Interactive Fly, Drosophila


Effects of Mutation or Deletion

Table of contents

Rhomboid and Malpighian tubules

The Drosophila Malpighian tubules (MTs), form a simple excretory epithelium comparable in function to kidneys in vertebrates. MTs function as the insect kidney both in the larva and the adult. They consist of two pairs of blind ending tubes that are composed of a single cell-layered epithelium made up of a tightly controlled number of cells. The tubules float in the hemolymph from where they take up nitrogenous waste that is excreted as uric acid. During embryogenesis, MTs evert as four protuberances from the hindgut primordium, the proctodeum. The everting tubules grow by cell proliferation, which takes place in a few cells along the tubules and extensively in a distal proliferation domain located in the tip region of the tubules. Cell ablation experiments and studies on the pattern of cell division have shown that a single large cell at the distal end of each tubule, termed the tip cell, is decisive for controlling the proliferation of its neighboring cells. The tip cell that differentiates into a cell with neuronal characteristics during later stages of development arises by division of a tip mother cell that is selected in the tubule primordium by lateral inhibition involving the Notch signaling pathway and the transcription factor Krüppel (Kr). It has been suggested that the tip cell sends a mitogenic signal to adjacent cells in the distal proliferation zone. It has remained elusive, however, what the signal is or what its target molecules in the signal-receiving cells could be and how cell proliferation during MT morphogenesis is regulated. Seven-up is shown to be a key component that becomes induced in response to mitogenic EGF receptor signaling activity emanating from the tip cell. Seven-up (Svp) in turn is capable of regulating the transcription of cell cycle regulators (Kerber, 1998).

To identify the nature of the mitogenic tip cell signal a screen was carried out for genes specifically active in the tip cells. The genes rhomboid (rho) and Star (S), which encode transmembrane proteins involved in epidermal growth factor receptor (EGFR) signaling, are expressed in the tip cells and both are required for MT growth. When the tubules start to evert, rho and S are expressed in the tip mother cell; subsequently rho is strongly expressed in the tip cell and S in the tip cell and its former sister cell. An analysis of the MTs in the corresponding amorphic mutants reveals a strong decrease of cells in rho mutants and a weaker decrease in S mutants. In a rho;S double mutant, the tubules are barely detectable, indicating that rho and S activities are essential (albeit redundant) components controlling MT growth. The tubule phenotype of rho;S double mutants is very similar to that of EGFR mutants, which also show a drastic decrease in the tubule cell number. As in svp mutants, the allocation and the differentiation of the tip cells are normal in the receptor mutants, indicating that receptor activity is not required for tip cell determination and differentiation. The reduction of the tubule cell number in EGFR mutants is due to a failure of proper cell divisions. No BrdU incorporation occurs in EGFR mutants in the outbudding tubules at the time when cells divide in wild-type embryos. However, BrdU incorporation occurs again much later during the endomitotic cycles, indicating that in EGFR muants, a specific defect in DNA replication exists in cells that would normally divide (Kerber, 1998).

Rho and S process a membrane-bound form of the activating ligand of the receptor, the TGFalpha-like Spi protein, to generate the secreted form of Spi (sSpi). sSpi is then proposed to diffuse to neighboring cells, bind to the receptor, and activate target genes via the Ras/Raf signaling cassette; these include the primary target gene pointedP1 (pntP1), encoding an ETS domain transcription factor, and the secondary target gene argos (aos), encoding a negatively acting ligand of the receptor. These downstream components of the pathway are also active during tubule development. pntP1 and aos are expressed during stage 10 in six to eight cells on one side of the MTs overlapping the rho and S expression domains and later, weakly in several cells in the tip region. In amorphic aos mutants a slightly larger number of tubule cells are observed, whereas amorphic pnt mutants show a decrease of tubule cells. These results indicate that for controlling cell proliferation and cell determination, the same key components of the EGFR cascade are required (Kerber, 1998).

These findings suggest that the EGFR pathway provides the mitogenic tip cell signal that activates svp expression and regulates cell division. To test this hypothesis, svp expression was analyzed in EGFR mutants and ectopic expression studies were performed with various members of the pathway using the UAS-Gal4 system. svp is absent in mutants for the Egfr. It is still expressed, however, in amorphic pnt mutants, suggesting that Svp is a transcriptional regulator that is likely to be activated in parallel to the primary transcription factor PntP1 in the signaling cascade. If sSpi activity is provided ectopically in all of the tubule cells, the svp expression domain becomes dramatically expanded and an increase of the tubule cell number is observed. Similar, although slightly weaker effects on svp transcription and the number of tubule cells could be observed upon ubiquitous expression of other components of the EGFR pathway, like Rho, activated Ras, or Raf. Conversely, when a dominant-negative Ras allele is ectopically expressed in all of the tubule cells, svp transcription became strongly reduced. Ectopic expression of svp in an Egfr mutant background restores the tubule cell number to a considerable extent. These results provide strong evidence that svp is a downstream target gene of EGFR signaling in the tubules (Kerber, 1998).

Rhomboid and the ventral cuticle

The spitz-group mutants (spitz, rhomboid, and pointed) are embryonic lethal and have similar cuticle phenotypes; they are shorter than wild type and have deletions of ventral cuticle. vein mutant are shorter and the Keilin's organs and ventral black dots are closer together than in wild-type. Ventral cuticle is deleted between Keilin's organs. The deletions occur in a similar region in spitz-group mutants; spitz and rhomboid have a larger portion of ventral cuticle deleted than vein mutants, but pointed embryos have similar deletions. In vein mutants sensory hairs surrounding the pit structure of Keilin's organs are missing. Unlike the spi-group genes, vein is not critical for embryonic survival and head skeleton and sense organs are normal. Most vein mutants die either as embryos or as larvae, but a small number do pupariate. Individuals that survive to pupariate secrete a pupal case with pattern abnormalities (Schnepp, 1996).

Rhomboid and chordotonal development

The selection of Drosophila sense organ precursors (SOPs) for sensory bristles is a progressive process: each neural equivalence group is transiently defined by the expression of proneural genes (proneural cluster), and neural fate is refined to single cells by Notch-Delta lateral inhibitory signalling between the cells. Unlike sensory bristles, SOPs of chordotonal (stretch receptor) sense organs are tightly clustered. It has been shown that for one large adult chordotonal SOP array (the adult femoral chordotonal sense organ), clustering results from the progressive accumulation of a large number of SOPs from a persistent proneural cluster. This is achieved by a novel interplay of inductive epidermal growth factor- receptor (EGFR) and competitive Notch signals. EGFR acts in opposition to Notch signaling in two ways: it promotes continuous SOP recruitment despite lateral inhibition, and it attenuates the effect of lateral inhibition on the proneural cluster equivalence group, thus maintaining the persistent proneural cluster. SOP recruitment is reiterative because the inductive signal comes from previously recruited SOPs (zur Lage, 1999).

The adult femoral chordotonal sense organ arises from a group of some 70-80 SOPs. A developmental analysis of Ato expression has revealed that these SOPs accumulate over an extended period of time in the dorsal region of each leg imaginal disc during the third larval instar and early pupa. The continued expression of Ato implies a sustained requirement for proneural function throughout the process of SOP accumulation. Unusually, Ato is persistently expressed in a group of ectodermal cells identified as the proneural cluster (PNC). From this PNC, cells are funnelled inward into a cavity formed by the folding of the disc. This invagination later becomes visible as a distinctive 2-cell wide intrusion, which is referred to as the 'stalk'. Cells at the deepest end of the stalk undergo shape changes to form an amorphous inner SOP mass. Invaginating cells are characterised by upregulation of Ato expression, a characteristic of SOP commitment. Surprisingly, SOP markers (Ase protein and the A101 enhancer trap line) are not expressed in all the stalk SOPs. Instead, these markers are only apparent in older cells, particularly at the time when they become part of the inner mass (which is therefore referred to as mature SOPs). Despite this, entry into the stalk seems to mark SOP commitment, since both the stalk and the mature SOPs are absent in discs from ato mutant larvae. This apparent intermediate stage may not have a counterpart in external sense organ precursor formation, although there is some evidence for multiple steps between the uncommitted cell and the SOP (the so-called pre-sensory mother cell state). Initially, Ato remains activated in all invaginated SOPs. This extended period of proneural gene expression is unusual since AS-C proneural expression is typically switched off in SOPs shortly after commitment. Later, at approximately 6 hours before puparium formation (BPF), Ato expression is switched off synchronously in the mature SOPs, although expression remains in the stalk SOPs and the PNC. At this point there is very little overlap between Ato and Ase or A101 (zur Lage, 1999).

The process of chordotonal SOP formation described above is at odds in several respects with the well-known paradigm of SOP selection for sensory bristles. In the latter, the solitary SOP expresses Delta, which triggers expression in the PNC of genes of the E(spl)-C, thereby preventing further SOP commitment and forcing loss of AS-C expression and neural competence. In the case of the femoral chordotonal organ, newly committed cells from the PNC are in contact with previously committed SOPs in the stalk, but are apparently not receiving (or not responding to) lateral inhibition signals from these to prevent their commitment. Likewise, the presence of committed SOPs does not switch off ato expression in the PNC. Nevertheless, components of the N-Dl pathway are expressed in patterns consistent with lateral inhibition. The newly formed SOPs express Dl, suggesting that they send inhibitory signals, while the PNC expresses mgamma, a member of the E(spl)-C, suggesting that these cells are responding to the Notch-Delta signal. Indeed, mgamma is coexpressed with ato in the PNC throughout the development of the SOP cluster. Chordotonal SOP formation is shown to be sensitive to N inhibitory signaling. Strong activation of N signaling or its effectors can inhibit chordotonal SOP formation. Thus, N signaling has an important role to play: it acts to limit the process of SOP selection from the PNC. Some mechanism, however, must prevent N signaling from completely inhibiting multiple SOP formation (zur Lage, 1999).

The progressive accumulation of chordotonal SOPs suggests that a recruitment mechanism could explain the clustering of SOPs. The Drosophila Egfr signaling pathway is involved in a number of recruitment processes in development, and a role for Egfr signaling has been demonstrated in the induction of embryonic chordotonal precursors (zur Lage, 1997). Although there appear to be significant differences in the process of SOP formation in imaginal discs, as compared with the embryo, it was asked whether Egfr signaling is also involved in forming the femoral chordotonal cluster. To address this question, the pathway was conditionally disrupted by expressing a dominant negative form of Egfr protein. Expression of UAS-Egfr DN results in a dramatic loss of chordotonal SOPs in late third instar imaginal leg discs (as judged by Ase protein expression or the A101 enhancer trap line). This demonstrates that Egfr signaling is required for the process of femoral chordotonal SOP formation. In contrast, the appearance of bristle SOPs is unaffected, arguing against the possibility of a nonspecific effect on SOPs in general (zur Lage, 1999).

To determine whether Egfr signaling controls SOP number, expression of components of the Egfr pathway that determine the level of signaling was forced, thus resulting in hyperactivation of the pathway. pointed (pnt) is an effector gene that encodes a transcription factor and is activated in cells responding to Egfr signaling. Both rho and pnt are expressed during chordotonal SOP formation. Indeed, forced expression of rho or pnt increases chordotonal SOP formation. Egfr could promote SOP formation by stimulating the commitment of PNC cells or by stimulating proliferation of SOPs. Both functions would be consistent with known Egfr roles, but the current investigations favour the former. Analysis of Ato expression in leg discs in which rho has been misexpressed reveals a large invagination of cells and a smaller PNC. Shrinking of the PNC was confirmed by the reduced extent of mgamma expression. These observations are consistent with an increased rate of SOP commitment upon Egfr hyperactivation. Moreover, this effect is reminiscent of the effect of N loss of function on Ato expression, suggesting that Egfr signaling supplies the mechanism that interferes with lateral inhibition of SOP commitment (zur Lage, 1999).

Although it seems that cells of the PNC and stalk are held in a state of mitotic quiescence throughout the time that SOP fate decisions are being made, BrdU is incorporated in the older (mature) SOPs. The experiments so far have indicated that Egfr signaling affects SOP commitment from the PNC. To determine more precisely the spatial patterning of Egfr activity required for SOP clustering and N antagonism, the expression patterns of key components of the pathway were characterized. Localized expression of rho appears to play a central role in spatial restriction of Egfr activity in cases where Spi is the ligand; in these cases it appears to mark the cells that are a source of signaling. During development of the femoral chordotonal organ, rho is expressed in a very restricted pattern: RHO mRNA is only detected in the SOPs, becoming confined in the late third instar larva to the youngest SOPs at the top of the stalk. To identify the cells responding to rho-effected signaling, an antibody that detects the dual-phosphorylated (activated) form of the ERK MAP kinase (dp-ERK) was used. In leg imaginal discs, dp-ERK is detected in a confined area corresponding to the uppermost (youngest) stalk SOPs. Thus, like rho, dp-ERK is expressed in the newly formed stalk SOPs. Double labelling for RHO RNA and dp-ERK confirms this, but also suggests that the overlap in expression is not complete: dp-ERK is detected above the uppermost rho-expressing cells of the stalk, probably in one or a few cells of the proneural cluster as they funnel into the stalk. This suggests that Egfr promotes SOP commitment as a consequence of direct signaling from previous SOPs to overlying PNC cells. Since rho expression is itself activated upon SOP commitment, this process occurs cyclically: the newly recruited SOPs are in turn able to signal to further overlying PNC cells. That is, recruitment is reiterative. Egfr signaling via Spitz has been shown to help to maintain neural competence by attenuation of Notch directed lateral inhibition. The opposing forces of Notch and Egfr signaling are thought to be played out through direct Notch and Egfr signaling between the epidermal proneural cells, which bear Notch, and the SOP, which sends inhibitory signals through the Delta ligand, and stimulatory signals through the Spitz ligand (zur Lage, 1999).

Reiterative recruitment alone cannot entirely explain the accumulation of SOPs. Such an accumulation also relies on the persistence of the competent pool of PNC cells from which SOPs can be recruited. For AS-C PNCs, this does not occur, because the mutual inhibition required for continued competence is unstable and resolves quickly to a state of lateral inhibition once the SOP emerges from the PNC. This results in rapid shutdown of AS-C expression and hence competence within the PNC. It is possible that the members of E(spl)-C that are expressed in the PNC (notably mgamma and mdelta) are less aggressive inhibitors of proneural gene expression than the E(spl)-C members expressed in AS-C PNCs (m5 and m8). The results obtained in the femoral SOP suggest, however, that Egfr has a role to play in maintaining the PNC by partially attenuating lateral inhibition on a PNC-wide scale. Thus, the PNC is not completely shut off by inhibition from SOPs, but instead kept in check, allowing continued mutual inhibition and maintenance of competence but not allowing general SOP commitment. Since neither rho nor dp-ERK are detected in the PNC as a whole, this function of Egfr could be indirect and achieved through partial attenuation of Dl signaling from the stalk SOPs themselves. The trans- or auto-activation of EGFR signaling between the stalk SOPs (as suggested by the co-expression of dp-ERK and rho) might be an indicator of this function. It is also possible, however, that Egfr signaling is direct and that the dp-ERK antibody is not sensitive enough to detect expression in the PNC cells (zur Lage, 1999).

Rhomboid and oocyte development

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).

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).

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 recognises 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 os 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).

Dynamic model for the coordination of two enhancers of broad by EGFR signaling

Although it is widely appreciated that a typical developmental control gene is regulated by multiple enhancers, coordination of enhancer activities remains poorly understood. This study proposes a mechanism for such coordination in Drosophila oogenesis>, when the expression of the transcription factor Broad (BR) evolves from a uniform to a two-domain pattern that prefigures the formation of two respiratory eggshell appendages. This change reflects sequential activities of two enhancers of the br gene, early and late, both of which are controlled by the epidermal growth factor receptor (EGFR) pathway. The late enhancer controls br in the appendage-producing cells, but the function of the early enhancer remained unclear. This study found that the early enhancer is essential for the activity of the late enhancer and induction of eggshell appendages. This requirement can be explained by a mechanism whereby the BR protein produced by the early enhancer protects the late enhancer from EGFR-dependent repression. This complex mechanism is illustrated using a computational model that correctly predicts the wild-type dynamics of BR expression and its response to genetic perturbations (Cheung, 2013).

Temporal control of transcription can be provided by changes in the levels of inductive signals, by cross-regulatory interactions between genes, and by dynamic use of different enhancers. For example, the dynamic expression of rho in the early Drosophila embryo results from sequential activities of two different rho enhancers, responding to two different inductive cues. In another control strategy, the early enhancer initiates expression, and the late enhancer maintains it through positive autoregulation. This mechanism controls Krox20 during the hindbrain segmentation in vertebrates. Both of these scenarios are different from the mechanism that coordinates br enhancers in Drosophila oogenesis. First, both the early and late enhancers respond to the same inductive signal. Second, the early enhancer is needed not to the initiate the expression of the late enhancer, but to protect it from ectopic and premature repression (Cheung, 2013).

In the wild-type egg chamber, brL, the late enhancer, is repressed only in cells exposed to the maximal levels of GRK. In the absence of brE, the early enhancer, signaling levels sufficient for repression are realized in the appendage primordia, due to amplification of EGFR activation resulting from ectopic expression of rho. This model is supported by eggshell defects induced by the RNAi-based disruption of BR expression by brE, and by ectopic expression of rho mRNA and PNT-dependent loss of brL activity in the absence of BR. The requirement for the rho-dependent amplification of EGFR signaling was tested computationally, by analyzing a simplified model in which BR and PNT repress each other directly, without feedback by rho. Extensive exploration of the parameter space in this model could not identify a set of parameters that would be consistent with both the wild-type expression of brL and its response to genetic perturbations. Based on this, it is argued that amplification of EGFR signaling by rho is essential for explaining the results (Cheung, 2013).

Going beyond br and rho, it is noted that dozens of genes regulated by GRK are expressed in dynamic patterns. Some of these patterns may be explained using the proposed computational model based on the interplay of multiple enhancers and dynamic signals. Although these models are more complex than existing models of developmental patterning, their analysis is essential for understanding temporal control of gene expression in development (Cheung, 2013).

Rhomboid and tracheal development

The Drosophila tracheal system is a network of epithelial tubes that arises from the tracheal placodes, lateral clusters of ectodermal cells in ten embryonic segments. The cells of each cluster invaginate and subsequent formation of the tracheal tree occurs by cell migration and fusion of tracheal branches, without cell division. The combined action of the Decapentaplegic (Dpp), Epidermal growth factor (EGF) and breathless/ branchless pathways are thought to be responsible for the pattern of tracheal branches. It is asked how these transduction pathways regulate cell migration and the consequences on cell behaviour of the Dpp and EGF pathways is examined. rhomboid (rho) mutant embryos display defects not only in tracheal cell migration but also in tracheal cell invagination unveiling a new role for EGF signaling in the formation of the tracheal system. These results indicate that the transduction pathways that control tracheal cell migration are active in different steps of tracheal formation, beginning at invagination (Llimargas, 1999).

Defects in tracheal migration are associated with defects in invagination in rho and vvl mutant embryos, but not in bnl and btl mutant embryos. This is consistent with the observation that EGF-dependent activation of MAP kinase (ERK) in the tracheal placode precedes ERK activation by the Bnl/Btl pathway. Thus the tracheal phenotype of mutations in the EGF pathway, which has been shown to result from impaired activity of the pathway in the trachea, is likely to originate before the onset of migration. It has been proposed that the EGF pathway might be required for tracheal cells in specific branches to follow the leading cell. Tracheal migration defects of rho mutant embryos are due, at least in part, to the failure of some tracheal cells to invaginate (Llimargas, 1999).

Invagination of the tracheal pits is dependent on trh. This process associates with an accumulation of actin in the cell surfaces facing the invagination and both actin accumulation and invagination are dependent on trh activity. Thus, the role of trh as an inducer of tubulogenesis could stem, at least in part, from its potential to reorganize the actin cytoskeleton. These results also indicate that induction of tracheal invagination by trh involves making cells competent to EGF signaling by regulating rho expression. However, there must be other targets of trh because tracheal invagination is only partially affected in rho mutant embryos. Interestingly, an interaction between EGF signaling and trh also occurs in salivary duct determination, suggesting that the coordinated activity of trh and the EGF pathway could be part of a more general mechanism for cell invagination and tubulogenesis (Llimargas, 1999).

How, then, can EGF signaling influence cell migration? In addressing this issue, it has to be noted that while EGF-dependent activation of ERK in the tracheal placodes is abolished in rho mutant embryos, tracheal invagination is only partially affected. One possibility is that EGF signaling is specifically required for the invagination of only a subset of the tracheal cells; this would reveal a regional subdivision among the cells in the tracheal placode. Accordingly, the cells invaginating in rho mutant embryos would be the Dpp-induced cells, where the EGF pathway is never activated. An alternative possibility is that the tracheal defects in rho mutant embryos could be due to a general decrease in the efficiency of cell invagination that would result in fewer cells invaginating. In that case, the branching defects in rho mutant embryos could be attributed to a change in the topological distribution of the tracheal cells that would alter their ability to receive a particular signal. For instance, as fewer cells invaginate in rho mutant embryos, almost all of them could be reached by the Dpp signal expressed in the dorsal and ventral side of the invaginating tracheal placode. This would explain why in rho mutant embryos the Dpp-induced branches are usually formed and the dorsal trunk (that is formed by cells where the Dpp pathway is not active) is completely or partially missing. This interpretation could also account for the observation that the rho tracheal phenotype is more variable and less specific than the tkv phenotype (Llimargas, 1999).

These observations also illustrate the role of vvl in tracheal formation. Since btl expression is normally initiated in vvl mutants, early but not sustained activity of the Btl pathway could cause the tracheal phenotype in vvl mutant embryos. Since vvl is also required for the tracheal expression of tkv and rho, failure to activate the Dpp and EGF pathways could also be the source of the cell shape phenotypes in vvl mutant embryos. This latter possibility is substantiated by the observation that vvl and rho mutant embryos show abnormalities in tracheal invagination that are not present in btl mutant embryos. Finally, the tkv;rho double mutant tracheal phenotype is very similar to the vvl phenotype (Llimargas, 1999).

Multiple signaling pathways interact to determine the formation of the different tracheal branches. However, even though they all affect the directed migration of the tracheal cells, they are active in different steps in the morphogenesis of the tracheal tree. In particular, the results show that while the Bnl/Btl pathway is specifically required for migration, EGF signaling is active in tracheal cell invagination. These observations also indicate that the accurate invagination of the tracheal cells inside the embryo is an important factor in order to follow a particular direction of migration. In particular, different levels of invagination could predetermine whether cells would migrate in one or the other direction. In this regard, it is worth noting that while in rho mutant embryos some cells remain at the embryonic surface and do not invaginate, in tkv mutant embryos some cells remain in an intermediate position, indicating that they are able to invaginate but do not reach their final location. Altogether, these observations suggest that the precise topology of the invaginating cells controlled by EGF and Dpp signaling could be determining how the tracheal cells will respond to guiding cues, such as Bnl (Llimargas, 1999).

Rhomboid and leg development

Arthropods and higher vertebrates both 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 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 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).

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).

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).

Rhomboid and bract induction

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

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

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

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

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

Table of contents

rhomboid: Biological Overview | Regulation | Protein Interactions | Developmental Biology | References

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