cells along the dorsal anterior boundary of the roof domain blocks tube formation. The next steps in increasing the scale of understanding regarding patterning and morphogenesis will require models that integrate geometry and mechanics with signaling dynamics, as well as quantitative approaches to validating model predictions (Zartman, 2011).

Evolutionary changes in TGFalpha distribution underlie morphological diversity in eggshells from Drosophila species

Drosophila eggshells display remarkable morphological diversity among species; however, the molecular origin of this structural diversification is mostly unknown. This study analyzed the dorsal ridge (DR), a lumen-like structure along the dorsal side of eggshells, from numerous Drosophila species. This structure varies in length and width across species, and is absent from D. melanogaster eggshells. DR formation with distinct spatiotemporal changes in Epidermal growth factor receptor (EGFR) activation, which acts as a key receptor in eggshell patterning. Changes in the distribution of the TGFalpha-like ligand Gurken (GRK), a crucial ligand for axis formation, underlies EGFR activation and DR formation in D. willistoni. Furthermore, this study demonstrates that GRK from D. willistoni rescues a grk-null D. melanogaster fly and, remarkably, it is also sufficient to generate a DR-like structure on its eggshell (Niepielko, 2014).

EGFR signaling promotes the identity of follicle stem cells via maintenance of partial cell polarity

Epithelial stem cells divide asymmetrically, such that one daughter replenishes the stem cell pool and the other differentiates. This study found that in the epithelial follicle stem cell (FSC) lineage of the Drosophila ovary, EGFR signaling functions specifically in the FSCs to promote the unique partially polarized state of the FSC, establish apical-basal polarity throughout the lineage, and promote FSC maintenance in the niche. In addition, a novel connection was identified between EGFR signaling and the cell polarity regulator, LKB1, which indicates that EGFR signals through both the Ras-Raf-MEK-Erk pathway and through the LKB1-AMPK pathway to suppress apical identity. The development of apical-basal polarity is the earliest visible difference between FSCs and their daughters, and these findings demonstrate that the EGFR-mediated regulation of apical-basal polarity is essential for the segregation of stem cell and daughter cell fates (Castanieto, 2014: PubMed).

Cell cycle withdrawal, progression, and cell survival regulation by Egfr and its effectors: Egfr coordinates partially independent processes as the eye differentiates

Receptor tyrosine kinases such as the EGF receptor transduce extracellular signals into multiple cellular responses. In the developing Drosophila eye, Egfr activity triggers cell differentiation. This study focuses on three additional cell autonomous aspects of Egfr function and their coordination with differentiation, namely, withdrawal from the cell cycle, mitosis, and cell survival. Whereas differentiation requires intense signaling, dependent on multiple reinforcing ligands, lesser Egfr activity maintains cell cycle arrest, promotes mitosis, and protects against cell death. Each response requires the same Ras, Raf, MAPK, and Pnt signal transduction pathway. Mitotic and survival responses also involve Pnt-independent branches, perhaps explaining how survival and mitosis can occur independently. These results suggest that, rather than triggering all or none responses, Egfr coordinates partially independent processes as the eye differentiates (Yang, 2003).

Signal transduction was evaluated downstream of the Egf receptor in Drosophila eye development. The developing Drosophila eye permits the study of cell populations with single-cell resolution because the fates of even neighboring cells differ and because a temporal gradient of differentiation, spreading across the retina from posterior to anterior over approximately 2 days, means that many sequential developmental stages are arrayed in each experimental preparation (Yang, 2003).

Egf receptor activation behaves as an all or none switch to trigger differentiation of retinal cells. Distinct cell types are recruited sequentially. The specific fate chosen depends on combinations of EGFR signaling with other classes of receptors and with the current competence of the recruited cell. For example, the activity of another receptor, Notch (N), is required as well as Egfr for cone cell specification. N and the RTK Sevenless (Sev) are required along with Egfr for R7 cell specification, whereas R1 and R6 cells require Egfr, but not Sev or N (Yang, 2003).

Egfr plays additional roles in cell survival, proliferation, and cell cycle arrest. These are cell autonomous responses to Egfr activity within each cell, not indirect effects of changed differentiation pattern in response to altered Egfr signaling. Each cell can make survival, cell cycle, or differentiation responses independently. Individual cells may divide with or without differentiating; survive with or without dividing, and divide but live or die. Such flexibility might arise from combinatorial action of Egfr with survival and mitotic inputs from other receptors, analogous to combinatorial fate specification. Alternatively, Egfr signals might be transduced by effectors other than the Ras/Raf/MAPK cascade to permit different kinds of response (Yang, 2003).

Focus was placed on three aspects of Egfr function that occur at two developmental stages. These are the withdrawal from the cell cycle that accompanies the first fate specifications and the mitosis and survival of cells that pass unspecified through a 'second mitotic wave' (SMW) before later recruitment to retinal cell fates. The onset of advancing differentiation is defined by a morphogenetic furrow, which sweeps anteriorly from the posterior part of the eye imaginal disc. Just anterior to the morphogenetic furrow, cells arrest in G1 of the cell cycle. Within the morphogenetic furrow some of the arrested cells are specified as individual R8 photoreceptor cells, the founders of each ommatidium. Each R8 cell then produces ligands that activate Egfr in four neighboring cells. These neighbors are recruited to become the R2, R3, R4, and R5 photoreceptor cells of each ommatidium. While these five cells maintain their G1 arrest and differentiate in response to Egfr activity, the surrounding cells in which Egfr is inactive reenter the cell cycle and begin S phase DNA synthesis. Entry into this SMW occurs around retinal column 1, also the stage at which 'preclusters' of R8, R2, R3, R4, and R5 cells first become morphologically recognizable. The SMW produces unspecified cells that will be recruited to take the remaining 14 retinal cell fates. Egfr is required for survival and G2/M progression of SMW cells as well as later cell fate specification (Yang, 2003).

R2-5 differentiation requires the Egfr ligand Spitz (Spi) and the Ras/Raf/MAPK pathway. Spitz is not required to maintain R2-5 in G1, even though experiments with a temperature-sensitive allele show that Egfr is required simultaneously to maintain G1 arrest and to initiate differentiation. Any function of the neuregulin-like Egfr ligand Vein (Vn) in cell cycle withdrawal was ruled out from the study of spi; vn double mutants. The role of another zygotic ligand, Keren (Krn), was evaluated through mutations in rhomboid1 (rho) and rhomboid3 (a.k.a. roughened [ru]), two proteases required to cleave transmembrane precursors of the Keren and Spitz proteins. Cell cycle progression was assessed in clones of homozygous ru; rho1 double mutant cells with Cyclin B as a marker. Cyclin B protein accumulates in cells that have passed the G1/S checkpoint but not yet divided. All cells except R8s reentered the cell cycle in ru; rho1 double mutant clones. It is inferred that Krn must be necessary and sufficient to maintain R2-5 cells in G1 in the absence of spi (Yang, 2003).

To investigate the difference between Egfr activation by Spi and by Krn, whether the Ras/Raf/MAPK/Pnt pathway is required to maintain G1 arrest was examined. Mosaic analysis of null alleles was used. For Ras and Raf the result was the same as that for Egfr. All these genes are required to prevent cells from reentering the cell cycle. It was not possible to generate clones of cells lacking MAPK using the FLP/FRT system for mosaic analysis because of the centromeric location of the MAPK gene rolled proximal to all FRT transgene insertion sites. The transcription factor Pnt is the next component downstream in photoreceptor differentiation. Only R8 cells remain in G1 in cells deleted for the entire pnt gene. Thus, keeping R2-5 in G1 requires Egfr signaling through the same Ras/Raf/Pnt pathway as taken during differentiation (Yang, 2003).

Since spi activates the Ras/Raf/MAPK/Pnt cascade, it was asked whether Spitz could also maintain cells in G1. A secreted Spi protein (sSpi) that does not require Rho function was overexpressed to test this. The GMRGal4 driver was used to target expression posterior to column 1 of the eye disc. The GMR>sSpi eye discs lacked any secondary mitotic wave (SMW). Neither mitotic figures nor cells reentering the cell cycle were seen. Thus, not only does maintaining R2-5 cells in G1 require the same effectors as differentiation, but the differentiation ligand Spi can also maintain G1 arrest (Yang, 2003).

Since signaling for cell cycle withdrawal and differentiation appears qualitatively similar, whether different levels of signaling were required was tested. Two hypomorphic Egfr mutant alleles, l(2)05351 and l(2)03033, each associated with insertions in Egfr regulatory sequences, were tested. Since neither insertion disrupts Egfr coding sequences, expression of the Egfr protein must be reduced. Differentiation of photoreceptors other than R8 was 95% abolished in clones mutant for l(2)05351. By contrast many cells remained in G1 in addition to the R8 cells. Similar results were obtained with l(2)03033. These results suggest that maintaining G1 arrest requires lower-level Egfr activity than does R2-5 differentiation (Yang, 2003).

In order to test whether differentiation requires more activity of Pnt than maintaining G1 arrest, small deletions were used that specifically mutate one of the two pnt transcripts that encode alternative forms of Pnt protein. Both transcripts encode proteins that bind to the same DNA sequences, but Pnt-P1 is a constitutive activator of transcription, while Pnt-P2 requires MAPK phosphorylation for activity. In clones mutant for Pnt-P1, only 4% of R2-5 differentiation remained, but three times as many cells remained in G1. This indicates that, in the absence of Pnt-P1, Pnt-P2 is able to maintain some cells in G1 but rarely triggers their differentiation. In clones mutant for Pnt-P2, R8 cells were the only cells to differentiate, but other cells remained in G1, in addition. Thus, in the absence of Pnt-P2, Pnt-P1 is able to maintain some cells in G1 but did not trigger their differentiation. These findings indicate that both Pnt-P1 and Pnt-P2 proteins are necessary for normal levels of G1 maintenance and R2-5 differentiation but that Pnt proteins are required more stringently for differentiation than for G1 arrest (Yang, 2003).

These findings demonstrate that Egfr signaling through Ras and Raf to Pnt can trigger different responses at different activity levels. Increased Egfr activity increases the activity of nuclear Pnt proteins to promote differentiation, in addition to G1 arrest. The level of Egfr signaling of each cell depends on multiple activating ligands (Yang, 2003).

A further phase of Egfr signaling begins about 6 hr later. Differentiating R2-5 cells provide an additional source of signals that now activate Egfr in surrounding unspecified cells. Egfr is required for such cells to progress from G2 phase into mitosis of the SMW and, subsequently, for survival and postmitotic cell fate specifications. G2 arrest in the absence of Egfr is not due to a prior defect in S phase DNA synthesis because it occurs independently of mei-41. mei-41 encodes an ATM homolog required for G2 arrest of cells with damaged DNA (Yang, 2003).

In contrast to the G1 arrest and differentiation of R2-5, G2/M progression is less synchronous, and cells are not specified simultaneously. Most mitosis occurs between columns 3 and 5. Cells recruited to the R1/6 fates begin to differentiate immediately. Twenty percent of the mitoses occur later and overlap with Egfr roles in survival as well as recruitment of R7 and cone cells (Yang, 2003).

Because mitosis often occurred earlier than differentiation, whether mitosis also depends on the Ras/Raf/Pnt pathway was investgated. It was critical to distinguish between direct and indirect effects of ras, raf, or pnt mutations. Indirect effects were anticipated because R2-5 cells are a source of Egfr ligands, so mutations that affect R2-5 differentiation will eliminate ligand sources and affect other cells indirectly. If Ras, Raf, or Pnt directly transduces mitotic signaling by Egfr, the respective gene should be required cell autonomously in the dividing cells; effects on ligand production will affect mitosis nonautonomously. An indirect effect on mitosis is illustrated by ru; rho-1 mutant clones, in which most cells remained in G2 arrest posterior to column 3, but both cells in early mitosis with nuclear Cyclin B and postmitotic cells lacking Cyclin B were seen near boundaries with wild-type cells. rho-1 and ru activities are not required autonomously in the mitotic cells themselves, but nonautonomously to activate ligands for mitotic signaling (Yang, 2003).

Cells in ras or raf mutant clones retain premitotic Cyclin B levels, and no mitoses are detected, showing that these mutant cells are cell autonomously arrested in G2 phase. Many cells null for pnt function also retained Cyclin B. In contrast to ras or raf, however, mitotic and postmitotic pnt mutant cells were sometimes observed. Histone H3 phosphorylation was assessed to confirm mitosis of pnt mutant cells. Whereas ras or raf mutant cells never label for phosphorylated H3, labeled mitotic cells are seen in pnt mutant clones at ~40% of the frequency of control, wild-type twin clones. It is concluded that pnt promoted mitosis but is not essential (Yang, 2003).

If the Egfr/Ras/Raf pathway could bypass Pnt to regulate G2/M progression, it would be expected that Ras activated by the Val12 mutation would induce mitosis in clones of cells null for pnt. As predicted, RasV12 expression increases the number of mitotic cells in pnt mutant clones, as expected if pnt-independent mitosis were a target of Egfr signaling. In these experiments mitosis could not be an indirect effect of Ras activation of photoreceptor differentiation because photoreceptor differentiation requires pnt activity (Yang, 2003).

The pathways downstream of activated RasV12 were explored with mutations in the Ras effector loop. Mitosis was also increased by RasV12S35, but not by RasV12G37 or RasV12C40. This was consistent with activation of Raf being the relevant target of Ras, since only RasV12S35 permits full activation of Raf (Yang, 2003).

Does Pnt have any direct role in mitosis? Although the mitotic index is reduced in pnt mutant clones, reduced ligand secretion due to absent R2-5 cells might account for this. However, wild-type regions barely elevate the mitotic index in adjacent pnt mutant cells, and mitoses is often delayed, even when RasV12 or RasV12S35 is expressed to restore the mitotic index in pnt mutant clones. These observations suggest that both Pnt and another pathway downstream of Raf contribute to mitosis (Yang, 2003).

During the SMW, Egfr becomes required for survival of both pre- and post-mitotic unspecified cells. The requirement begins around column 6, while mitosis and differentiation are ongoing about 8 hr later than in column 1. Despite the common dependence on Egfr, mitosis does not always predict survival, and some cells survive without dividing or differentiating. It is thought that Egfr promotes survival through the Ras/Raf/MAPK pathway and antagonizes the proapoptotic gene hid (Yang, 2003).

Ras, Raf, and Pnt should act cell autonomously if they promote survival directly. They could still affect survival nonautonomously if Egfr promotes survival through a distinct transduction pathway, since photoreceptor differentiation would be altered. Autonomy of gene function for survival in clones of mutant cells was assessed using an antibody called CM1, which reacts with the activated caspase ICE from Drosophila and thus labels cells deficient in survival signals. Expression of p35 was used to block apoptosis and thus to preserve the arrangement of cells lacking survival signals. The cell autonomous apoptosis of ras, raf, and pnt mutant cells is seen, indicating that, like Egfr, ras, raf, and pnt are required directly for cell survival, not indirectly through their role in photoreceptor differentiation and ligand production. Caspase activation begins 2-3 hr later in pnt mutant clones than in egfr, ras, or raf clones, suggesting that a pnt-independent pathway downstream of Raf can postpone apoptosis. Consistent with this, pnt mutant clones are often larger than egfr, ras, or raf clones (Yang, 2003).

Why do some cells perform mitosis yet fail to survive? One key question is whether any cell death is unrelated to Egfr. Previous work establishes that lack of the Egfr pathway is the cause of apoptosis in cells mutated for Egfr pathway components. By contrast it is less certain why some wild-type cells die in normal development. Such death may reflect inadequate Egfr activity or loss of another essential pathway (Yang, 2003).

To investigate why cells die in normal development whether activation of the Egfr pathway restored survival was tested. Apoptosis is completely suppressed when activated RasV12 or RasV12S35 is expressed posterior to column 1 with the GMRGal4 driver. This is consistent with low Ras and Raf activity as the cause of normal cell death. Since photoreceptor differentiation is dramatically increased, however, this is equally consistent with release of another signal by photoreceptor cells. Conditions were therefore sought where Egfr signaling was elevated without inducing excess differentiation (Yang, 2003).

Elevated Egfr expression reduces normal apoptosis by 50% without inducing differentiation, supporting the notion that cell death occurs because cells lacked Egfr activity. RasV12 carrying a second G37 mutation rescues all eye disc apoptosis without inducing differentiation. In Drosophila, RasV12G37 has reduced capacity to activate Raf and predominantly activates PI3Kinase. PI3K activity was manipulated directly to distinguish whether PI3K or Raf is relevant to survival. Expression of the catalytic subunit Dp110 does not reduce cell death, as it should have if death were due to inadequate PI3K activity. Expression of a dominant-negative variant of the adaptor protein p60 does not increase cell death, consistent with previous conclusions that the Insulin Receptor/PI3K pathway is dispensable for retinal cell survival. For a more sensitive assay, the same genotypes were examined 2-3 days later, when many cells of the pupal retina undergo apoptosis due to inadequate Egfr activity. Both elevated Egfr expression and RasV12G37 reduces cell death. Ectopic photoreceptor cells were observed, consistent with Raf-mediated differentiation after such sustained expression. Elevated expression of the Drosophila Insulin Receptor also reduces cell death and promotes differentiation in both eye discs and pupal retina. These observations indicate that, when ectopically expressed, both the Insulin Receptor and RasV12G37 promote cell survival as does Egfr, by activating Raf to compensate for inadequate endogenous Egfr activity (Yang, 2003).

Since differentiation, division, and survival affect overlapping, but distinct, sets of cells, the role of Egfr in each response must be distinct in some way. Differentiation requires higher levels of Egfr signaling than G2/M progression. It has been argued that survival requires less Ras activity than does differentiation. Ras acts cell autonomously through Raf and Pnt; a deficit in this pathway causes the cell death that occurs in the wild-type. If survival required less activity than does G2/M progression, this could explain the survival of most cells that remain undivided. It would be necessary to propose declining Egfr activity to account for the death of some cells that have already divided (Yang, 2003).

Survival and G2/M progression were compaired to determine whether they occurred with different thresholds. The expression of Egfr, RasV12G37, or InR reduces apoptosis and also increases the number of SMW mitoses, although not as rapidly as RasV12 expression. It appears that both mitosis and survival are achieved at lower signaling levels than differentiation, but some cell death often remains under conditions when G2/M progression is already stimulated. An Egfr activity level has not been found that promotes survival without mitosis or vice versa, should such a level exist (Yang, 2003).

These data provide some insight into cellular responses to RTK activity. Eye development shares features with the yeast pheromone response, where a MAPK cascade activates transcription of target genes in a graded fashion. Multiple outputs are thought to differ in pheromone dose response because of phosphorylation of multiple substrates by the MAPK Fus3p. By contrast Xenopus oocyte maturation exemplifies an all or none response. In Drosophila, simultaneous differentiation and withdrawal from the cell cycle of R2-5 cells provide an example of tightly coupled responses to Egfr activity, but the mechanism differs from that in Xenopus. Simultaneous differentiation and cell cycle withdrawal depend on enough exposure to ligands Spi and Krn to activate both responses reliably. The responses uncouple if inadequate ligand is present. Other outputs of the Egfr also depend, in part, on different thresholds, and differing dpERK levels exist in individual eye disc cells. Responses might not be separable if the Egfr/Ras/Raf/MAPK pathway provided only all or none output, as when variable progesterone levels are amplified to maximal dpERK levels in Xenopus oocyte maturation (Yang, 2003).

Egfr signalling and ommatidial orientation 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/ru 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).

All known cases of Egfr signalling in Drosophila are transmitted through the canonical Ras/Raf/MAPK pathway, and through a transcriptional output. The transcription factor Pointed is involved in most circumstances: PointedP2 is directly phosphorylated and activated by MAPK, and upregulates the expression of PointedP1; both factors mediate the transcription of downstream genes. In the case of rotation, which is envisaged as being a specialized case of cell motility or tissue remodelling, it seemed possible that Egfr signalling might influence the cytoskeleton directly, rather than exerting its effects by transcriptional control. Therefore whether a pointed hypomorph shows rotational defects was tested. Although many ommatidia show under-recruitment of photoreceptors, rotational defects are frequent in those ommatidia that are correctly specified, indicating that this function of the Egfr pathway relies on Pointed-mediated transcription (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 argosrlt (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 spiscp1 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 spiscp1. 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).

At what stage in ommatidial development does the Egfr control rotation? Several markers were used to look at rotation in the eye disc: anti-Bar, which stains R1 and R6, svp-lacZ, which is strongly expressed in R3 and R4 and more weakly in R1 and R6, and mDelta0.5-lacZ, which highlights R4 only. The first two markers enable visualization of the rotational angle during disc development, and the third shows which cell of the R3/R4 pair develops R4 fate, thus providing a marker for chirality. mDelta0.5-lacZ staining of discs misexpressing keren shows no defects in R3/R4 specification, which correlates well with the lack of chiral defects in the adults. Surprisingly, rotational defects are also very minor in the third instar disc. The vast majority of ommatidia reach 45° as expected, and by the back of the disc have turned to 90°. This is in stark contrast to the adult eye, in which approximately 28% have rotated less than 90°, and 6.2% less than 45°, as well as 65% being rotated greater than 90°. Occasional misrotations can be seen in the larval disc, but analysis shows that the frequency of these is not significantly different from WT. This result demonstrates that the eye defects seen in the adult must arise at a stage later in development than the third instar imaginal disc (Brown, 2003).

In order to try and determine when Egfr signalling affects rotation, two approaches were taken. First, eyes were examined at stages intermediate between the third instar larva and the adult. In the WT disc, ommatidia have reached 90° and stopped rotating several rows before the back of the disc. If disrupting Egfr signalling leads to a failure to stop rotation, then defects should be obvious by this stage. However, discs misexpressing keren looked indistinguishable from WT, even at the posterior of the disc, implying that the effects of perturbing Egfr signalling are only apparent later than 6 hours post-pupariation. By 30 hours post-pupariation, rotational defects are clearly visible in the retina, indicating that rotation becomes disrupted between 6 and 30 hours post-pupariation (Brown, 2003).

The second approach taken was to ask when Egfr signalling is required in order to influence rotation. HS-Gal4 driving a dominant-negative form of the Egfr was used to disrupt the pathway at specific times through development, and then the effects of these heatshocks in the adult eye were examined. The wave of rotational defects shown by these heatshock experiments can be used to deduce the time of susceptibility to loss of Egfr signalling. In the case of white prepupae, the band of misrotation spreads from approximately 14 rows from the posterior margin, to approximately 21 rows. At this stage, the morphogenetic furrow (which moves approximately one row every two hours) has progressed approximately 28 rows from the posterior. Given the likelihood of a delay between time of heatshock and expression of the dominant-negative construct, this suggests that the period of sensitivity to loss of Egfr signalling corresponds to approximately 10-15 rows behind the furrow. This coincides with the second 45° rotation: ommatidia reach 45° at approximately row 6, and 90° at approximately row 15-16. Data from other time points are remarkably consistent with this analysis, both in regard to the initiation and duration of susceptibility (Brown, 2003).

These results demonstrate that Egfr signalling is required for the maintenance through eye development of the correct orientation of ommatidia. It was speculated that rotation may rely at least partly on the adhesive properties of the cells. In an initial attempt to examine this hypothesis, genetic interactions between components of the Egfr pathway and various adhesion molecules were sought. A Star heterozygote, in which Egfr signalling is slightly reduced, was used as a background in which to look for interactions, because this phenotype is very weak, allowing any enhancement of rotational defects to be easily recognized. Halving the dose of alpha-laminin (wing blister) and the integrin ß subunit (myospheroid) does not modify the Star/+ phenotype. In contrast, alleles of E-cadherin (shotgun) shows a significant interaction with Star, with many more misrotated ommatidia. Under the strongest condition, there is also an enhancement of the rare misrecruitment defects seen in Star/+ eyes, but the enhancement of the rotational defect is independent of this by two criteria. First, the rotational defects were only measured in correctly specified ommatidia; and second, the weaker alleles of shotgun affected rotation without enhancing recruitment. On the basis of these results, it is concluded that the control of rotation by Egfr signalling is linked to cadherin-based adhesion (Brown, 2003).

A model that might account for these results is proposed that suggests that the role of Egfr signalling is to establish a 'locking' mechanism that ensures that ommatidia remain in their final orientation. Such a mechanism might be necessary to protect the ommatidia against positional disruption during later events in eye development. Signalling would therefore be required during or at the end of normal rotation in order to set in place this hypothetical 'lock', although defects might not arise until significantly later than this, when processes occur that would cause ommatidia to reorient in the absence of such a lock (Brown, 2003).

Egfr signaling is evolutionarily conserved and controls a variety of different cellular processes. In Drosophila these include proliferation, patterning, cell-fate determination, migration and survival. Evidence is provided for a new role of Egfr signaling in controlling ommatidial rotation during planar cell polarity (PCP) establishment in the Drosophila eye. Although the signaling pathways involved in PCP establishment and photoreceptor cell-type specification are beginning to be unraveled, very little is known about the associated 90° rotation process. One of the few rotation-specific mutations known is roulette (rlt) in which ommatidia rotate to a random degree, often more than 90°. rlt is shown to be a rotation-specific allele of the inhibitory Egfr ligand Argos; modulation of Egfr activity shows defects in ommatidial rotation. The data indicate that, beside the Raf/MAPK cascade, the Ras effector Canoe/AF6 acts downstream of Egfr/Ras and provides a link from Egfr to cytoskeletal elements in this developmentally regulated cell motility process. Evidence is provided for an involvement of cadherins and non-muscle myosin II as downstream components controlling rotation. In particular, the involvement of the cadherin Flamingo, a PCP gene, downstream of Egfr signaling provides the first link between PCP establishment and the Egfr pathway (Gaengel, 2003).

Since ommatidial rotation is a cell motility process requiring cytoskeletal rearrangements, it was of interest to determine if effectors of Egfr other than the Raf/MAPK cascade play a role in this process. The Ras GTPase, the main transducer of Egfr signaling, can utilize distinct effectors in different contexts. In addition to nuclear signaling, mediated by the Raf/MAPK/Pnt cascade, Ras can affect cell growth and cytoskeletal rearrangements via its effectors Rgl/Ral, Phospho-inositol-3-Kinase (PI3K) and Canoe, whose human homolog (AF6) is known as the critical partner of ALL1 in a chimeric protein associated with myeloid leukemia (Gaengel, 2003).

Several point mutations have been identified within the Ras effector loop (amino acids 34-41) that abrogate the binding to and activation by Ras of specific effectors. The specificity of the existing Ras-effector loop mutations has been thoroughly tested in Drosophila imaginal discs (Prober, 2002). RasV12[S35], able to interact with Raf in cell culture, can activate ERK/Rolled (via Raf) and induce Ras/Raf/ERK-specific transcriptional responses in wing and eye imaginal discs (Prober, 2002). In contrast, RasV12[G37], unable to bind Raf in cell culture, cannot activate these responses, but is still capable of activating PI3K-specific read-outs (Prober, 2002). Therefore the Ras effector loop mutations were tested in constitutively activated RasV12 for their effects on ommatidial rotation (Gaengel, 2003).

Expression of RasV12 in developing photoreceptor precursor cells using common eye-specific drivers causes induction of many extra photoreceptors, and thus does not allow the analysis of rotation (the orientation of individual ommatidial clusters cannot be determined unambiguously in the presence of extra photoreceptor cells). To circumvent this problem, RasV12 and its effector loop isoforms were expressed in a limited photoreceptor subset after these had been determined as photoreceptors using the mDelta0.5-Gal4 driver. RasV12 effects on photoreceptor number and fate were thus strongly reduced. To determine the full effect of activated Ras expression under the control mDelta0.5-Gal4, constitutively active RasV12, that activates all known Ras-effectors, was expressed. This gave rise to eyes with some gain and loss of photoreceptors and severe misrotations. The equivalent expression of RasV12[S35], thought to activate mainly Raf, also resulted in rotation abnormalities and occasional gain or loss of photoreceptors, again supporting a requirement of the Raf/MAPK cascade (Gaengel, 2003).

Strikingly, expression of RasV12[G37] and RasV12[C40] also caused misrotations, suggesting an involvement of additional Ras-effectors in this process. In particular, RasV12[G37], in which Raf activation is abolished (or at least strongly reduced), results in severe rotation defects, suggesting that PI3K, Rgl/Ral or Canoe might play a role in this cell motility process. Similarly, RasV12[C40], eliminating Raf activation, but maintaining weaker activation of other effectors, also shows rotation abnormalities, albeit weaker than RasV12[G37]. Moreover, expression of RasV12[C40] under the control of the sevenless (sev) promoter (in R3/R4, R1/R6 and R7) results in strong rotation defects that were comparable to those seen with RasV12[G37] under sev control. Taken together, these data suggested an involvement of PI3K, Rgl/Ral or Canoe in ommatidial rotation (Gaengel, 2003).

To confirm the RasV12[G37] effect and determine which of the three known effectors activated by RasV12[G37] is required in ommatidial rotation, PI3K, Ral and Canoe were analyzed directly. UAS-PI3K expressed under mDelta0.5-Gal4 has no effect on rotation, suggesting that PI3K is not required in this process. This is further supported by the lack of rotation defects in dPI3K mutant clones. In contrast, expression of activated Ral (Sawamoto, 1999) as well as mDelta0.5-Gal4>UAS-Cno exhibits rotation defects (Gaengel, 2003).

Next, a direct canoe (cno) requirement in ommatidial rotation was tested using LOF alleles. First, it was asked whether cno heterozygosity interacts with the Star48-5/+ rotation phenotype. Strikingly, similar to the enhancement observed with Egfr or Ras, the cno2/+ and cno3/+ genotypes enhance the S48-5/+ rotation phenotype. cno is required for cone cell and photoreceptor differentiation and thus clones of null and strong alleles cause a general disorganization of the eye and are difficult to analyze for rotation defects. However, the hypomorphic cnomis1 allele is subviable in trans to the strong alleles cno2 and cno3 with mildly rough eyes, allowing an analysis of ommatidial rotation. Eye sections of such transheterozygous cno flies (e.g. cnomis1/cno2) reveal severe rotation defects. To test whether such defects are already observed at the time when rotation takes place, cno mutant third instar eye discs were analyzed. Strikingly, rotation defects, comparable in strength to the stronger aos alleles, are apparent in cno eye imaginal discs. The discs were counterstained with anti-Elav to ensure that the photoreceptor complement is normal in such cno mutant discs and the observed rotation abnormalities are primary defects, which was indeed confirmed. A similar analysis of Ral/Rgl is precluded by the lack of suitable alleles. In summary, these data indicate that cno plays a critical role in ommatidial rotation and acts as an effector of Egfr/Ras signaling in this context (Gaengel, 2003).

Since ommatidial rotation is a cell biological event, it is probable that among the main read-outs affected are cell-adhesion properties of the precluster cells and effects on cytoskeletal elements. This is further supported by observations that (1) Raf/MAPK-independent and thus transcription-independent Egfr/Ras signaling pathways are important, and (2) that canoe is required in this context. To address this further, two sets of experiments were performed. First, tests were performed for genetic interactions between the dosage-sensitive Star/+ rotation phenotype and selected factors required in cell adhesion and cytoskeletal regulation; and second, whether cell-adhesion components such as cadherins and integrins are normally localized in aosrlt and cnoMis1 mutant backgrounds was directly analyzed (Gaengel, 2003).

To specifically test the involvement of cytoskeletal elements and adhesion as well as junctional components, candidate genes were tested for dominant interaction of the mild Star rotation phenotype. These genetic data argue for an involvement of E-Cadherin/shotgun, the atypical cadherin Flamingo (Fmi), the adherens junction protein canoe, non-muscle myosin II (zipper), the septin peanut, and capulet, a protein with actin and adenylate cyclase-binding ability (Gaengel, 2003).

Next, the expression of Fmi and Shotgun in ommatidial preclusters was examined during rotation. Strong LOF alleles of Egfr and its signaling components also affect cell proliferation, fate specification and survival, making the analysis of cell adhesion and junctional components in the context of rotation rather difficult. Thus localization of the cadherins and Arm/ß-catenin was examined in imaginal discs of the rotation-specific aosrlt allele (Gaengel, 2003).

Although the overall expression and localization of Shotgun and Arm/ß-catenin are largely unaffected, the localization of Fmi is changed in aosrlt discs. In WT, Fmi is initially present apically in all cells of the morphogenetic furrow and subsequently becomes asymmetrically enriched in the R3/R4 precursor pair. In and posterior to column 6, Fmi is expressed at the membrane of R4, and largely depleted from R3 membranes that do not touch R4, forming a horseshoe-like R4-specific pattern. In contrast, in aosrlt discs, Fmi restriction to the R4 precursor is generally delayed, and often not established even in columns 8-12, where high levels of Fmi are still seen around the apical membrane cortex of R3 and R4. Since Fmi is thought to act as a homophyllic cell-adhesion molecule, its increased presence on R3 membranes should have a direct effect on Fmi localization in neighboring cells and thus possibly the adhesive properties of the precluster. It is worth noting that although Fmi is required during PCP establishment and R3/R4 cell-fate specification, the delay in Fmi restriction to R4 has no significant effect on the R3/R4 cell-fate decision. Although Fmi interacts with Fz and Notch in this context, the R4-specific mDelta-lacZ marker does not differ significantly from WT and adult aosrlt eyes also display no defects in R3/R4 specification. Thus, it appears that the delay in Fmi localization specifically affects ommatidial rotation, probably through adhesion, and possibly explains the broad range of rotation angles in aosrlt and other Egfr pathway mutants (Gaengel, 2003).

The Egfr/Ras/Cno link is intriguing for several reasons. The cno gene was originally identified as a mutation affecting the dorsal closure process during embryogenesis. Cno shows a genetic and molecular link to Ras: it contains two Ras-interacting domains and binds both WT Ras and activated Ras-V12. In addition, Cno has been postulated to link cytoskeletal elements to cellular junctions via its ability to bind actin, its interaction with ZO-1/Pyd and its homology with kinesin and myosin-like domains. Thus Cno could directly mediate an Egfr/Ras signal to cytoskeletal and cell architecture elements through its association with adherens junctions and its kinesin and myosin-like domains. Interestingly, Zipper does not only show a similar interaction with Star, like Cno, but it is also required during embryonic dorsal closure, and thus a more general Cno-Zipper link might exist in cell motility contexts (Gaengel, 2003).

A second interesting feature of cno is that it has been genetically linked to sca and Notch signaling. First, the phenotype of the sca1 allele is strongly enhanced by cno/+. Second, cno alleles also display Notch-like phenotypes in the wing and a GOF Notch allele, NotchAbruptex, is suppressed by cno. Although the biochemical role of Sca remains obscure, it has been linked to Notch, possibly as a Notch ligand, in several contexts. Thus, since sca has recently been implicated in ommatidial rotation, the link between Cno and Sca/Notch is intriguing. Taken together, Cno could serve as a factor integrating signaling input from different pathways, e.g., Egfr and Notch in this process, and relaying this to cytoskeletal elements. The Canoe link is also interesting from a disease point of view since its human homolog AF6 is the critical partner of ALL1 in a chimeric protein associated with myeloid leukemia. Thus, taken together, Cno could serve as a factor integrating signaling input from different pathways, e.g., Egfr and Notch in ommatidial rotation, and relaying this to the regulation of cell adhesion and cytoskeletal elements in the context of a developmental patterning process or disease (Gaengel, 2003).

Thus Egfr/Ras signaling plays a general role in the regulation of ommatidial rotation. Canoe has been identified as an effector of Ras in this context. Although much is known about how ommatidial chirality and the associated R3/R4 cell-fate decision are regulated (Fz/PCP-Notch signaling), no clear link between the mechanistic aspects of ommatidial rotation and Fz/PCP signaling previously existed. This is the first link to be demonstrated between Egfr signaling and PCP genes, namely Fmi. A further connection between Egfr signaling and PCP establishment is provided by Zipper, which acts downstream of Fz/Dsh and Rok in wing PCP and modifies the Star rotation phenotype. The identification of the Egfr pathway and its regulation of Fmi/cadherin-mediated cell adhesion will serve as an important entry point to further such studies (Gaengel, 2003).

Extracellular signals responsible for spatially regulated proliferation in the differentiating Drosophila eye

Spatially and temporally choreographed cell cycles accompany the differentiation of the Drosophila retina. The extracellular signals that control these patterns have been identified through mosaic analysis of mutations in signal transduction pathways. All cells arrest in G1 prior to the start of neurogenesis. Arrest depends on Dpp and Hh, acting redundantly. Most cells then go through a synchronous round of cell division before fate specification and terminal cell cycle exit. Cell cycle entry is induced by Notch signaling and opposed in subsets of cells by EGF receptor activity. Unusually, Cyclin E levels are not limiting for retinal cell cycles. Rbf/E2F and the Cyclin E antagonist Dacapo are important, however. All retinal cells, including the postmitotic photoreceptor neurons, continue dividing when rbf and dacapo are mutated simultaneously. These studies identify the specific extracellular signals that pattern the retinal cell cycles and show how differentiation can be uncoupled from cell cycle exit (Firth, 2005).

The EGFR holds R2-R5 cells in G1 phase and promotes G2/M progression of other cells during the second mitotic wave (SMW). Earlier regulation is now found to depend on longer-range signaling by the Hh, DPP, and N signals already known to drive the progression of the morphogenetic furrow. These studies exclude other models that show that Hh, Dpp, or N act indirectly by releasing other, cell cycle-specific signals from differentiating cells, or that patterned cell cycle withdrawal or reentry occur independent of extracellular signals, such as by synchronized growth. Instead, specific signals are necessary or sufficient for each aspect of cell cycle patterning (Firth, 2005).

G1 arrest ahead of the morphogenetic furrow depends on posterior-to-anterior spread of Hh and Dpp. Hh is secreted from differentiating cells, starting at column 0 in the morphogenetic furrow. Dpp is transcribed in ~6 ommatidial columns in the morphogenetic furrow in response to Hh. Cells accumulate in G1 about 16-17 cell diameters anterior to column 0, suggesting an effective range of ~13-17 cells for Hh and Dpp (Firth, 2005).

The contribution of Dpp to this cell cycle arrest is known already, but that of Hh was not suspected. Both Dpp and Hh signaling can promote proliferation in other developmental contexts (Firth, 2005).

S phase entry in the SMW depends on another signal, N. Expression of the N ligand Dl begins at the anterior of the morphogenetic furrow. The first S phase cells are detected 6-8 cell diameters more posteriorly, just behind column 0. The transmembrane protein Dl must act more locally or more slowly than the secreted Hh and Dpp proteins, to explain gaps between S phases (Firth, 2005).

Although N activity has been associated with growth through indirect mechanisms involving the release of other secreted growth factors and also regulates endocycles, this appears to be the first report of a specific role of N in G1/S in diploid Drosophila cells. Notably, deregulated N signaling contributes to at least two human cancers and is oncogenic in mice (Firth, 2005).

At the same time that N promotes S phase entry in the SMW, EGFR activity ensures that R2-R5 cells remain in G1. N is still required in the absence of EGFR, so N activity is a positive signal and is not required only to counteract EGFR activity. Instead, EGFR activity interferes with S phase entry in response to N (Firth, 2005).

Ligands for the EGF receptor are thought to be released from R8 precursor cells, although EGFR-dependent MAPK phosphorylation is detected one ommatidial column before the column where R8 precursor cells can be identified, which is in column 0. This means that EGFR activation begins after Dl expression but before S phase DNA synthesis starts. Later, ligands released from differentiating precluster cells activate EGFR in surrounding cells to permit SMW mitosis around columns 3-5 (Firth, 2005).

Hh and Dpp together promote expression of Dl and of EGFR ligands; in part, this occurs indirectly through Atonal and the onset of differentiation. EGF receptor activity also promotes Dl expression (Firth, 2005).

At least three genetic mechanisms arrest distinct retinal cells in G1. Arrest ahead of the morphogenetic furrow depends on Dpp and Hh. During the SMW, R2-R5 cells are held in G1 by EGFR, which counteracts the SMW-promoting N activity. In addition, R8 cells, which are defined by the proneural gene atonal, remain in G1 independent of EGFR. After the SMW, all cells remain in G1 indefinitely, independent of EGFR. Although cell cycle withdrawal roughly correlates with differentiation, many of the cells that arrest after the SMW are still unspecified (Firth, 2005).

Loss of rbf and dap together overcome all cell cycle blocks, even though cell differentiation continues. This redundancy indicates that Cyclin E/Cdk2 targets other than Rbf are needed for proliferation, consistent with many other studies. Dap may be regulated by EGFR in R2-R5 cells. If rbf regulates the normal SMW, where Cyclin E expression seems not to be limiting, then other E2F targets may be involved. Some cell cycle arrest can also be overridden by forced expression of Cyclin E, E2F/DP, dRef, and ORC1, or by mutation of the Cyclin A antagonist rux (Firth, 2005).

The results show that mechanisms that assure both short- and long-term arrest of retinal cells must operate upstream of (or parallel to) Rbf and Cyclin E activities. They might resemble the barriers to transformation and regeneration that exist in mammals (Firth, 2005).

The tumor suppressor gene fat modulates the EGFR-mediated proliferation control in the imaginal tissues

Molecules involved in cell adhesion can regulate both early signal transduction events, triggered by soluble factors, and downstream events involved in cell cycle progression. Correct integration of these signals allows appropriate cellular growth, differentiation and ultimately tissue morphogenesis, but incorrect interpretation contributes to pathologies such as tumor growth. The Fat cadherin is a tumor suppressor protein required in Drosophila for epithelial morphogenesis, proliferation control and epithelial planar polarization, and its loss results in a hyperplastic growth of imaginal tissues. While several molecular events have been characterized through which fat participates in the establishment of the epithelial planar polarity, little is known about mechanisms underlying fat-mediated control of cell proliferation. Evidence is provided that fat specifically cooperates with the epidermal growth factor receptor (EGFR) pathway in controlling cell proliferation in developing imaginal epithelia. Hyperplastic larval and adult fat structures indeed undergo an amazing, synergistic enlargement following EGFR oversignalling. Such a strong functional interaction occurs downstream of MAPK activation through the transcriptional regulation of genes involved in the EGFR nuclear signalling. Considering that fat mutation shows di per se a hyperplastic phenotype, a model is suggested in which fat acts in parallel to EGFR pathway in transducing different cell communication signals; furthermore fat function is required downstream of MAPK for a correct rendering of the growth signals converging to the epidermal growth factor receptor (Garoia, 2005).

These results support the hypothesis of a relevant functional interaction between ft and genes of the EGFR pathway. When EGFR signalling is raised in fact, an amazing, non-additive increase in ft-induced proliferation is observed. ft clones in UAS-rho or UAS-rafGOF wings where an EGFR oversignalling is induced show the same distinctive features of those induced in a wild-type background (tissue hyperplasia, reduced cell size, loss of planar polarity), with a phenotype much more severe concerning outgrowth number and dimensions. On the contrary, in experiments where the EGFR signalling is reduced, ft-induced hyperproliferation results are partially suppressed. The same trend is observed in the adult eye; the ft head-capsule where rho or rafGOF were ectopically expressed was particularly enlarged (Garoia, 2005).

The most dramatic effects were however observed in the eye and wing imaginal discs where the EGFR signalling was increased in the presumptive dpp expression domains. The controls did not show significant phenotypes; conversely, in ft discs severe effects were observed, including non-autonomous aberrations in the disc morphology. In the ft rafDN eye disc a strong non-autonomous reduction of the eye presumptive territory was observed that may be caused by decrease of proliferation rate and/or by increase of cell death. Non-autonomous cell death may result from cell competition, ectopic cell proliferation or experimentally induced apoptosis. An increase in non-autonomous cell death was observed not only in ft rafDN discs but in all the ft/UAS combinations analyzed. Thus, cell death does not seem to be the mechanism through which the growth deficit occurs in the ft rafDN eye disc, suggesting a more complex interaction between ft and EGFR activity in the modulation of eye disc growth (Garoia, 2005).

Therefore ft interacts with the EGFR pathway modulating its proliferative signal; ft function is therefore involved ingrowth regulation, allowing the structures to correctly interpret signals incoming from EGFR that are essential for eye and wing to acquire the final shape. No interaction was found with mutants involved in pathways other than the EGFR cascade (N, dpp and wg), making the role of ft in the control of the imaginal disc growth specifically dependent on its interaction with the EGFR pathway (Garoia, 2005).

The proximal ft clone allocation along the wing blade arises from an alteration in the growth direction and not from a different viability of the cells relative to their layout in the proximo-distal axis; it is then obvious that ft cells show a greater 'affinity' for the proximal region of the wing. The ft mutant phenotype seems anyway to be influenced by EGFR signalling also with respect to the proximalization of the clones; the distribution is indeed more homogeneous along the wing blade if the EGFR signalling is altered. It is interesting to notice that an EGFR signalling reduction or increase produces, in this case, the same biological effect, while the proliferative phenotype is directly correlated to the activity of the EGFR effectors. The activity of the EGFR cascade is spatially and temporally modulated during development, and in the wing disc it is gathered in the hinge and vein presumptive regions. Even if there are no evidences that the EGFR signalling plays a role in the P/D patterning of the wing blade, recent studies have shown that a gradient of EGFR activity is required for the correct P/D development of the leg. In the experimental conditions used, the MS1096-GAL4 driver creates an almost homogeneous EGFR signal along the wing blade, determining a quasi wild-type distribution of ft clones. This role in the modulation of the differential distribution of ft mutant cells along the P/D wing axis suggests for the EGFR pathway an involvement in the morphogenetic events that control the final shape of the wing (Garoia, 2005).

The ft-induced hyperplasia is associated with an abnormal pattern of gene expression, as visualized in two-dimensional protein gels of ft mutant imaginal discs. Although no data is available to hypothesize a cytoplasmic interaction between ft and the EGFR signalling, an alteration in MAPK expression could not be excluded. The ft mutant phenotypes, however, do not include the differentiation defects typical of the EGFR pathway genes, whose activity is indeed not altered since no significant modifications of the activated dpERK levels or patterns were detected in the ft tissues with respect to the wild-type (Garoia, 2005).

The results shown in this paper suggest that the interaction between ft and EGFR takes place at the proliferation level, while differentiation signals controlled by the EGFR pathway appear unaffected. With the aim to find some mechanisms that could explain the synergic phenotype of ft and EGFR mutations, the transcriptional levels of yan, dmyc and pnt, genes involved in proliferation control whose function is regulated by the EGFR cascade, were studied in ft and wild-type imaginal tissues. The results of semi-quantitative RT-PCR trials showed in ft tissues an increase of the transcription levels of yan and dmyc, whereas pnt was unaffected. The Dmyc transcription factor, the unique Drosophila homologue of the Myc family of proto-oncogenes, plays a central role in the control of cell growth in Drosophila. Overexpression of ras is capable to increase post-transcriptionally the Dmyc protein levels, promoting the G1-S transition via the increase of CycE translation. The increase in the Dmyc levels, however, affects growth rate but not proliferation, since the shortening of the G1 phase is balanced by the compensatory lengthening of G2, resulting in an increase in cell size but not in cell number. ft mutation otherwise induces an increase of cell proliferation without altering the cell size. Taken together, these results indicate that ft mutation affects not only the G1-S transition via Dmyc but also the G2-M transition, since the coordinated stimulation of the two cell-cycle checkpoints is necessary to increase the proliferation rate in Drosophila imaginal discs. Interestingly, the transcription level of pnt was unaffected in ft mutant discs. pnt is an ETS transcriptional activator that plays a central role in the mitosis control mediated by the EGFR signalling cascade; several studies however suggest the presence of additional Pnt-independent effectors in EGFR-mediated mitosis control. The ft control of the G2–M transition may involve EGFR effectors other than pnt, or molecules functioning through different signalling pathways. The yan gene is another component of the ETS transcriptional regulator family involved in the EGFR signalling. Phosphorylation by MAPK affects stability and subcellular localization of Yan, resulting in a rapid down-regulation of its activity. Yan functions as a fairly general inhibitor of differentiation, allowing both neuronal and non-neuronal cell types to choose between cell division and differentiation in multiple developmental contexts and recent studies indicate that the mammalian homologue of the Drosophila yan, TEL, is overexpressed in tumors. In the Drosophila developing eye yan is expressed in all undifferentiated cells and is down regulated as cells differentiate, so a high yan activity in ft mutant discs is correlatable with the observed proliferative advantage of ft cells (Garoia, 2005).

There are several indications that EGFR signalling can trigger different responses by different activity levels: in the Drosophila eye disc, differentiation requires high signalling levels, whereas lesser EGFR activity promotes mitosis and protects against cell death. These findings indicate that EGFR signalling may coordinate partially independent processes, transferring graded activity to the nucleus, rather than triggering 'all or none' responses. The simultaneous increase of activity in both growth promoters (dmyc) and differentiation repressors (yan) in ft mutant imaginal discs suggests the presence of a mechanism that shifts the EGFR nuclear equilibrium towards a level insufficient to induce differentiation but adequate for promoting cell growth and proliferation (Garoia, 2005).

These results indicate that, in the Drosophila imaginal discs, ft function is necessary for the correct interpretation of the multiple EGFR signals that coordinate proliferation, and that its loss causes misinterpretation of proliferation stimuli leading to tissue overgrowth. This effect may be due, at least in part, to the transcriptional regulation of genes involved in EGFR signalling. Nevertheless, the hyperplastic phenotype of ft mutations cannot be completely ascribed to its role in modulating signals transduced by EGFR, according to the very partial rescue observed utilizing dominant-negative alleles of the pathway. These results suggest that the ft function is not restricted to the modulation of EGFR signals, but controls different developmental events involved in imaginal discs morphogenesis (Garoia, 2005).

Several findings indicate that cadherin-catenin complexes may interact with growth factor receptors. The association of cadherins with growth factor receptors allows the assembly of a locally active apparatus that is essential for the generation of correct cell-cell signalling, as suggested by the downregulation of E-cadherins observed in mammalian tumors. Furthermore, E-cadherins were found to be a direct biochemical target of the EGFR pathway, suggesting a close relation of these molecules with the modulation of cell-cell communication. The only partial homology between the Ft protocadherin and the classic E-cadherins, and the lack of data about interactors for the cytoplasmic domain of ft makes a direct comparison of their function very difficult. Taken together, the data suggest a novel mechanism through which ft tumor suppressor gene and EGFR pathway cooperate in the control of proliferation and morphogenesis in Drosophila imaginal tissues (Garoia, 2005).

Identification of the death zone: a spatially restricted region for programmed cell death that sculpts the fly eye

Programmed cell death (PCD) sculpts many developing tissues. The final patterning step of the Drosophila retina is the elimination, through PCD, of a subset of interommatidial lattice cells during pupation. It is not understood how this process is spatially regulated to ensure that cells die in the proper positions. To address this, PCD of lattice cells in the pupal retina was observed in real time. This live-visualization method demonstrates that lattice cell apoptosis is a highly specific process. In all, 85% of lattice cells die in exclusive 'death zone' positions between adjacent ommatidia. In contrast, cells that make specific contacts with primary pigment cells are protected from death. Two signaling pathways, Drosophila epidermal growth factor receptor (Egfr) and Notch, that are thought to be central to the regulation of lattice cell survival and death, are not sufficient to establish the death zone. Thus, application of live visualization to the fly eye gives new insight into a dynamic developmental process (Monserate, 2006).

The Drosophila pupal retina was used as a model system to study spatial regulation of PCD. Apoptosis of a subset of lattice cells in the mid-pupal retina is the final patterning step in sculpting the fly eye. The static nature of ultrastructural studies made it impossible to determine whether dying lattice cells occupied specific positions between ommatidia, Live visualization of the pupal retina is advantageous because it not only allows snapshots to capture specific events but also allows one to identify an apoptotic cell, and essentially look back in time to assign the cell's position before death (Monserate, 2006).

Using time-lapse imaging of the eye of the living pupa, dying cells were identified as they lost their apical footprint, one of the earliest death events. It was determined that 50% of apoptotic lattice cells occupied a position next to the bristle group and in the horizontal face between ommatidial units. Furthermore, 35% of dying lattice cells were located next to the bristle group along an oblique face. Thus, a remarkable 85% of apoptotic lattice cells died in two very specific regions between ommatidial units and adjacent to bristle groups (Monserate, 2006).

However, bristle groups cannot be the origin of the death signal; removal of bristle groups does not block apoptosis. Instead, in the absence of bristle groups, the pattern of lattice cell death changed dramatically. Lattice cells at either the anterior or posterior portion of the horizontal face now had roughly an equal chance of dying. Together, the two horizontal regions accounted for 85% of the lattice cells undergoing PCD. It is concluded that lattice cell PCD is regulated spatially so that apoptosis removes cells occupying the anterior and posterior horizontal positions between ommatidia. This region was termed the 'death zone'. Between these two points of position-specific PCD, a single cell is protected from apoptosis. Apoptosis in the death zone ends when only this cell remains in the horizontal face between ommatidia (Monserate, 2006).

How could bristle groups so dramatically affect the pattern of PCD? One possibility is that the bristle groups attract cells to their death in the death zones. It has been shown that bristle groups secrete Spi and the data demonstrate that lattice cells respond by surrounding the source of secreted Spi. Just prior to the cell death period, there are on average 1.6 times more cells in the regions around the bristle groups than in other vertices. Additionally, in the absence of bristle groups, more cells appear to populate the horizontal regions prior to death (at the expense of the oblique regions) (Monserate, 2006).

Survival of lattice cells depends on a signal, hypothesized to be secretion of Spi, from primary pigment cells and/or cone cells. The experiments confirm that lattice cells transduce a dEgfr signal, but the interpretation is complicated by the fact that P-MAPK wss not detected in lattice cell nuclei, suggesting that none of the cells respond transcriptionally to Egfr signaling. Instead, P-MAPK was identified in the cytoplasm of all lattice cells, including those in the death zone. It was recently reported that retaining P-MAPK in the cytoplasm in cells in the larval eye disc is important for normal development. In the current study, it is unlikely that blocking of nuclear MAPK signaling by 'cytoplasmic hold' is required to induce apoptosis because ectopic transcriptional repression of activated MAPK targets by expression of the activated form of aop had no effect on apoptosis in the pupal retina. Taken together, these experiments argue that Egfr signaling in lattice cells serves solely to activate P-MAPK in the cytoplasm. Cytoplasmic P-MAPK may suppress apoptosis through phosphorylation of cytoplasmic proteins such as HID (Monserate, 2006).

In light of the fact that Notch is upstream of Egfr, a new model was put forth: primary pigment cells and/or cone cells promote survival by inhibiting Notch activation and its disruption of Egfr signaling in lattice cells. It was possible to directly test this model because the live-visualization data predicted the pattern of death signals. The data do not support this hypothesis as all lattice cells, whether living or dying, are transducing a Notch signal; surviving lattice cells show no downregulation or inhibition of Notch signaling. Instead it is proposed that Notch dampens the Spi-activated dEgfr survival signaling in all lattice cells. Thus, experimental removal of Notch (i.e., heat shock of Nts1) would cause a general increase of Egfr signaling (and survival of all cells). Conversely, loss of Egfr signaling would lead to PCD, the default pathway. This model predicts that Notch transcriptionally activates a gene whose protein product keeps dEgfr signaling poised. A second scenario is one in which the Notch protein may itself influence the life or death decision of a cell. It has been suggested that Notch acts through a noncanonical pathway. In this model, Notch might function to activate PCD only in death zone cells, acting as a third signal (Monserate, 2006).

How is the death zone molecularly defined? Cells in the horizontal region are more sensitive to a reduction of Egfr signaling, but, as demonstrated by P-MAPK levels, this is unlikely to be owing to a lower level of Egfr signaling. Thus, if the death zone is defined by Egfr signaling, this may be owing to a qualitative change in cytoplasmic P-MAPK activity. A more likely possibility is that Egfr signaling (dampened by Notch) merely poises the cell to allow a third signal to induce PCD specifically in the death zone. In support of this idea is the finding that cells in the death zone regions are more likely than cells in other regions to die in response to a brief interruption in dEgfr signaling (Monserate, 2006).

Localization of apoptosis in the lattice to death zone regions brings additional complexity to the problem of PCD regulation. Clearly, the signal that regulates this final cell fate must be localized. The most likely candidate cell to produce the regulating signal is the primary pigment cell. However, each primary pigment cell contacts numerous lattice cells; some that will survive and some that will die. Thus, a model is suggested in which the dorsal and ventral regions of each primary pigment cell localize a death-inducing factor. Evidence for such a subcellular domain has been seen in primary pigment cells. The contact regions at which the two primary pigment cells meet must either block this signal or localize a strong survival signal (Monserate, 2006).

Placed in the context of other examples in which the architecture of a tissue is shaped through selective PCD, the Drosophila retina is a unique model in that it can be manipulated genetically, microscopically and development can be visualized in the living animal. The determination that apoptosis is spatially regulated in the pupal eye directs future experiments designed to identify how the death zone is created (Monserate, 2006).

Epithelial cell adhesion in the developing Drosophila retina is regulated by Atonal and the EGF receptor pathway

In the Drosophila retina, photoreceptor differentiation is preceded by significant cell shape rearrangements within and immediately behind the morphogenetic furrow. Groups of cells become clustered into arcs and rosettes in the plane of the epithelium, from which the neurons subsequently emerge. These cell clusters also have differential adhesive properties: adherens junction components are upregulated relative to surrounding cells. Little is known about how these morphological changes are orchestrated and what their relevance is for subsequent neuronal differentiation. This study reports that the transcription factor Atonal and the canonical EGF receptor signalling cascade are both required for this clustering and for the accompanying changes in cellular adhesion. In the absence of either component, no arcs are formed behind the furrow, and all cells show low Armadillo and DE-cadherin levels, although in the case of EGFR pathway mutants, single, presumptive R8 cells with high levels of adherens junction components can be seen. Atonal regulates DE-cadherin transcriptionally, whereas the EGFR pathway, acting through the transcription factor Pointed, exerts its effects on adherens junctions indirectly, at a post-transcriptional level. These observations define a new function for EGFR signalling in eye development and illustrate a mechanism for the control of epithelial morphology by developmental signals (Brown, 2006).

The discovery that EGFR signalling regulates cellular morphology in the morphogenetic furrow adds to the multiple functions already ascribed to this pathway in the eye. The process of cluster formation is the earliest detectable stage of ommatidial development and is tightly coordinated with subsequent photoreceptor recruitment. However, the results presented in this study clearly demonstrate that clustering is a separable process from photoreceptor differentiation. Most directly, the fact that spitz mutant clones do not show defects in clustering, whereas they fail to differentiate any photoreceptors beyond the founding R8 cell, shows that the functions of the EGFR pathway in clustering and in recruitment are distinct. In addition, these roles are spatially and temporally separable. The initial source of the Spitz signal for photoreceptor recruitment is the R8 cell. However, at the time at which the first MAPK activation is seen, in the furrow, the R8 does not yet exist. atonal expressing cells in the furrow upregulate rhomboid levels, and presumably it is these cells that release the activating ligand to control clustering. Since spitz clones do not show aberrant clustering, it is believed that Keren may be the ligand required to activate the EGFR in this process (either alone or redundantly with Spitz), since it has been conjectured that Keren is also involved in the control of ommatidial spacing, cell survival behind the morphogenetic furrow and ommatidial rotation. Evidence is available supporting the role of Keren in several aspects of eye development (Brown, 2006).

There are clearly strong similarities between the function for the EGFR described in this study and its function in the control of ommatidial spacing. In both cases, the pathway is activated in the morphogenetic furrow, under the control of Atonal, and in both, it appears as though Keren, rather than Spitz, may be the principal activating ligand. Indeed, it seems likely that the same signalling event may be responsible for coordinating both these processes. In the case of R8 spacing, it has been hypothesised that activation of the EGFR pathway leads to the secretion of an as yet unidentified inhibitory molecule, which acts non-autonomously to repress atonal expression between proneural clusters. In clustering, the signalling pathway seems to be required for two related purposes: firstly, to regulate the cell shape changes that accompany rosette and arc formation, and secondly, to maintain high levels of AJ proteins in these cells. In contrast to R8 spacing, this function appears to be cell-autonomous—no significant rescue is seen of clustering close to the borders of mutant clones. It is proposed that the same EGFR signalling event that results in the expression and secretion of the spacing inhibitory factor also causes an autonomous change in the transcriptional program of cells and leads to their maintaining strong AJs and to undergoing the cell shape changes that are required for rosette and arc formation (Brown, 2006).

Although the results demonstrate that both Atonal and the EGFR signalling cascade act to control the adhesive and morphological changes of cells behind the morphogenetic furrow, they implicate two independent mechanisms by which they act. Atonal exerts a transcriptional effect upon DE-cadherin. On the contrary, shg-lacZ and arm-lacZ levels are unaffected in ras mutant clones, indicating a function for the pathway in controlling either translation of these components, or in regulating the subcellular distribution or stability of AJs. However, it should be noted that, while the effects upon adhesion proteins are post-transcriptional, the EGFR pathway is acting through its canonical pathway and via the transcription factor Pointed, which must therefore control the expression of some downstream factor (Brown, 2006).

The following course of events is proposed. As cells enter the morphogenetic furrow, they all upregulate their levels of Armadillo and DE-cadherin. This change in the adhesive properties of the cells is independent of either Atonal or the EGFR signalling pathway, and presumably occurs as a result of the earlier signals responsible for driving the progression of the furrow, such as Hedgehog or Dpp. Since shg-lacZ expression does not appear to be upregulated at this early stage, it is speculated that this increase in protein levels is the result of some post-transcriptional mechanism, for example by stabilisation of AJs, although this has not been investigated this further. Behind the furrow, cells not fated to differentiate as photoreceptors downregulate the levels of AJ components to a low basal level. However, in the rosettes and arcs, high levels of Armadillo and DE-cadherin are maintained. This, it is suggested, is due to transcriptional upregulation of shotgun by Atonal, which is expressed in clusters of cells within the furrow before the R8 is selected, leading to the initial, broad stripe of shg-lacZ. atonal expression is then refined to the R8 precursor and presumably continues to exert its transcriptional effect on DE-cadherin in this cell, which shows the highest levels of AJ proteins. However, transcriptional upregulation of DE-cadherin cannot fully account for the maintenance of adhesion in cluster cells since over-expression of Atonal is unable to compensate for the loss of EGFR pathway activity. The results demonstrate a necessary role for the EGFR pathway in the post-transcriptional regulation of adherens junctions—presumably by promoting translation or stabilising junctional complexes to maintain strong cell–cell adhesion. Thus, these two mechanisms in concert act to promote adhesion between cells of the cluster, which are fated to form photoreceptors, while surrounding cells, which will go on to divide again, become less tightly connected (Brown, 2006).

One question that arises from the current work is the extent to which the changes in adhesive properties and the cell shape changes accompanying cluster formation are interdependent. Are the high levels of AJ proteins between cells of the cluster sufficient to reorganise the cells into distinct rosettes and arcs, or are there other mechanisms involved? Recent work has demonstrated that, later in eye development, the morphology of cone cell clusters can be accounted for solely by homophilic cadherin interactions between them; it is possible that a similar process may be occurring at this early stage (Brown, 2006).

In addition to orchestrating the cell shape changes that precede neuronal differentiation, modulation of adhesion might also be important for regulating the actual process of ommatidial cell recruitment. The observation that reduction in DE-cadherin enhances the recruitment defects caused by reduced EGFR signalling in Star mutants is consistent with a model where proper levels of DE-cadherin-mediated adhesion are required for efficient EGFR signalling. A number of previous reports are consistent with this idea. Firstly, it has been shown, both in tissue culture and in Drosophila embryos, that the EGFR co-immunoprecipitates with DE-cadherin, suggesting that cadherin might modulate EGFR activity, either directly or simply by regulating its localisation. Secondly, in mammalian tissue culture experiments, AJ formation has been shown to be capable of inducing EGFR dependent MAPK activation in a ligand independent manner. Further investigation will be required to determine details of the relationship between DE-cadherin and the EGFR, but it is interesting to consider that not only might cell signalling regulate adhesion, but that adhesion may also feed back to modulate signalling (Brown, 2006).

Split ends antagonizes the Notch and potentiates the EGFR signaling pathways during Drosophila eye development

The Notch and Epidermal Growth Factor Receptor (EGFR) signaling pathways interact cooperatively and antagonistically to regulate many aspects of Drosophila development, including the eye. How output from these two signaling networks is fine-tuned to achieve the precise balance needed for specific inductive interactions and patterning events remains an open and important question. The gene split ends (spen) functions within or parallel to the EGFR pathway during midline glial cell development in the embryonic central nervous system. This study shows that the cellular defects caused by loss of spen function in the developing eye imaginal disc place spen as both an antagonist of the Notch pathway and a positive contributor to EGFR signaling during retinal cell differentiation. Specifically, loss of spen results in broadened expression of Scabrous, ectopic activation of Notch signaling, and a corresponding reduction in Atonal expression at the morphogenetic furrow. Consistent with Spen's role in antagonizing Notch signaling, reduction of spen levels is sufficient to suppress Notch-dependent phenotypes. At least in part due to loss of Spen-dependent down-regulation of Notch signaling, loss of spen also dampens EGFR signaling as evidenced by reduced activity of MAP kinase (MAPK). This reduced MAPK activity in turn leads to a failure to limit expression of the EGFR pathway antagonist and the ETS-domain transcriptional repressor Yan and to a corresponding loss of cell fate specification in spen mutant ommatidia. It is proposed that Spen plays a role in modulating output from the Notch and EGFR pathways to ensure appropriate patterning during eye development (Doroquez, 2007).

This study demonstrates that loss of spen perturbs the normal balance between the EGFR and Notch pathways as evidenced by the patterning disruptions and aberrant expression of multiple pathway components. These findings raise the question of whether Spen functions primarily in the Notch pathway, primarily in the EGFR pathway, or as a critical component of both. Although definite resolution is difficult given the extensive and intricate feedback regulation within and between these two signaling networks, a model is proposed in which Spen-mediated antagonism of the Notch pathway regulates the signaling flow through the EGFR pathway to achieve proper retinal cell fate specification (Doroquez, 2007).

Loss of spen results in hyperactivation of the Notch pathway as evidenced by elevated levels of both Notch and its transcriptional targets, the E(spl)-bHLHs. Therefore, a normal function of Spen in the developing eye is to limit the activity of Notch. Consistent with this model, heterozygous reduction of spen was shown to be sufficient to suppress the heterozygous Notch wing margin phenotype. However, loss of spen does not lead to the anti-neurogenic phenotypes typically associated with overexpression/overactivation of canonical members of the Notch pathway, suggesting that although Notch signaling output is elevated, the increase is below the threshold needed to achieve such phenotypes. Consistent with this interpretation, recruitment of the initial R8 photoreceptor neuron, a process influenced at multiple stages by Notch signaling, occurs normally in the absence of spen (Doroquez, 2007).

Where might Spen interface with the Notch signaling pathway? The striking increase in Sca expression in spen mutant clones at the MF is consistent with Spen regulating Notch activation by limiting the expression of sca either through transcriptional repression or by destabilizing the transcript. This suggests that in the Drosophila eye Spen may have an upstream role in the Notch pathway in contrast to the downstream role described for Spen mammalian orthologs. In contrast, because of extensive feedback regulation in Notch signaling, it is plausible that Spen interfaces with the network at a more downstream point. For example, ectopic expression of Notchintra was shown to promote Sca expression, which in turn activates Notch signaling. Additionally, although no such role was detected with respect to yan, it is possible that Spen limits Notchintra/Su(H)-mediated transactivation at the level of transcriptional repression of other Notch pathway targets, including the E(spl)-bHLHs, as is the case for the mammalian Spen orthologs. This latter mechanism might also be relevant posterior the MF, where Notch signaling remains elevated as judged by increased levels of both Notch and the E(spl)-bHLHs in spen mutant clones, but where Sca is no longer expressed (Doroquez, 2007).

Although pinpointing where Spen interfaces with the Notch signaling pathway remains a challenge, the simplest interpretation of the data is that at the MF, Spen either directly or indirectly regulates Sca expression to restrict Notch pathway output. Posterior to the MF, as discussed below, mutual antagonism between the Notch and EGFR pathways may stabilize the initial signaling imbalance independent of Sca, leading to sustained up-regulation of Notch and down-regulation of EGFR output in spen mutant tissue (Doroquez, 2007).

What might the consequences of a moderate increase in Notch pathway output be? Given the extensive functional antagonism that has been reported between the Notch and EGFR pathways in the eye, a likely outcome is that the increased Notch signaling in spen mutant tissue would dampen EGFR pathway output. Supporting the idea that spen plays a positive role with respect to EGFR signaling, the cell fate specification defects observed in spen mutant clones are highly reminiscent of phenotypes associated with hypomorphic mutants in positive components of the EGFR pathway. Thus, the defective specification of neuronal and non-neuronal cell types and the perturbed R8 spacing adjacent to the MF all suggest reduced, but not ablated, EGFR pathway function in spen clones (Doroquez, 2007).

Lending further support to a model in which elevated Notch signaling in spen clones dampens EGFR pathway output, both Ato and dpERK expression at the MF are reduced. Because previous work has shown that Ato is required for activation of the EGFR pathway at the MF, one possibility is that Spen stabilizes dpERK levels at the MF by antagonizing Notch-mediated lateral inhibition to ensure appropriate Ato expression. Another plausible mechanism for Spen-mediated regulation of dpERK activity would be downstream of or in parallel to Ras. In this scenario, Spen might mediate transcriptional repression of an inhibitor such as a MAPK phosphatase. However, qRT-PCR analysis in imaginal discs predominantly mutant for spen do not indicate a role for Spen in regulating the expression of two characterized Drosophila MAPK phosphatases - dMKP3 and PTP-ER. Thus, validation of such a mechanism will require identification of other MAPK phosphatases or pathway inhibitors that might be regulated by Spen (Doroquez, 2007).

It should be noted that the results of this analysis of spen function in the eye appear contradictory to those from a prior study that suggested spen antagonizes EGFR output and promotes Notch signaling during embryonic neural development (Kuang, 2000). Specifically, elevated EGFR signaling was reported in spen maternal/zygotic null embryos, as evidenced by increased numbers of midline glial cells and loss of Yan expression. However, these results could not be reproduced (F. Chen and I. Rebay, unpublished data reported in Doroquez, 2007). On the contrary, analysis of spen function during midline glial cell development in the embryonic central nervous system was entirely consistent with a role for spen as a positive contributor to EGFR signaling. Thus, at least with respect to EGFR signaling, it is believed that Spen serves an analogous role in multiple developing tissues (Doroquez, 2007).

With respect to Notch signaling, Kuang reported a strong reduction in E(spl)-bHLH expression throughout the embryo but no change in Notch levels, exactly opposite to the current findings in the eye. Additional work will be needed to determine whether and how spen interfaces with the Notch pathway during embryogenesis, and whether distinct or identical mechanisms operate in retinal versus embryonic neural development (Doroquez, 2007).

It is not yet clear whether spen's role in Notch-EGFR interactions posterior to the MF is identical to its role in events occurring at the MF. The failure to down-regulate Yan and the resulting cell fate specification defects show that EGFR signaling posterior to the MF is compromised in spen mutant tissue. Given that Yan up-regulation in spen clones does not result from loss of Spen-mediated transcriptional repression, but rather reflects loss of post-translational control, two models for Spen function seem likely. First, if the inability to detect changes in dpERK protein levels posterior to the MF in situ accurately indicates unaltered dpERK levels, then the ability of dpERK to phosphorylate Yan must be compromised in spen mutants. Alternatively, dpERK levels may be sufficiently reduced to increase Yan stability, but the change may be below the immunohistochemical detection threshold (Doroquez, 2007).

In terms of the signals that impinge on dpERK, whereas Notch signaling and Ato expression are critical for proper dpERK expression in the MF, reiterative EGFR signaling takes over posterior to the MF to maintain dpERK activity. Thus, it is possible that a Spen-dependent, Notch-independent mechanism may regulate EGFR output posterior to the MF. Alternatively, because Notch, E(spl) and Yan expression are all elevated in spen mutant tissue both in and posterior to the MF, Spen-mediated antagonism of Notch signaling may be relevant to EGFR regulation in both contexts. An extension of this idea that results in perhaps the most appealing model is that the initial increase in Notch output at the MF dampens EGFR signaling, which in turn leads to elevated Notch signaling in more posterior regions resulting in reduced EGFR output. In this way, the initial signaling imbalance created by loss of spen at the MF could be maintained over the entire eye disc through mutual antagonism and feedback regulation between the Notch and EGFR pathways (Doroquez, 2007).

In summary, this study analyzed the requirement for spen in regulating the EGFR and Notch pathways during Drosophila eye development, and it is proposed that increased Notch pathway activity upon loss of spen may be sufficient to dampen EGFR signaling, but not to disrupt other downstream effects of Notch signaling. Therefore, because the effects of spen loss appear to be at a threshold below the production of bona fide Notch-related phenotypes, it is suggested that Spen plays a subtle role in the regulation of the Notch pathway or functions redundantly alongside other components. An equally likely hypothesis is that Spen regulates the Notch and EGFR pathways separately and that the phenotypes reported reflect a composite of independent disruptions to both signaling networks (Doroquez, 2007).

Although much of the literature focuses on a primary role for Spen family proteins as co-repressors, recent findings suggest members of this family may also regulate non-coding RNA sequestration, mRNA export, RNA splicing, and proteolysis. Therefore, future identification of the precise molecular mechanisms by which Spen interfaces with the EGFR and Notch pathways may reveal novel modes of interaction between these two critical and conserved signaling networks (Doroquez, 2007).

Atonal and EGFR signalling orchestrate rok- and Drak-dependent adherens junction remodelling during ommatidia morphogenesi

Morphogenesis of epithelial tissues relies on the interplay between cell division, differentiation and regulated changes in cell shape, intercalation and sorting. These processes are often studied individually in relatively simple epithelia that lack the complexity found during organogenesis when these processes might all coexist simultaneously. To address this issue, this study makes use of the developing fly retinal neuroepithelium. Retinal morphogenesis relies on a coordinated sequence of interdependent morphogenetic events that includes apical cell constriction, localized alignment of groups of cells and ommatidia morphogenesis coupled to neurogenesis. Live imaging was used to document the sequence of adherens junction (AJ) remodelling events required to generate the fly ommatidium. In this context, it was demonstrated that the kinases Rok and Drak function redundantly during Myosin II-dependent cell constriction, subsequent multicellular alignment and AJ remodelling. In addition, it was shown that early multicellular patterning characterized by cell alignment is promoted by the conserved transcription factor Atonal (Ato). Further ommatidium patterning requires the epidermal growth factor receptor (EGFR) signalling pathway, which transcriptionally governs Rho-kinase (rok) and Death-associated protein kinase related (Drak)-dependent AJ remodelling while also promoting neurogenesis. In conclusion, this work reveals an important role for Drak in regulating AJ remodelling during retinal morphogenesis. It also sheds new light on the interplay between Ato, EGFR-dependent transcription and AJ remodelling in a system in which neurogenesis is coupled with cell shape changes and regulated steps of cell intercalation (Robertson, 2013).

In Drosophila, Rok seems to be the main kinase responsible for phosphorylating the Myosin regulatory light chain (Sqh) during epithelial patterning and apical cell constriction. This is the case for the activation of MyoII during intercalation as germband extension proceeds, but also during various instances of compartment boundary formation and cell sorting situations in the embryo and in the wing imaginal disc. The current work reveals that in the constricting cells of the MF, Rok functions redundantly with Drak, a kinase recently shown to phosphorylate Sqh both in vitro and in vivo (Neubueser, 2010). It is noteworthy that previous work has shown that RhoGEF2 is not required for cell constriction in the MF, suggesting that perhaps another guanine exchange factor (GEF) might function redundantly with RhoGEF2 to promote cell constriction. These data on Drak reinforce the idea that redundancies exist in this context. Because the RhoA (Rho1 -- FlyBase) loss of function abolishes this cell response entirely, it would be expected that Drak function is regulated by RhoA. In addition, the current data indicate that Drak acts redundantly with Rok during MyoII-dependent multicellular alignment and AJ remodelling during ommatidia patterning. It will be interesting to test whether Drak functions in other instances of epithelial cell constriction or MyoII-dependent steps of AJ remodelling in other developmental contexts in Drosophila (Robertson, 2013).

This study demonstrates a two-tiered mechanism regulating the planar polarization of MyoII and Baz. In the constricting cells in the posterior compartment, MyoII and Baz are segregated from one another and this is exacerbated by the wave of cell constriction in the MF. Upon Ato-dependent transcription in the MF cells, this segregated pattern of expression is harnessed and these factors become planar polarized at the posterior margin of the MF. This is independent of the core planar polarity pathway including the Fz receptor and is accompanied by a striking step of multicellular alignment. Previous work has demonstrated that Ato upregulates E-Cad transcription at the posterior boundary of the MF. In addition, apical constriction leads to an increase in E-Cad density at the ZA. The current data are therefore consistent with both hh-dependent constriction and ato-dependent transcriptional upregulation of E-Cad promoting differential adhesion, thus leading to a situation in which the ato+ cells maximize AJ contacts between themselves and minimize contact with the flanking cells that express much less E-Cad at their ZA. This typically leads to a preferential accumulation of cortical MyoII at the corresponding interface. Such actomyosin cables are correlated with increased interfacial tension, and it is proposed that this is in turn responsible for promoting cell alignment. Unfortunately, the very small diameter of these constricted cells precludes direct measurements of the AJ-associated tension using laser ablation experiments (Robertson, 2013).

Supra-cellular cables of MyoII have been previously associated with cell alignment in various epithelia and have also been observed at the boundary of sorted clones, whereby cells align at a MyoII-enriched interface. Interestingly, this study found that the actomyosin cable defining the posterior boundary of the MF is also preferentially enriched for Rok, a component of the T1, MyoII-positive AJ in the ventral epidermis (Simoes Sde, 2010). This indicates an important commonality between actomyosin cable formation during cell sorting and the process of cell intercalation. However, unlike during intercalation, this study found that in the developing retina baz is largely dispensable for directing the pattern of E-Cad and actomyosin planar polarization. Further work will therefore be required to understand better the relationship between Baz and E-Cad at the ZA during ommatidia morphogenesis. It is speculated that the creation of a high E-Cad versus low E-Cad boundary in the wake of the MF might be sufficient to promote Rok and MyoII enrichment at the posterior AJs. This posterior Rok and MyoII enrichment might perhaps prevent E-Cad accumulation by promoting E-Cad endocytosis, as has been recently shown in the fly embryo (Robertson, 2013).

This study has used live imaging to define a conserved step of ommatidia patterning that consists of the coalescence of the ommatidial cells' AJs into a central vertex to form a 6-cell rosette. The corresponding steps of AJ remodelling require Rok, Drak, Baz and MyoII, a situation compatible with mechanisms previously identified during cell intercalation in the developing fly embryo. The steps of AJ remodelling required to transform lines of cells into 5-cell pre-clusters are transcriptionally regulated downstream of EGFR in a ligand-dependent manner. Interestingly, in the eye EGFR signalling is activated in the cells that form lines and type1-arcs in the wake of the MF and, thus, are undergoing AJ remodelling. Previous work examining tracheal morphogenesis in the fly has demonstrated that interfaces between cells with low levels versus high levels of EGFR signalling correlate with MyoII-dependent AJ remodelling in the tracheal placode. This situation resembles that which is described in this study in the wake of the MF. In the eye, however, it was found that EGFR signalling is not required to initiate cell alignment. Nevertheless, taken together with work in the tracheal placode and previous studies related to multicellular patterning in the developing eye, this work indicates a conserved function for the EGFR signalling pathway in promoting MyoII-dependent AJ remodelling. This leaves open several interesting questions; for example, it is not presently clear how EGFR signalling can promote discrete AJ suppression and elongation. It is, however, tempting to speculate that previously described links between EGFR signalling and the expression of E-Cad or Rho1 might play a role during this process (Robertson, 2013).


Activation of EGFR and ERK by rhomboid signaling regulates the consolidation and maintenance of sleep in Drosophila

Epidermal growth factor receptor (EGFR) signaling in the mammalian hypothalamus is important in the circadian regulation of activity. This study examined the role of the EGF pathway in the regulation of sleep in Drosophila. The results demonstrate that rhomboid (Rho)- and Star-mediated activation of EGFR and ERK signaling increases sleep in a dose-dependent manner, and that blockade of rhomboid (rho) expression in the nervous system decreases sleep. The requirement of rho for sleep localized to the pars intercerebralis, a part of the fly brain that is developmentally and functionally analogous to the hypothalamus in vertebrates. These results suggest that sleep and its regulation by EGFR signaling may be ancestral to insects and mammals (Foltenyi, 2007).

The findings reported here show a previously unknown role for EGFR and ERK signaling in sleep regulation and consolidation in Drosophila. In the adult fruit fly, EGFR is expressed ubiquitously throughout the nervous system, where its only known role is in the maintenance and survival of neurons. The current results demonstrate that the overexpression of EGFR pathway signaling components Rho and Star in Drosophila causes an acute, reversible and dose-dependent increase in sleep that tightly parallels an increase in phosphorylated ERK in the head. The ability of a dominant-negative EGFR to block the activation of ERK, as well as the known selectivity of Rho for these ligands, argues that the manipulation is specific to the EGFR pathway. In contrast to the increase in sleep amount after Rho overexpression, inhibiting its expression led to a significant decrease in sleep. Notably, this decrease in sleep was due to a marked shortening of the duration of sleep episodes accompanied by an elevation of sleep bout number. This observation suggests that flies have an increased need for sleep, but are unable to stay asleep, which is perhaps analogous to insomnia in humans. Therefore, it is proposed that the EGFR pathway is essential for sleep maintenance (Foltenyi, 2007).

The brain regions that appear to be involved in the influence of signaling by Rho, EGFR and ERK on sleep are the pars intercerebralis, median bundle and tritocerebrum. The cells of the pars intercerebralis contain Rho and generate EGFR ligand that activates ERK in the receiving cells in the tritocerebrum. The pars intercerebralis was identified as the region that is responsible for EGFR ligand secretion by demonstrating that inhibiting Rho in this region resulted in decreased sleep, and that the cells in that region expressed endogenous Rho. The tritocerebrum was identified though the system-wide overexpression of the EGFR ligand-processing components Rho and Star, which resulted in a localized hyperactivation of ERK. This is presumably because an ectopic presence of Rho and Star will only result in heightened EGFR signaling if the cells contain endogenous ligand precursor (Foltenyi, 2007).

Although the mushroom body is the only region of the Drosophila brain that has been reported to have an effect on sleep, no Rho expression was detected in the mushroom body, nor did inhibiting Rho with UAS-rhoDN in this structure have any effect on sleep levels. However, it is reasonable to expect that the regulation of sleep would involve multiple brain regions and pathways, and that the regulation, versus the function, of sleep could be two distinct, but linked, processes (Foltenyi, 2007).

Cells of the pars intercerebralis send out axonal projections though the median bundle and then bifurcate, innervating the tritocerebrum or running alongside the esophageal canal to innervate the endocrine gland corpora cardiaca. The results indicate that the pars intercerebralis cells innervating the corpora cardiaca are not the ones responsible for the observed decrease in sleep; Gal-4 drivers that are active in these cells did not produce a significant drop in sleep levels when expressing rho RNAi. Developmental studies have led to the postulate that the pars intercerebralis and the corpora cardiaca are the developmental equivalent of the mammalian hypothalamic-pituitary axis. The hypothalamus is a major center in the mammalian brain for the regulation of arousal, and the SCN, which is a part of the hypothalamus, has already been shown to regulate circadian activity through EGFR signaling (Foltenyi, 2007).

Vertebrate studies have only investigated EGFR signaling in the subparaventricular zone, a region located immediately adjacent to the SCN, and this region was shown not to affect total sleep levels, but does alter its timing. In addition, evidence in mammals for a role of EGF in sleep per se is equivocal. These results directly demonstrate that the disruption of EGFR ligand production affects sleep though the pars intercerebralis and not though the circadian control center of the Drosophila brain. It also suggests that the pars intercerebralis shares some functional, as well as developmental, homology with the mammalian hypothalamus through its crucial and conserved involvement in regulating sleep and its maintenance with neural hormones such as the EGFR ligands (Foltenyi, 2007).

In the fly, a single member of the EGFR family binds both the TGF-α-like family of ligands (Spitz, Gurken and Keren) and the neuregulin-like ligand Vein. In vertebrates, these ligands bind to specific ErbB family members, with ErbB-1 (EGFR) binding EGF and TGF-α, whereas ErbB-3 and ErbB-4 bind the neuregulins. In mammalian systems, ErbB-2 and ErbB-4 cofractionate, coimmunoprecipitate and colocalize in cultured rat hippocampal neurons with the postsynaptic density protein PSD-95 (also known as SAP90), and show exclusion from presynaptic terminals. Similarly, ERK colocalizes with, and directly phosphorylates, PSD-95, as is the case with the ErbB receptor-family members. In the fly, EGFR interacts with the postsynaptic density protein Discs Large (Dlg), the Drosophila homolog of PSD-95 (Foltenyi, 2007).

ERK has a role in synaptic plasticity that is conserved among Aplysia, Drosophila and mammals. A recent study shows that ERK directly phosphorylates the pore-forming α subunit of the A-type potassium channel Kv4.2, a member of the Shal-type (Shaker-like) family. This broadens the role of ERK beyond the realm of cell proliferation, differentiation, and even long-term memory consolidation, and suggests that it may also contribute to the more immediate alterations of the electrical properties of the neuronal membrane (Foltenyi, 2007).

On the basis of the current findings and the published reports on the functions of EGFR, the following cellular mechanism is proposed for sleep regulation in Drosophila. Star and Rho in the pars intercerebralis produce and secrete ligand to EGFR located at the postsynaptic membrane of neurons in the tritocerebrum, leading to the activation of ERK in these cells. The difference in staining patterns between inactive ERK clustering near synapses and active ERK located out in the axons indicates that the activated ERK, at least in part, translocates from the postsynaptic membrane and spreads out into the axons that fill out the tritocerebrum and other locations to which these cells project. As a result of a lack of ppERK in the cell bodies of these neurons and the reversible nature of the sleep behavior, it is unlikely that these cells are undergoing long-term synaptic structural changes associated with changes in gene expression. Instead, it is proposed that the action of ppERK occurs at the synapse or in the axon (or both), where it is possibly altering the gating of a neural receptor or channel, and thus changing the membrane properties of the cells. This modification results in an altered brain state that ultimately manifests itself in the sleep behavior of the animal. Such a model is consistent with a previously described mutation in the potassium channel shaker (Kv1.4), which has been shown to be incapable of getting much sleep (Foltenyi, 2007).

EGFR signaling modulates synaptic connectivity via Gurken

Synaptic target selection is critical for establishing functional neuronal circuits. The mechanisms regulating target selection remain incompletely understood. This study describes a role for the EGF receptor and its ligand Gurken in target selection of octopaminergic Type II neurons in the Drosophila neuromuscular system. Mutants in happyhour, a regulator of EGFR signaling, form ectopic Type II neuromuscular junctions. These ectopic innervations are due to inappropriate target selection. It was demonstrated that EGFR signaling is necessary and sufficient to inhibit synaptic target selection by these octopaminergic Type II neurons, and that the EGFR ligand Gurken is the post-synaptic, muscle-derived repulsive cue. These results identify a new pathway mediating cell-type and branch-specific synaptic repulsion, a novel role for EGFR signaling in synaptic target selection, and an unexpected role for Gurken as a muscle-secreted repulsive ligand (Naylor, 2011).

Synaptic target selection is a critical step in establishing functional neural circuits. The molecular mechanisms governing this selection have not yet been fully explored. The observation that hppy mutants have an increased frequency of ectopic octopaminergic Type II NMJs has resulted in identification of a pathway critical for synaptic target recognition in these neurons. EGFR signaling pathway was found to be required to prevent the development of inappropriate synaptic contacts. This inhibitory signal is mediated by muscle-derived EGFR ligand, Gurken, working through the EGF receptor in type II motoneurons. This mechanism sculpts the neuronal wiring pattern in a cell-type and branch-specific manner (Naylor, 2011).

There are many signaling pathways that influence the innervation pattern of Drosophila motor neurons. These findings identify a novel role for EGFR signaling in mediating a repulsive guidance cue to Type II neurons. EGF has previously been demonstrated to regulate axon growth and guidance. For example, EGF positively regulates Sema-3a levels in the cornea and interacts with NCAM-180 to promote neuritogenic activity. However, these data are the first to demonstrate that an EGF receptor and ligand provide a synaptic targeting signal (Naylor, 2011).

Ectopic Type II NMJs were found in hppy mutants, as well as when a dominant negative EGFR was expressed in Type II neurons or its ligand Gurken was knocked down in the muscle targets. This presents an apparent contradiction. hppy is described as a negative regulator of EGFR (Corl, 2009), and its phenotype in this system is suppressed by a hypomorphic mutation in rolled (ERK), consistent with hppy functioning as a negative regulator of EGFR signaling. Why then does loss of hppy have the same phenotype as inhibition of the receptor or ligand? It was posited that the well-described strong negative feedback induced by EGFR signaling may be the explanation. A model is proposed in which activation of the EGFR pathway mediates a signal that inhibits the formation or stabilization of Type II NMJs. In hppy mutants, however, loss of negative regulation would allow for excess activation of the EGFR that would induce a quick, strong and long-lasting negative feedback activity early in development, essentially turning off EGFR signaling in cells expressing happyhour. The result would be loss of the synaptic inhibitory signal mediated by EGFR at the stage when these Type II neurons are extending to their targets and the promotion of ectopic synapse formation. It is appreciated that this model is speculative, and that an alternative is that hppy and rolled are regulating a pathway that is distinct from the EGFR/gurken pathway (Naylor, 2011).

By what mechanism does EGFR signaling affect synapse formation in Type II neurons? Presumably, there is a molecular program downstream of EGFR activation that modifies the Type II neuron such that it does not form and/or maintain an NMJ with an inappropriate target. These changes could occur at the level of the cell body or locally within individual branches. It is unlikely that cell body changes are central to the phenotype because such neuron-wide mechanisms could not easily be translated into branch-specific behavior. In contrast, local effects of EGFR signaling within neurites could explain such specificity. The cell biological mechanism mediating the branch-specific inhibition is not known. Possibilities include alterations in the local translation or membrane insertion of synaptogenic molecules, local modulation of cytoskeletal dynamics, or failure to properly prune Type II connections (Naylor, 2011).

Not only is the EGFR mediating a branch-specific effect, but it is also cell-type specific. The Type II motoneuron MNSNb/d-II and the Type Is motoneuron MNSNb/d-Is travel together and presumably encounter the same cues across the hemisegment, however they generate different innervation patterns. This implies that these two types of neurons have developed cell-type specific repertoires of receptors or signaling pathways that shape their target choices (Naylor, 2011).

While the data indicate a role for EGFR signaling in Type II synaptic target selection it is also likely that Type II target selection has multiple components. The phenotypes occur at a relatively low penetrance, so it is likely that complementary and combinatorial guidance cues function with the EGFR pathway to shape target selection of Type II neurons (Naylor, 2011).

Gurken has been studied exclusively in the developing oocyte and has no known roles in other tissues. Hence, it is surprising that Gurken conveys the repulsive signal from muscle to the octopaminergic Type II neurons. In support of a function in muscle, the modEncode RNA-seq project has found that Gurken transcript is enriched 3.5 fold in larval body wall muscles. While Gurken may be secreted from all muscles, a model is preferred in which localized expression in a muscle subset shapes the branching pattern of the innervating motoneuron. In this model, Gurken released from muscles 6 and 7, as well as other targets that should not be innervated, would locally inhibit synaptogenesis, blocking the formation of ectopic connections while allowing for the normal innervation at muscles 12 and 13. This model is consistent with findings that knockdown of Gurken in muscle results in ectopic NMJs while localized overexpression in the normal target cell inhibits formation of appropriate NMJs. While these functional data are strong, the model must remain speculative because it has not been possible to determine the localization of Gurken using currently available reagents. Future studies will investigate how this Gurken/EGFR pathway is integrated with the recently defined semaphorin- and activity-dependent mechanisms that also play an important role in shaping synaptic target selection in these neurons (Naylor, 2011).

Regulation of neurogenesis and epidermal growth factor receptor signaling by the Insulin receptor/Target of rapamycin pathway in Drosophila

Determining how growth and differentiation are coordinated is key to understanding normal development, as well as disease states such as cancer, where that control is lost. Growth and neuronal differentiation are coordinated by the insulin receptor/target of rapamycin (TOR) kinase (InR/TOR) pathway. The control of growth and differentiation diverge downstream of TOR. TOR regulates growth by controlling the activity of S6 kinase (S6K) and eIF4E. Loss of s6k delays differentiation, and is epistatic to the loss of tsc2, indicating that S6K acts downstream or in parallel to TOR in differentiation as in growth. However, loss of eIF4E inhibits growth but does not affect the timing of differentiation. This study shows that there is crosstalk between the InR/TOR pathway and epidermal growth factor receptor (EGFR) signaling. InR/TOR signaling regulates the expression of several EGFR pathway components including pointedP2 (pntP2). In addition, reduction of EGFR signaling levels phenocopies inhibition of the InR/TOR pathway in the regulation of differentiation. Together these data suggest that InR/TOR signaling regulates the timing of differentiation through modulation of EGFR target genes in developing photoreceptors (McNeill, 2008).

Tight coordination of growth and differentiation is essential for normal development. InR/TOR signaling controls the timing of neuronal differentiation in the eye and leg in Drosophila. This study demonstrates that the InR/TOR pathway regulates neuronal differentiation in an S6K-dependent, but 4EBP/eIF4E-independent manner. It has previously been impossible to determine whether InR/TOR signaling was acting downstream or in parallel to the EGFR/MAPK pathway. Using argos and rho as reporters this study shows that the InR/TOR pathway is able to regulate EGFR/MAPK signaling downstream of MAPK. Moreover, pntP2 expression is up- and downregulated by activation or inhibition of InR/TOR signaling, respectively, and InR/TOR and EGFR pathways interact through pntP2. Taken together these data suggest that temporal control of differentiation by the InR/TOR pathway is achieved by modulation of EGFR pathway transcriptional targets in differentiating PRs (McNeill, 2008).

TOR is part of two multimeric complexes (TORC1 and TORC2) and is a core component of the InR pathway. TORC1 activity is regulated by nutrient and energy levels providing a conduit for hormonal and catabolic cellular inputs. Growth is regulated by two downstream targets of TORC1: S6K and 4EBP. The current data demonstrate that upstream of TORC1, differentiation and growth are regulated by the same factors. Downstream of TORC1, differentiation and growth differ significantly in that loss of s6k, but not eIF4E (or overexpression of 4EBP) affects differentiation. eIF4E regulates 7-methyl-guanosine cap-dependent translation and is the rate-limiting factor in translation initiation. The finding that eIF4E does not affect differentiation suggests that the temporal control of differentiation is not based on a translation initiation-dependent mechanism. Strikingly, loss of s6k blocks the precocious differentiation induced by loss of tsc2. Given the relatively weak effects of loss of s6k this may seem surprising. However, the degree of suppression is similar to the effect of loss of s6k on the overgrowth phenotype caused by loss of tsc2, namely, tsc2, s6k double-mutant cells are the same size as wild-type cells. Although loss of eIF4E has no affect on differentiation it may act redundantly with another factor, such as s6k. Testing this hypothesis though is technically challenging since the Drosophila genome contains eight different eIF4E isoforms. It will be interesting in future to test whether any of these isoforms regulate differentiation or alternatively whether eIF4E and s6k act redundantly. Although further work is required to determine the precise relationship between S6K and the InR/TOR pathway, the data point to a critical role of S6K in coordinating neuronal differentiation and growth (McNeill, 2008).

As in other neuronal systems, differentiation of PRs in the Drosophila eye occurs in a stereotyped manner. The advantage of the Drosophila retina as an experimental system is that the PRs differentiate spatiotemporally. Using this feature, as well as a series of cell-type-specific antibodies, this study has demonstrated that InR/TOR signaling is selective in the cell-types that it affects. The differentiation of PRs 2/5, 3/4, and 8 are unaffected by perturbations in InR/TOR signaling, whereas PRs 1, 6, and 7 and cone cells are dependent on this pathway for temporal control of differentiation. Interestingly the affected cells all differentiate after the second mitotic wave. However, regulators of the cell cycle do not affect the temporal control of differentiation. Why then are PRs 1, 6, and 7 and cone cells specifically affected? In cells with increased InR/TOR signaling, the expression of argos, rho, and pntP2 is precocious and increased throughout the clone, suggesting that the upregulation of EGFR signaling occurs in all cells. However, decreasing EGFR activity using a hypomorphic pntP2 allele specifically affects the differentiation of PRs 1, 6, and 7 and cone cells. Interestingly, pntP2 expression in differentiated cells is also restricted to PRs 1, 6, and 7 and cone cells. These observations suggest that differentiation of PRs 1, 6, and 7 and cone cells is critically dependent on EGFR levels signaling through pntP2. Therefore, although activation of InR/TOR signaling causes upregulation of EGFR transcriptional targets in all cells as they differentiate, the phenotypic effect is seen only in PRs 1, 6, and 7 and cone cells since these cells are highly sensitive to EGFR activity signaling through pntP2. This possibility is supported by the fact that precocious differentiation caused by overexpression of Dp110 can be suppressed by the simultaneous reduction of pntP2 levels. The complete suppression of the Dp110 differentiation phenotype by simultaneous reduction of pntP2 strongly suggests that pntP2 acts downstream of Dp110 and InR/TOR signaling in a pathway that regulates the temporal control of differentiation. It has been suggested that later differentiating PRs require higher levels of EGFR activity than their earlier differentiating neighbors. In particular, the activation of PR 7 requires both EGFR and Sevenless RTKs. In the case of InR/TOR pathway activation it may be that, through its regulation of EGFR downstream targets, the 'second burst' of RTK activity is enhanced causing PRs 1, 6, and 7 and cone cells to differentiate precociously. There may also be other as yet unidentified factors through which the InR/TOR pathway controls the expression of Aos and rho in PRs 2-5 and 8 (McNeill, 2008).

Activation of insulin and insulin-like growth factor receptors in mammalian systems is well known to elicit a response via the Ras/MAPK pathway. However, loss of the InR in the Drosophila eye does not result in a loss of PRs, a hallmark of the Ras pathway, nor does mutation of the putative Drk binding site in chico affect the function of the Drosophila IRS. In accordance with these data no change is seen in dpERK staining when the InR/TOR pathway is activated in the eye disc. Rather than a direct activation of Ras signaling by the InR, the data suggest that in the developing eye crosstalk between these pathways occurs at the level of regulation of the expression of EGFR transcriptional outputs. The most proximal component of the EGFR pathway that is regulated by InR/TOR signaling is pntP2. However, the data suggest that temporal control of PR differentiation requires concerted regulation of EGFR transcriptional outputs, since overexpression of pntP2 alone is not sufficient to cause precocious differentiation, whereas overexpression of activated EGFR is sufficient. Interestingly, microarray analyses of Drosophila and human cells have shown that the InR/TOR pathway regulates the expression of hundreds of genes. The mechanism by which this transcriptional control is exerted has yet to be elucidated. It will be interesting in future to determine the extent of transcriptional crosstalk between InR/TOR and EGFR pathways in developing neurons (McNeill, 2008).

EGFR signaling regulates the proliferation of Drosophila adult midgut progenitors

In holometabolous insects, 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, as visualized using esgGal4-driven GFP expression, 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).

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. Studies have suggested that AMP proliferation might precede the onset of metamorphosis. However, the extensive proliferation of the AMPs that is seen in this study has not been reported and the early larval proliferative phase when the AMPs divide and disperse has not been reported. 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).

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 seen 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 Shot-mediated concentration of Vn on AMPs activates EGFR signaling in the AMPs during early larval development. Consistent with this, dpERK staining is only seen 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 seen when sSpi, sKrn, lambdaTOP (activated Egfr) or RasV12 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 that 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 (Reich, 2002; 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). RasV12-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, it was shown 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 went 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 remained small and diploid and maintained strong esgGal4 expression. For several reasons, it is thought that these esg-positive cells are the future adult intestinal stem cells (ISCs). (1) esgGal4 expression marks AMPs, including adult ISCs and enteroblasts. (2) Mitoses in the adult midgut are only observed in ISCs, and this study observed mitoses only in the esg-positive cells during metamorphosis. (3) esg-positive cells migrated to the basal side of the midgut epithelium, the location of adult ISCs. (4) 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 contained 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 lacked 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 the 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 were induced at mid-third instar, the mosaic clusters always contained 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 were never observed, as judged by their ploidy (diploid) and lack of expression of the enteroendocrine marker Prospero. 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 and ISCs in adult midgut homeostasis, it is edexpect 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 (RasV12), 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. (1) 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. (2) The structure of the midgut epithelium with basal AMPs or ISCs is similar in larval and adult stages. (3) 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. This study 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).

EGF signaling regulates the proliferation of intestinal stem cells in Drosophila

Precise control of somatic stem cell proliferation is crucial to ensure maintenance of tissue homeostasis in high-turnover tissues. In Drosophila, intestinal stem cells (ISCs) are essential for homeostatic turnover of the intestinal epithelium and ensure epithelial regeneration after tissue damage. To accommodate these functions, ISC proliferation is regulated dynamically by various growth factors and stress signaling pathways. How these signals are integrated is poorly understood. This study shows that EGF receptor signaling is required to maintain the proliferative capacity of ISCs. The EGF ligand Vein is expressed in the muscle surrounding the intestinal epithelium, providing a permissive signal for ISC proliferation. The AP-1 transcription factor FOS serves as a convergence point for this signal and for the Jun N-terminal kinase (JNK) pathway, which promotes ISC proliferation in response to stress. These results support the notion that the visceral muscle serves as a functional 'niche' for ISCs, and identify FOS as a central integrator of a niche-derived permissive signal with stress-induced instructive signals, adjusting ISC proliferation to environmental conditions (Biteau, 2011).

These findings establish a crucial role for EGF signaling in the regulation of ISC proliferation, and thus support the notion that the visceral muscle surrounding the intestinal epithelium has the characteristics of a functional niche. vein expression in the muscle maintains the competence of ISCs to enter rapid proliferation in responses to stress and JNK signaling, and is thus expected to regulate epithelial homeostasis. Interestingly, it was found that both the EGFR-mediated permissive signal and the JNK-derived inductive signal are relayed by FOS, establishing an integrated molecular mechanism for the control of ISC proliferation (Biteau, 2011).

Many stem cell populations are regulated by their microenvironments, and larval ISC progenitors are regulated by a transient niche (Mathur, 2010). However, ISCs in adult flies apparently lack such a closely associated cell population within the intestinal epithelium. By contrast, control of ISC maintenance by muscle-derived Wingless suggested this tissue as a potential functional niche for adult ISCs. The current results support and extend this idea by identifying a second growth factor derived from the visceral muscle that controls ISC proliferation. In its regulation of stem cell function through Wingless and Vein, and in the close association of ISCs and muscle cells, the muscle thus shares characteristics of stem cell niches in other systems, yet it also differs from these in important ways. In mammals, as well as in the Drosophila and C. elegans gonads, the niche of most stem cell populations maintains stem cell quiescence and prevents differentiation. The EGF signal originating from the muscle, however, maintains the capacity of ISCs to divide, allowing these cells to respond to stimulating signals while not affecting ISC differentiation. Interestingly, EGFR signaling has not been described so far as crucial for interactions between the niche and stem cell populations in other systems, and these findings raise the possibility that this signaling pathway might also regulate the function of other stem cell populations in both invertebrates and vertebrates (Biteau, 2011).

Whereas knocking down the expression of vein in the muscle partially affects the ability of ISCs to proliferate under normal conditions and in response to stress, the inhibition of EGFR completely abolishes stem cell division. This might reflect the inefficiency of the veinRNAi constructs used in this study, but might also suggest a contribution of other EGFR ligands to the regulation of ISC function. Accordingly, a genome-wide analysis of the transcriptional response of the adult intestine to bacterial infection suggests that expression of vein, as well as of two other genes encoding EGFR ligands, Keren and spitz, is increased after immune challenge. However, the potential role for these additional EGF-like ligands in regulating ISC function remains to be investigated and the cells expressing spitz and Keren in the adult intestine have yet to be identified (Biteau, 2011).

ISC function is regulated by systemic insulin-like peptides expressed by neurosecretory cells in the brain, muscle-derived vein and wingless, local unpaired cytokine expressed by ECs, and cell-intrinsic signals. These multiple signals are integrated in ISCs to adapt their proliferation rate and differentiation program to environmental and physiological challenges. To fully understand stem cell regulation in this high-turnover tissue, the molecular structure of this signaling network has to be unraveled. The findings of this study introduce the transcription factor FOS as a crucial regulator of ISC proliferation that integrates mitogenic and stress signals, and indicate that JNK and ERK regulate FOS activity directly by phosphorylation on distinct residues, controlling ISC proliferation in a combinatorial fashion. This signal-specific mode of FOS regulation by ERK and JNK in Drosophila had previously been described in the context of morphogenetic movements (in which FOS is regulated by JNK) and of eye and wing growth during development (in which it is regulated by ERK and JNK) (Biteau, 2011).

How FOS promotes ISC proliferation remains unclear. In developing imaginal discs, inhibition of FOS causes an accumulation of cells in the G2/M phase of the cell cycle, probably owing to a loss of Cyclin B expression, an essential regulator of the G2/M transition. Interestingly, in ISCs, expression of FosRNAi not only inhibits stress-induced accumulation of pH3+ cells, but also represses BrdU incorporation, indicating that FOS regulates entry into S phase. In these cells, FOS might thus regulate the transcription of essential S phase components. Further studies will be required to identify such ISC-specific FOS target genes (Biteau, 2011).

The maintenance of stem cells in a primed state, ready to respond to inductive mitogenic stress signals, is likely to be crucial for high-turnover tissues like the intestinal epithelium, which require rapid activation of stem cell division for an efficient regenerative response to tissue damage. At the same time, this enhanced mitogenic potential of ISCs might contribute to the loss of tissue homeostasis in the aging gut, and contribute to cancer formation in mammalian intestinal epithelia. Interestingly, a conserved role of AP-1 transcription factors and JNK signaling in the regulation of intestinal stem cell proliferation and intestinal cancer is emerging in mice. JNK activation is sufficient to induce cell proliferation in the intestinal crypt and increases tumor incidence and tumor growth in an inflammation-induced colon cancer model. These effects of JNK signaling are mediated by the FOS binding partner JUN, as shown by the requirement for JNK-mediated phosphorylation of JUN for APCmin/+-induced tumorigenesis. Strikingly, ISC-specific activation of WNT signaling, by mutating APC or expressing an active form of ß-catenin or wingless itself, is sufficient to induce the formation of tumor-like stem cell clusters in the fly intestine. A potential interaction of WNT signaling with JNK and JUN or FOS in ISCs remains to be tested in Drosophila. Interestingly, increased FOS activity has also recently been shown to be sufficient to promote hematopoietic stem cell self-renewal in mice, further illustrating the conserved function of FOS in the regulation of stem cell function (Deneault, 2009). AP-1 transcription factors are thus emerging as conserved essential regulators of stem cell function and the current findings provide an important starting point for further studies characterizing stem cell-specific signaling networks that integrate mitogenic, survival and stress signals to control stem cell maintenance, quiescence and proliferation, and thus influence the balance between regeneration and tumor suppression in high turnover tissues (Biteau, 2011).

Hole-in-one mutant phenotypes link EGFR/ERK signaling to epithelial tissue repair in Drosophila

Epithelia act as physical barriers protecting living organisms and their organs from the surrounding environment. Simple epithelial tissues have the capacity to efficiently repair wounds through a resealing mechanism. The known molecular mechanisms underlying this process appear to be conserved in both vertebrates and invertebrates, namely the involvement of the transcription factors Grainy head (Grh) and Fos. In Drosophila, Grh and Fos lead to the activation of wound response genes required for epithelial repair. ERK is upstream of this pathway and known to be one of the first kinases to be activated upon wounding. However, it is still unclear how ERK activation contributes to a proper wound response and which molecular mechanisms regulate its activation. In a previous screen, mutants were isolated with defects in wound healing. This study describes the role of one of these genes, hole-in-one (holn1), in the wound healing process. Holn1 is a GYF domain containing protein that is required for the activation of several Grh and Fos regulated wound response genes at the wound site. Evidence is provided suggesting that Holn1 may be involved in the Ras/ERK signaling pathway, by acting downstream of ERK. Finally, it was shown that wound healing requires the function of EGFR and ERK signaling. Based on these data, it is concluded that holn1 is a novel gene required for a proper wound healing response. A model is proposed whereby Holn1 acts downstream of EGFR and ERK signaling in the Grh/Fos mediated wound closure pathway (Geiger, 2011).

Holn1 is not required for the initial rapid response to wound infliction, i.e. the formation of the actomyosin cable within minutes of wounding and the phosphorylation of ERK, which is also detectable soon after wounding. This observation is consistent with Holn1 playing an indirect role in the mechanics of wound closure by regulating the mRNA levels of genes required for this process, such as those involved in rapid and productive cable contraction. Interestingly, the actin cable was present in all the wound closure mutants isolated in the previous screen, suggesting that regulatory events downstream of cable formation dominate the wound closure process. In any case, it is clear that Holn1 is required to perform some additional function needed to sustain the closure process, as holn1 mutants take on average 1.5 times longer to close a wound compared to wild type embryos. A similar delay in wound closure was previously reported for rho1 GTPase mutants, which do not form an actin cable, but can still close small wounds, albeit 2 times slower than wild type embryos]. Aside from its possible role in the epithelial hole closure process, Holn1 could also be involved in cuticle repair. Grh and ERK activity are required for the re-establishment of the epithelial permeability barrier after injury. Thus, Holn1 might be involved in this process by regulating the ERK/Grh pathway (Geiger, 2011).

In the future, just as the holn1 mutation uncovered a connection with the EGF/Ras/ERK signaling pathway and wound healing, microarray analysis of wounded holn1 embryos would identify genes that are likely activated downstream of this wound closure pathway. Performing the same experiment using an alternative splicing array as in would further reveal if Holn1 plays a role in wound dependent splicing events (Geiger, 2011).

Antagonistic feedback loops involving rau and sprouty in the Drosophila eye control neuronal and glial differentiation

During development, differentiation is often initiated by the activation of different receptor tyrosine kinases (RTKs), which results in the tightly regulated activation of cytoplasmic signaling cascades. In the differentiation of neurons and glia in the developing Drosophila eye, this study found that the proper intensity of RTK signaling downstream of fibroblast growth factor receptor (FGFR) or epidermal growth factor receptor require two mutually antagonistic feedback loops. A positive feedback loop was identified mediated by the Ras association (RA) domain-containing protein Rau (CG8965) that sustains Ras activity and counteracts the negative feedback loop mediated by Sprouty. Rau has two RA domains that together show a binding preference for GTP (guanosine 5'-triphosphate)-loaded (active) Ras. Rau homodimerizes and is found in large-molecular weight complexes. Deletion of rau in flies decreases the differentiation of retinal wrapping glia and induces a rough eye phenotype, similar to that seen in alterations of Ras signaling. Further, the expression of sprouty is repressed and that of rau is increased by the COUP transcription factor Seven-up in the presence of weak, but not constitutive, activation of FGFR. Together, these findings reveal another regulatory mechanism that controls the intensity of RTK signaling in the developing neural network in the Drosophila eye (Sieglitz, 2013).

During development, often single bursts of RTK activity suffice to direct important cellular decisions. In other cases, multiple rounds of RTK activation are required to trigger a certain reaction profile, and in yet other cases, such as in the developing eye imaginal disc, a sustained low level of EGFR activity is needed. This study identified the Drosophila RA domain containing protein Rau, which constitutes the first cell-autonomous positive feedback regulator acting on both EGFR- and FGFR-induced signaling. In the developing fly compound eye, it was found that sustained RTK activity is modulated through a positive feedback loop initiated by Rau, which is counterbalanced by the negative regulator Sprouty. The balance of these two regulatory mechanisms ensures the correct activity of EGFR- and FGFR-dependent signaling pathways in the developing eye (Sieglitz, 2013).

Within the RTK signaling pathway, different positive and negative feedback mechanisms have been identified. A prominent negative feedback mechanism is triggered by the secreted protein Argos. Argos expression is induced by RTK activation, and secreted Argos protein can sequester the activating ligand Spitz. In addition, intracellular proteins have been identified to exert a negative feedback function. Sprouty is the most prominent inhibitor of RTK activity and was shown in this study to act downstream of FGFR signaling as well as downstream of EGFR signaling. However, the precise point at which Sprouty intercepts RTK signaling is variable. In the developing fly eye, Sprouty acts upstream of Ras, whereas in the developing wing, Sprouty functions at the level of Raf. An additional negative feedback loop is mediated by the cell surface protein Kekkon, which is specific to EGFR signaling (Sieglitz, 2013).

Positive feedback loops are less frequent and may act through the transcriptional activation of genes that encode activating ligands. This is found in the ventral ectoderm of Drosophila embryos or in follicle epithelium, where the activity of the EGFR pathway is amplified by induction of the expression of its ligand Vein. In addition, the expression of rhomboid may be triggered, which subsequently facilitates the release of activating ligands. Together, these mechanisms ensure a paracrine-mediated amplification of the RTK signal and are thus likely not as effective in regulating RTK activity in single cells (Sieglitz, 2013).

Rau is a previously unidentified positive regulator of RTK signaling that acts within the cell. This study found that Rau function sustains both EGFR and FGFR signaling activity. Rau is a small 51-kD protein that harbors two RA domains, which are found in several RasGTP effectors such as guanine nucleotide–releasing factors. Pull-down experiments demonstrated that Rau preferentially binds GTP-loaded (activated) Ras. The Rau protein is characterized by two RA domains. Although both RA domains are able to bind Ras individually, single RA domains do not show any selectivity toward the GTP-bound form of Ras. Thus, the clustering of two RA domains promotes the selection of RasGTP. In agreement with this notion, it was found that Rau can form dimers or, possibly, multimers. In lysates from embryos, Rau is found in high–molecular weight protein complex, suggesting that it could interact with other components of the RTK signalosome. This way, RA domains are further clustered, and thus, GTP-loaded Ras may be sequestered. In addition, this may also contribute to the clustering of Raf, which is more active in a dimerized state. Moreover, it was recently shown that Ras signaling depends on the formation of nanoclusters at the membrane. This local aggregation may further promote interaction of Ras with Son of sevenless, which can trigger additional activation of the RTK signaling cascade. In addition, Rau harbors a class II PDZ-binding motif, suggesting that Rau can integrate further signals to modulate RTK signaling (Sieglitz, 2013).

The activity of the EGFR and the FGFR signaling cascades is conveyed in part through the transcription factor Pointed. Heterozygous loss of pointed significantly increases the rough eye phenotype evoked by loss of Rau function. Moreover, upon overexpression of the constitutively active PointedP1, rau expression was also increased. In line with this notion, CG8965/rau was also identified in a screen for receptor tyrosine signaling targets. Thus, the data suggest that Rau activation occurs after initial RTK stimulation through direct transcriptional activation through Pointed, which is similar to the activation of the secreted EGFR antagonist Argos (Sieglitz, 2013).

This study has dissected the role of Rau in differentiating glial cells of the fly retina. These glial cells are borne out of the optic stalk and need to migrate onto the eye imaginal disc where some of these cells differentiate into wrapping glial cells upon contacting axonal membranes. The development of these glial cells is under the control of FGFR signaling. Initially, low activity of FGFR signaling in these glial cells is permissive for expression of seven-up, which encodes an orphan nuclear receptor of the COUP-TF (COUP transcription factor) family that suppresses sprouty, but not rau, expression. Activation of Rau requires greater activity of FGFR, which is achieved only through interaction with axons. High activation of FGFR signaling also inhibits seven-up expression and thus relieves the negative regulation of sprouty. This negative regulation of COUP-TFII transcription factors by RTKs is also seen during photoreceptor development in the fly eye and appears to be conserved during evolution (Sieglitz, 2013).

In conclusion, the Rau/Sprouty signaling module provides effective means to sustain a short RTK activation pulse, for example, during cellular differentiation. It is proposed that Rau dimers or multimers assemble a scaffold that favors the recruitment of RasGTP, which then could more efficiently activate the MAPK cascade. Thus, ultimately, Rau may promote the formation of Raf dimers, which might confer robustness and increased signaling intensity. Future studies will reveal the precise conformations and complexes that enable Rau to modulate RTK signaling in fly development (Sieglitz, 2013).

The Drosophila Arf GEF Steppke controls MAPK activation in EGFR signaling

Guanine nucleotide exchange factors (GEFs) of the cytohesin protein family are regulators of GDP/GTP exchange for members of the ADP ribosylation factor (Arf) of small GTPases. They have been identified as modulators of various receptor tyrosine kinase signaling pathways including the insulin, the vascular epidermal growth factor (VEGF) and the epidermal growth factor (EGF) pathways. These pathways control many cellular functions, including cell proliferation and differentiation, and their misregulation is often associated with cancerogenesis. In vivo studies on cytohesins using genetic loss of function alleles are lacking, however, since knockout mouse models are not available yet. Recently studies have identified mutants for the single cytohesin Steppke (Step) in Drosophila, and an essential role of Step in the insulin signaling cascade has been demonstrated. The present study provides in vivo evidence for a role of Step in EGFR signaling during wing and eye development. By analyzing step mutants, transgenic RNA interference (RNAi) and overexpression lines for tissue specific as well as clonal analysis, it was found that Step acts downstream of the EGFR and is required for the activation of mitogen-activated protein kinase (MAPK) and the induction of EGFR target genes. It was further demonstrated that step transcription is induced by EGFR signaling whereas it is negatively regulated by insulin signaling. Furthermore, genetic studies and biochemical analysis show that Step interacts with the Connector Enhancer of KSR (CNK). It is proposed that Step may be part of a larger signaling scaffold coordinating receptor tyrosine kinase-dependent MAPK activation (Hahn, 2013).

The proper development of multicellular organisms requires the coordination of proliferation and differentiation, which is a particular challenge during the formation of the tissues and organs of the body. Numerous studies have shown that receptor tyrosine kinases such as the vascular growth factor receptor (VEGFR) epidermal growth factor receptor (EGFR) and insulin/insulin-like growth factor receptors (InR/IGF-Rs) play prominent roles in signaling cell proliferation and differentiation. Misregulation of both pathways is often causative for tumor development and progression through their effects on uncontrolled cell growth, inhibition of apoptosis, angiogenesis, and tumor-associated inflammation. Determining how growth and differentiation are coordinated by these pathways is thus essential to understanding normal development, as well as disease states such as cancer (Hahn, 2013).

Steppke (Step) has been identified as a new and essential component of the insulin signaling pathway in Drosophila. The insulin signaling cascade is conserved from flies to humans and was shown to regulate cell and organismal growth in response to extrinsic signals such as growth factors and nutrient availability. In Drosophila, activation of a unique insulin-like receptor (InR) stimulates a conserved downstream cascade that includes the Phosphatidylinositol-3-kinase (PI3K) and Protein Kinase B (PKB or AKT). AKT is involved in enhancement of glucose absorption and glycogen synthesis, and regulates the activity of the Forkhead box O (FoxO) transcription factor, a negative regulator of cell growth. Step acts downstream of the insulin receptor and upstream of PI3K in the insulin/IGF-like signaling (IIS) cascade (Fuss, 2006). Step is a member of the cytohesin family of guanine nucleotide exchange factors (GEFs) which regulate small GTPases of the ADP-ribosylation factor (ARF) family. Small ARF GTPases are involved in the regulation of many cellular processes including vesicle transport, cell adhesion and migration. Studies in mice have confirmed an evolutionary conserved role of cytohesin family members in IIS (Hahn, 2013 and references therein).

Whereas previous studies focused on the role of Step in IIS-dependent larval growth control, this study examined its function in the Drosophila wing, which develops from an epithelial sheet during larval and pupal stages. The wing is an ectodermal structure formed by a dorsal and ventral epithelium, interspersed with cuticular ectodermal tubes, the so called wing veins. Stereotypical arrangement of wing veins is determined in the imaginal wing disc in late larval and pupal stages by several signaling pathways including the EGFR cascade. EGFR activation by EGF-like ligands Spitz or Vein results in the activation of the small GTPase RAS by its loading with guanosine triphosphate (GTP), which as a result triggers the activation of a number of downstream effector proteins including the Ser/Thr-kinase RAF [mitogen-activated protein kinase (MAPK) kinase kinase]. Once activated, RAF phosphorylates and activates MEK (MAPK kinase), which in turn phosphorylates and activates MAPK/ERK. Phosphorylated MAPK exerts its role in the cytoplasm as well as in the nucleus, where it controls expression of EGFR target genes like pointed (pnt), argos (aos), rhomboid (rho) and ventral nervous system defective (vnd). The scaffolding protein Connector enhancer of KSR (CNK) has been described to facilitate RAS/RAF/MAPK signaling by providing a protein scaffold at the plasma membrane that integrates Src and RAS activities to enhance RAF and MAPK activation (Claperon, 2007). EGFR/MAPK signaling is crucial quite early during wing vein differentiation, where phosphorylation of MAPK determines the positioning of proveins and later during development for maintenance of longitudinal veins. In addition to patterning, both EGFR/RAS/MAPK signaling and IIS control general cell proliferation and cell growth during wing development. Thus, EGFR/RAS/MAPK signaling controls both cell fate (vein versus intervein) and general cell proliferation along with IIS at similar times within the wing tissue (Hahn, 2013).

Recent studies in human lung and breast adenosarcoma cancer cell lines indicated a function of cytohesins in ErbB (EGFR) signaling, where they facilitate signaling by stabilizing an asymmetric ErbB receptor dimer (Bill, 2010). This study provides the first in vivo model that the cytohesin Step, in addition to its previously characterized function as component of IIS, regulates EGFR signaling dependent wing growth and vein differentiation. Genetic, immunohistochemical and biochemical experiments indicate that Step acts downstream of the EGF receptor in the EGFR signaling cascade and is necessary and sufficient for MAPK activation and the induction of EGFR target genes. Whereas step transcription is negatively regulated by IIS (Fuss, 2006), it is induced by EGFR signaling. Evidences are further provided that Step might directly interact with the Connector Enhancer of KSR (CNK) protein that is part of a protein scaffold known to coordinate RAS-dependent RAF and MAPK signaling from tyrosine kinase receptors (Hahn, 2013).

This study demonstrates an in vivo function of the Arf GEF Step as an essential component of the EGFR signaling pathway which acts downstream of the EGFR. Step is necessary and sufficient for activation of MAPK and the induction of EGFR target genes in the Drosophila wing. Based on biochemical, immunohistochemical and the genetic data a mechanistic model is proposed in which Step and dCNK interaction is important for EGFR signaling. dCNK is the single member of the CNK protein family in Drosophila. CNK proteins are scaffolding proteins that have been linked with RAS, Rho, Rac, Ral and Arf GTPases and are proposed to act as general regulators of GTPase-mediated events downstream of receptor tyrosine kinases, including EGFR and InR/insulin-like growth factor receptors (Claperon, 2007). Together with the kinase suppressor of RAS (KSR), CNK was shown to assemble a signaling complex including RAF and MEK which promotes RAS-dependent RAF activation and the subsequent phosphorylation of MAPK. It is suggested that Step is a functional part of this scaffolding complex via its direct interaction with CNK. This is also consistent with recent data in HeLa and 393T cells showing that human CNK1 directly interacts with cytohesin-2 to coordinate PI3K/AKT signaling downstream of InR/IGF-R. It was proposed that CNK1 recruits cytohesin-2 to the plasma membrane, where activity of plasma membrane bound GTPases leads to a PIP2 rich microenvironment, which enhances IRS1 recruitment and hence facilitates PI3K/AKT signaling. Similarly, Drosophila cytohesin Step was shown to be required for PI3K activation. Together, several lines of evidence support a role of cytohesins and CNK in similar signaling contexts (RAS/RAF/MAPK and PI3K/AKT signaling), where a direct interaction of both proteins as part of a signaling platform might promote downstream signaling events like MAPK phosphorylation and PI3K activation. This does not exclude other functions of cytohesins, e.g. the stabilization of asymmetric ErbB (EGFR) dimers, as shown recently in human lung and breast adenosarcoma cancer cell lines. The data indicate, however, that a major function of the Drosophila cytohesin Step in EGFR signaling resides downstream of the EGFR and upstream of MAPK (Hahn, 2013).

Crosstalk between epithelial and mesenchymal tissues in tumorigenesis and imaginal disc development

Cancers develop in a complex mutational landscape. Interaction of genetically abnormal cancer cells with normal stromal cells can modify the local microenvironment to promote disease progression for some tumor types. Genetic models of tumorigenesis provide the opportunity to explore how combinations of cancer driver mutations confer distinct properties on tumors. Previous Drosophila models of EGFR-driven cancer have focused on epithelial neoplasia. This study reports a Drosophila genetic model of EGFR-driven tumorigenesis in which the neoplastic transformation depends on interaction between epithelial and mesenchymal cells. Evidence is provided that the secreted proteoglycan Perlecan (Drosophila Trol) can act as a context-dependent oncogene cooperating with EGFR to promote tumorigenesis. Coexpression of Perlecan in the EGFR-expressing epithelial cells potentiates endogenous Wg/Wnt and Dpp/BMP signals from the epithelial cells to support expansion of a mesenchymal compartment. Wg activity is required in the epithelial compartment, whereas Dpp activity is required in the mesenchymal compartment. This genetically normal mesenchymal compartment is required to support growth and neoplastic transformation of the genetically modified epithelial population. This study reports a genetic model of tumor formation that depends on crosstalk between a genetically modified epithelial cell population and normal host mesenchymal cells. Tumorigenesis in this model co-opts a regulatory mechanism that is normally involved in controlling growth of the imaginal disc during development (Herranz, 2014).

Accumulating evidence indicates that tumor progression results from the interaction between tumor cells and the surrounding normal cells that make up the tumor microenvironment. This study has used the Drosophila wing imaginal disc to dissect the crosstalk between tumor cells and surrounding normal cells, in tumors of epithelial origin. In this model, interaction between the two cell populations is required for tumor growth, neoplastic transformation of the epithelium, and metastasis, even though the genetic modifications were introduced into only one of the two cell populations (Herranz, 2014).

Carcinomas express growth factors involved in the communication between cancer cells and tumor-associated normal cells. The role of TGF-β and Wnt signaling pathways in tumor initiation is well known, but their role as mediators of the interaction between tumor cells and stromal cells has been less well studied. This study observed that EGFR overexpression induced expression of the endogenous Wg and Dpp genes in the epithelial compartment of the tumors. Wg, together with EGFR, is needed in the epithelial cells to drive tumorous growth. The role of the Dpp pathway is different. The findings indicate that Dpp, produced by the epithelial cells, acts on the mesenchymal stromal cells. Dpp signaling activity was not required in the epithelial cells themselves for tumorous growth. Instead, downregulation of the Dpp pathway in mesenchymal cells blocked tumorous growth of the epithelial population. This suggests that Dpp signaling elicits a feedback response from the mesenchymal population. As a consequence, the resulting tumors are composed of a mix of mutant epithelial cells and genetically normal mesenchymal cells, resembling organization observed in human tumors (Herranz, 2014).

EGFR is upregulated in many carcinomas. EGFR is able to promote tissue overgrowth, but additional mutations are required for malignant transformation and invasion. The findings of this study have shown that upregulation of Perlecan is sufficient to cooperate with EGFR to produce neoplastic transformation. Perlecan is a secreted HSPG of the ECM that is overexpressed in many human tumor types. TGF-β ligands have been shown to promote changes in the tumor microenvironment in mammals. Perlecan has also been reported to stabilize Wg and promote Wg activity in Drosophila. Thus, Perlecan production could potentiate the effects of Dpp and Wg produced by the epithelial cells. These findings raise the possibility that Perlecans might have a fundamental role in mediating interactions between epithelial tumor cells and mesenchymal stromal cells, in addition to their known roles in tumor angiogenesis (Herranz, 2014).

The crosstalk between tumor and microenvironment determines the phenotype of the tumor. Signaling from the tumor microenvironment can suppress the malignant tumor phenotype, yet the tumor microenvironment can also promote malignant transformation. The finding that ablation of the mesenchymal cell population reverted the tumor phenotype in this model suggests that signals from the mesenchymal cells are required for tumor progression. This same cell population was required to support growth of the epithelial population in a nontumorous normal tissue context. It is postulated that signals from the adepithelial mesenchymal cells sustain proliferation of the epithelial cells and likewise that signals from the epithelia drive proliferation of the mesenchymal cells. This normal feedback mechanism can be coopted to drive growth of the two tissues, as, for example, when EGFR and Perlecan were overexpressed. A remarkable, unexpected aspect of these findings is that this feedback loop appears to be sufficient to drive the epithelial tissue beyond hyperplasia, through neoplastic transformation, and into metastasis (Herranz, 2014).

EGF receptor : Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Effects of mutation | References

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