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).

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-Hth^1-430) 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-Hth^1-430 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).

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

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

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

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

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

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

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

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

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

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

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

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

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 Spi^S (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 Spi^S 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 Spi^S 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).

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-Egfr^DN 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 (rl^sem) 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).

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).

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 neu^A101 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