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).
RNA in situ hybridisation to wild-type embryos shows that cv-c is not maternally supplied. cv-c mRNA is detected in stage 8 embryos in the head region, including the head mesoderm, at the tip of the cephalic furrow, in the amnioserosa, and in distinct regions of the hindgut and posterior midgut anlagen in the amnioproctodeal invagination. At stage 11, cv-c transcripts can be found at high levels in the tracheal pits, in the mesoderm, and at lower levels in the cells that will later become the leading edge cells during dorsal closure. Transcripts are first detected in the Malpighian tubules during germband retraction and accumulate to high levels in this tissue by stage 13. At this stage, cv-c is also expressed in many tissues including the peripheral nervous system, posterior spiracle, the somatic mesoderm, the longitudinal visceral muscles (LVM) of the midgut, the lymph gland and in the head. By stage 16, cv-c is expressed strongly in both the LVM and circular visceral muscles of the gut and throughout the ectoderm (Denholm, 2005).
In a series of screens designed to isolate mutations in genes controlling morphogenesis, two EMS alleles were identified of a gene located between the third chromosome markers curled and stripe. Further analysis using single nucleotide polymorphism (SNP) markers refined the map position to 88B-88C, between SNP-NK7.1 and SNP-88C -- a region of ~116 kb that, according to the Drosophila genome database, contains nine predicted genes. A P-element insertion, l(3)06951, is allelic to these mutants. The cv-c locus also maps to chromosomal band 88C. This locus is represented by three alleles, cv-c1, cv-cc and cv-cM1, that are characterised by the loss of the posterior crossvein (PCV) and a detachment of the anterior crossvein (ACV) (Diaz-Benjumea, 1990; Edmondson, 1952; Stern, 1934). Both EMS alleles and l(3)06951, when transheterozygous with each other, are lethal, but they are viable and exhibit PCV defects in trans with cv-c1. Further alleles of cv-c were isolated, including a set showing embryonic lethality (Denholm, 2005).
In addition to the nine predicted genes, a number of expressed sequence tags (ESTs), indicate that two other transcription units map to the cv-c region (EST clot numbers: 13471 and 13975). Plasmid rescue of the P-element in l(3)06951 shows that it is inserted close to SNP-88C and within the transcription unit defined by ESTs in clot 13975. However, the transcription unit defined by clot 13957 does not contain an ORF of appreciable size and it is believed that the lethal mutation caused by the P-element insertion is unrelated to it (Denholm, 2005).
No mutants have been reported for any of the nine predicted genes flanked by the SNPs, precluding complementation analysis. However, a UAS-RNAi construct directed to RhoGAP88C (CG31319) has been generated (Billuart, 2001). Tests were performed to see whether expression of this RNAi construct could recapitulate the cv-c phenotypes observed. To do this, Gal4 driver lines were used that express in the developing wing throughout pupal development and are active during the period of PCV specification at 24-26 hours APF. The four Gal4 driver lines tested produce a phenotype indistinguishable from the cv-c1 phenotype. No other wing structures, including the longitudinal veins, are affected. This result contrasts with previous data reporting that expression of UAS-RhoGAP88C in the developing wing pouch (using T80-Gal4) results in a reduction or absence of wing vein L2 (Billuart, 2001). These results suggest that cv-c encodes RhoGAP88C, and encouraged a search for molecular lesions in the coding sequence of RhoGAP88C in the EMS alleles (Denholm, 2005).
Molecular lesions were identified in the coding region of three of the cv-c EMS alleles. Two of these, cv-cC524 and cv-cM62, contain nonsense mutations at amino acid positions 369 and 666, respectively, resulting in protein products truncated either before the GAP domain for cv-cC524, or within it for cv-cM62. The absence of a complete GAP or START domain in either mutant product suggests they are likely to be amorphic mutations. In cv-c7, arginine 601 is substituted for glutamine. This arginine is highly conserved at the corresponding position in RhoGAP88C homologues in other species and is also conserved in GAP proteins more distantly related to the RhoGAP family, underlining the importance of this residue for GAP function. A combination of biochemical and structural studies have shown that the conserved arginine residue projects into the active site of the GTPase and stabilises the transition state of the hydrolytic reaction. Mutational analysis reveals that substitution of this so-called 'arginine finger' with another residue dramatically reduces the catalytic activity of the GAP protein. The finding that the arginine finger is specifically mutated in cv-c7 indicates that GAP activity is central to the function of cv-c. Surprisingly, however, cv-c7 homozygous embryos have consistently stronger phenotypes than either cv-cC524 or cv-cM62 homozygotes, suggesting that cv-c7 is an antimorphic allele. The interpretation of these findings is that substitution of arginine 601 to glutamine not only abolishes the catalytic activity of cv-c, but also impedes the weak, intrinsic GTPase activity of its substrate. Therefore, the balance between GTP-bound and GDP-bound states of the substrate GTPase will be shifted even further towards the active, GTP-bound form in cv-c7 homozygous embryos (Denholm, 2005).
The P-element insertion in cv-cl(3)06951 is ~60 kb upstream to the start of transcription. The reporter activity faithfully recapitulates the endogenous expression of cv-c. Precise excision of the P-element reverts the lethality and cv-c phenotype. Both cv-cl(3)06951 and the new imprecise excision alleles generated are homozygous lethal but are weaker than those of the amorphic mutations. In one of these excision alleles, cv-cJ17, cv-c transcripts are absent from most, but not all tissues. These results indicate that sequences controlling cv-c transcription reside at a considerable genomic distance from the transcription unit (Denholm, 2005).
cv-cl(3)06951 is likely to disrupt regulatory sequences. To test this hypothesis, the expression of cv-c in cv-cl(3)06951 and the jump-out allele cv-cJ17 were examined. Although it was not possible to detect any major differences in cv-c expression in cv-cl(3)06951, cv-c expression is reduced in all tissues apart from the LVM in cv-cJ17 embryos. This confirms that the cv-cJ17 lesion removes cv-c regulatory elements (Denholm, 2005).
The mutant phenotypes of cv-cM62 homozygotes are described since this allele is thought to be an amorphic. Analysis of cv-c germline clones confirms that cv-c is not supplied maternally and for this reason zygotic mutants were used for the phenotypic analysis (Denholm, 2005).
The cells that contribute to the larval mouth skeleton of Drosophila are internalised during development in a process known as head involution. In cv-c mutants, cells fail to move into the embryo so that structures such as the medial tooth, the H piece and dorsal bridge remain on the surface of the embryo. cv-c7 consistently shows stronger defects, in which the head does not involute normally so that the remnants of the whole head skeleton lie on the surface (Denholm, 2005).
The posterior spiracle forms by invagination of surface ectodermal cells into the interior to form the spiracular chamber. In cv-c mutants, invagination of these cells is aberrant, causing defects ranging from branching of the spiracular chamber to complete failure of invagination, so that the entire internal cuticular structure (the Filzkörper) is later found on the surface (Denholm, 2005).
During dorsal closure, sheets of epithelial cells on either side of the embryo move dorsally over the amnioserosa, so that their epithelial fronts meet and fuse at the dorsal midline, enclosing the embryo. Although embryos mutant for strong cv-c alleles complete dorsal closure, staining with an antibody against the apical membrane marker Stranded at second (Sas) shows that closure is delayed, and puckering of the dorsal cuticle is frequently observed. The phenotypes observed do not result from abrogation of Dpp signalling, that underlies dorsal closure, suggesting defects occur in the morphogenetic movements themselves (Denholm, 2005).
In wild-type embryos, the midgut becomes subdivided into four compartments by three constrictions that result from interactions between the visceral mesoderm and the underlying endoderm. The formation of these constrictions involves changes in cell shape and movement that occur without cell division. In cv-c mutant embryos, the anterior (and occasionally also the posterior) midgut constriction fails to develop. Failure of the gut constrictions is not due to defects in visceral mesoderm specification or signalling and is therefore likely to result from defects in the normal cell movements (Denholm, 2005).
In cv-c embryos, the Malpighian tubules form a single large cyst- or ball-like structure instead of four elongated tubules. This phenotype results from the failure of tubule remodelling, which normally occurs by a series of convergent extension movements, such that each tubule extends along its proximodistal axis and narrows around its circumference. The cyst-like structure in mutants sometimes, but not always, exhibits a central lumen, which is revealed by the accumulation of secreted urates. In a small proportion of embryos, the distal regions of one and, occasionally, two tubules undergo normal convergent extension movements, with the distal tips finding their correct location within the body cavity (Denholm, 2005).
As part of the analysis to confirm that cv-c encodes RhoGAP88C, a UAS-RhoGAP88C construct was generated, and it was asked whether this could rescue the cv-c phenotype. Expression of the construct in cv-cM62 mutant embryos using the CtBGal4 driver, which is specific for the Malpighian tubules, rescues the tubule phenotype, confirming that cv-c encodes RhoGAP88C. Furthermore, rescue of the Malpighian tubule phenotype in an embryo otherwise mutant for cv-c demonstrates that the requirement for Cv-c activity is cell autonomous in this tissue at least. In summary, the phenotypic analysis shows that the tissues most sensitive to loss of Cv-c function are those actively undergoing morphogenetic movement or remodelling. It is proposed that the cv-c gene product functions autonomously to perform a fundamental role in the control of morphogenesis of multiple tissues (Denholm, 2005).
The morphogenetic defects observed could result from alterations in cell polarity or cell shape through cytoskeletal reorganisation. In order to distinguish between these, both planar and apicobasal polarity of cells were analyzed in cv-c embryos. Planar polarity in epidermal cells during dorsal closure was examined because it is not known whether the Malpighian tubules exhibit planar polarity. To do this, the redistribution of Canoe (Cno) was visualised. In wild-type embryos, Cno is expressed at the cell cortex of leading edge cells, but as dorsal closure proceeds it clears from sites of apposition with the amnioserosa and refines to distinct puncta where adjacent epidermal cells meet. This redistribution depends on the planar cell polarity pathway. This redistribution of Cno occurs normally in cv-c mutant embryos, indicating that planar polarity is not disrupted in the absence of Cv-c activity. Whether cv-c tubule cells have normal apicobasal polarity was examined. Using CD8-GFP to label the entire membrane of Malpighian tubule cells and staining for the apical membrane marker Bazooka (Baz) reveals that apicobasal polarity is established normally and maintained in cv-c 'tubules' until the end of embryogenesis. In addition, the presence and position of the adherens junction marker dE-Cadherin relative to apical markers such as Sas are normal in mutant tubule cells (Denholm, 2005).
Attention was directed towards the actin cytoskeleton. To do this, use was made of the UAS-GMA construct, which encodes the actin-binding region of moesin fused to GFP and provides a faithful readout of filamentous actin localisation. Using the CtB-Gal4 driver, UAS-GMA was expressed in the Malpighian tubules throughout embryonic development. In wild-type embryos at stage 13, F-actin is found localised to the cell cortex of all tubule cells and is particularly enriched at the apical (luminal surface). A similar distribution of F-actin is maintained throughout the remainder of embryogenesis. F-actin distribution is more diffuse in stage 13 cv-c mutant embryos, and although it is present around the cell cortex, its levels here are significantly lower than in wild-type embryos. Similarly, apical accumulation of actin is either absent or, at best, very weak. Although F-actin becomes localised to the cell cortex in later stages of development, its distribution is less compact than in wild type and diffuse staining throughout the cytoplasm suggests that attachment of the subcortical actin network to the membrane is disrupted. Although apical accumulation of F-actin is never observed in cv-c cystic Malpighian tubules, those in which the distal end does become tubular possess weak apical F-actin accumulation at the luminal membrane. These data show that Cv-c is required to regulate the spatial organisation of F-actin in tubule cells during morphogenesis. Furthermore, apical accumulation of F-actin, which is largely absent in cv-c tubules, is crucial for the maintenance of tubular integrity; in its absence the Malpighian tubules collapse to form cyst-like sack structures (Denholm, 2005).
In order to analyse the gain-of-function phenotype, Cv-c was overexpressed in the Malpighian tubules throughout embryogenesis. Tubules in which Cv-c is ectopically expressed are significantly shorter in length and fatter around their circumference, suggesting that elevated Cv-c activity also causes defects in convergent extension movements. The most obvious phenotype observed, however, affects the tip cell, a specialised cell that protrudes from the distal end of each of the four tubules. The length of the tip cell stalk is ~7 µm in both wild-type tubules and in the tubules of cv-c loss-of-function embryos. However, when Cv-c is overexpressed, the stalk length can increase to over 50 µm. Analysis using 22C10, which labels the tip cell membrane, as well as observations of F-actin, reveals that the extended stalk is associated with underlying F-actin. Staining for Sas reveals that the stalk is an extension of the basolateral, rather than apical membrane. It was reasoned that if the normal function of Cv-c is to downregulate the activity of RhoGTPases, then overexpression of a dominant-negative RhoGTPase might be expected to produce a similar phenotype. This is indeed the case: ectopic expression of the dominant-negative Rho1 construct UAS-Rho.N19 (but not the dominant-negative Rac1 construct UAS-Rac1.N17) resulted in an increase in tip cell stalk similar to the phenotype in cv-c overexpression. These data, taken together with the loss-of-function analysis, are consistent with a role for Cv-c in regulating or stabilising links between the cell membrane and the cortical actin cytoskeleton. Furthermore, in agreement with genetic interaction experiments, these data further suggest that this function for Cv-c, in the Malpighian tubules at least, is mediated at the level of Rho1 and not Rac1 activity (Denholm, 2005).
RhoGAPs normally function to stimulate the intrinsic GTP hydrolysing capacity of the GTPases, thereby converting them to the inactive GDP-bound form. Thus, the phenotypes observed in the absence of Cv-c function are likely to be caused by elevated activities of the cognate GTPase substrate(s) of Cv-c. Such relationships can be tested by genetic interactions; a reduction in the activity of a substrate GTPase would rescue the cv-c phenotype, while an increase in GTPase activity would enhance it. To identify the substrate(s) of Cv-c, genetic interactions between cv-c and candidate GTPases were analysed for which mutants are available. Embryos homozygous for cv-cM62 and homozygous or hemizygous for mutations in the GTPases Rho1, Rac1, Rac2, Mtl and Cdc42 were analysed for both the Malpighian tubule and embryonic cuticle phenotypes (Denholm, 2005).
Removal of any of the Rac candidates, Rac1, Rac2 or Mtl, and reduction or removal of Cdc42 failed to modify the cv-cM62 phenotype in the Malpighian tubules. By contrast, 50% of cv-cM62 mutant embryos additionally homozygous for Rho172R and 20% of cv-cM62 mutant embryos heterozygous for Rho172R had a phenotype significantly less severe than that of the cv-cM62 homozygote alone. The Malpighian tubules of doubly mutant embryos undergo convergent extension movements to some extent, such that they resemble weaker alleles of cv-c. These data strongly suggest that Rho1 is a substrate for Cv-c in the Malpighian tubules (Denholm, 2005).
Genetic interations were examined in the embryonic epidermis. Dorsal closure defects occur in 82% of embryos doubly mutant for Rac1 and Rac2. However, if Rac1,Rac2 embryos are additionally mutant for cv-cM62, 37% of these embryos are rescued, indicating that Rac GTPases are substrates for Cv-c during dorsal closure (Denholm, 2005).
The posterior spiracle phenotype in cv-c embryos was not suppressed by any of the RhoGTPase mutants, possibly because maternal contribution of the substrate GTPase is sufficient to provide full activity in this tissue. However, it was unexpectedly found that embryos mutant for both Rac1 and Rac2 do not decrease, but enhance the cv-cM62 posterior spiracle phenotype. One possible explanation for this interaction comes from observations in cell culture, where it has been shown that Rac activity downregulates Rho activation. If Rac normally functions to inhibit Rho in the posterior spiracle, then loss of Rac and Cv-c together, would lead to hyperactivity in Rho1 and this would lead to the enhancement of the cv-c phenotype. In support of this, cv-c-like posterior spiracle phenotypes were seen with low penetrance in Rac1 Rac2 embryos (Denholm, 2005).
In summary, the data from these genetic analyses in the embryo indicate that cv-c interacts with Rho1, Rac1 and Rac2. Furthermore, these data suggest that interactions between Cv-c and its substrates might be regulated in a tissue-specific manner (Denholm, 2005).
Rho GTPase and its upstream activator, guanine nucleotide exchange factor 2 (RhoGEF2), have emerged as key regulators of actin rearrangements during epithelial folding and invagination (Nikolaidou, 2004). A Rho-GTPase-signaling pathway is used reiteratively in epithelial folding and potentially selects the outcome of Rho activation. This study shows that Drosophila 18 wheeler (18W), a Toll-like receptor protein, is a novel component of the Rho-signaling pathway involved in epithelial morphogenesis. 18w mutant embryos have salivary gland invagination defects similar to embryos that lack components of the Rho pathway, and ubiquitous expression of 18W results in an upregulation of Rho signaling. Transheterozygous genetic interactions and double mutant analysis suggest that 18W affects the Rho-GTPase-signaling pathway not through Fog and RhoGEF2, but rather by inhibiting Rho GTPase activating proteins (RhoGAPs). RhoGAP5A and RhoGAP88C/Crossveinless-c (CV-C) are required for proper salivary gland morphogenesis, implicating them as potential targets of 18W (Kolesnikov, 2007).
Based on microarray experiments, 18w is downregulated in Scr mutants, implicating it in salivary gland development. To follow 18w expression in more detail, RNA in situ hybridizations were performed. The results show that in wild-type embryos 18w RNA is not maternally contributed but is expressed in salivary gland cells prior to and throughout their invagination. Within the salivary placode, 18w expression first becomes evident at stage 11 as a small spot in the dorsal posterior region. During early stage 12, following the beginning of invagination, 18w expression spreads throughout the placode. During the remainder of stage 12, 18w transcripts can be detected in both gland cells that have yet to invaginate as well as those that have already internalized, albeit at a reduced intensity. At stage 13, 18w transcripts cease to be expressed in salivary gland cells but are evident in salivary duct cells. In addition to salivary glands and ducts, 18w RNA is also detected in other tissues undergoing morphogenesis, including the tracheal placodes and the hindgut. As anticipated from microarray experiments, 18w transcripts are absent exclusively from parasegment two in Scr mutants while expression in the rest of the embryo remains unaltered. Overall, performing microarray experiments with Scr mutant embryos has proven to be an effective method for identifying salivary gland genes (Kolesnikov, 2007).
While 18w is expressed in several tissues undergoing morphogenesis, embryonic defects have yet to be identified in 18w mutant embryos. The striking 18w expression within the invaginating salivary gland prompted a more carefully investigation of the role of 18w in embryonic salivary gland development. In examining a null allele of 18w, 18wΔ21, it was discovered that initiation of invagination in the dorsal posterior region of the placode appears normal. However, during the next phase of invagination, in which the remaining cells of the salivary placode normally internalize in a strict sequential order, defects become apparent. In 18w mutants invagination is less synchronized; too many placode cells invaginate simultaneously rather than sequentially, thereby causing a wider lumen in 66% of 18w mutants when compared to wild-type embryos. Thus, it is the timely progression of invagination, not the invagination process itself, that is affected in 18w mutants. Due to the lack of proper coordination in 18w, the internalization of the gland cells appears to be delayed causing the cells that internalize last to remain near the ventral surface instead of reaching their final, more dorsal, position within the embryo. Eventually all of the salivary gland cells do internalize in 18w mutants, but the proximal part of the gland, which remains abnormally close to the ventral surface, becomes caught in the anterior movement of the ectoderm during head involution. As a result, the glands end up much closer to the anterior end of the embryo than in wild-type embryos. Similar defects are seen in mutants homozygous for the loss of function 18w allele, 18wΔ7–35, and in 18wΔ21, 18wΔ7–35 transheterozygous mutant embryos. Based on these results, 18w is an important component in coordinating salivary gland invagination (Kolesnikov, 2007).
Activation of the Rho-signaling pathway results in the phosphorylation of the myosin II regulatory light chain, encoded by the spaghetti squash (sqh) gene. The phosphorylated form of Sqh can interact with actin and cause actomyosin-based contractility at the apices of cells. To verify that 18W is part of the Rho pathway, whether overexpression of 18W results in an upregulation of Rho signaling, evident as an increase in phosphorylation of Sqh, was checked. Immunoblot analysis on embryonic extracts using anti-phospho-Sqh antibody reveals that overexpressing 18W throughout the embryo results in a two-fold increase of P-Sqh when compared to wild type. Similar results are seen when a constitutively active form of Rho is overexpressed ubiquitously in the embryo. Moreover, introducing one copy of a phosphomimetic sqh transgene, sqhE20E21, into an 18w mutant rescues the 18w invagination defects, indicating that 18W acts upstream of Sqh phosphorylation. Thus, both genetic and biochemical evidence indicate that 18W is a novel component of the Rho signaling pathway (Kolesnikov, 2007).
To determine whether 18W negatively regulates the RhoGAP branch of the Rho pathway, it was necessary to identify the RhoGAPs involved in salivary gland development. Of the 20 distinct RhoGAPs encoded by the Drosophila genome, 17 UAS-RhoGAP dsRNA lines were examined. Each of these lines was crossed to flies containing a salivary gland-expressing driver, scabrous-GAL4, and the progeny were screened for salivary gland defects. Only RhoGAP5A dsRNA expression resulted in salivary gland defects. Invagination defects are evident from more anteriorly placed glands, while migration defects resulted in wavy glands. Similar defects are seen upon expressing high levels of constitutively active Rho in the salivary gland with the scabrous-GAL4 driver. Overexpressing low levels of Rho in the gland results in mostly migratory defects, indicating that gland migration is more sensitive to the levels of Rho signaling than is the process of invagination. Invagination defects are seldom accompanied by migration defects, presumably because glands that have not properly internalized do not reach the visceral mesoderm upon which they normally migrate (Kolesnikov, 2007).
Since the GAL4/UAS dsRNA system may not result in a complete loss-of-function phenotype, strong alleles of two GAPs, RhoGAP68F and RhoGAP88C/Cv-c, which will be referred to simply as Cv-c. These have previously been shown to regulate Rho activity but lack salivary defects using the dsRNA interference technique. While neither rhoGAP68F nor cv-c mutations cause invagination defects, the cv-c mutants do display migration defects similar to those seen in embryos expressing RhoGAP5A dsRNA. To determine whether RhoGAP5A and Cv-c act redundantly within the salivary gland to regulate Rho activity, RhoGAP5A dsRNA within the gland was examined in a cv-c mutant. These embryos should have reduced activity of both RhoGAPs. They have gland invagination and migration defects that are more severe and penetrant than embryos that lack just one of them, indicating that these RhoGAPs are, in fact, partially redundant during salivary gland development (Kolesnikov, 2007).
In situ hybridization in wild-type embryos shows that cv-c RNA is not maternally contributed but is expressed in several tissues undergoing morphogenesis. In addition to its expression within the developing trachea and mesoderm, cv-c, similar to 18w, is expressed in the salivary glands prior to and during their invagination. Unlike 18w, however, cv-c expression does not appear to originate at the initial invagination site but rather initiates expression throughout most of the placode. During the onset of invagination at stage 12, cv-c expression intensifies and continues to be expressed within cells that have internalized until the conclusion of invagination at stage 13 (Kolesnikov, 2007).
To examine whether 18W regulates Rho activity by inhibiting Cv-c, several overexpression and genetic interaction experiments were performed. As might be expected if 18W negatively regulates Cv-c, overexpressing 18w within the salivary gland results in migratory defects similar to those seen in cv-c mutant embryos. In addition to migratory defects, however, some embryos overexpressing 18w also exhibit invagination defects, suggesting that Cv-c may not be the only RhoGAP negatively regulated by 18W. Moreover, lowering the dose of cv-c enhances the defects caused by 18w overexpression and suppresses the 18w mutant invagination defects, further supporting the role of 18W as a negative regulator of RhoGAP signaling. Therefore, genetic interaction experiments indicate that 18W regulates Rho signaling in the salivary gland by inhibiting at least one known RhoGAP (Kolesnikov, 2007).
Previous studies have shown that the Fog ligand activates RhoGEF2 through an as yet unidentified receptor, leading to the apical constriction of cells that form the ventral furrow and posterior midgut. Similar to salivary gland cells in 18w mutants, cells of the ventral furrow and posterior midgut in fog mutants do eventually invaginate but in an uncoordinated and delayed fashion. Since 18w and fog mutants have similar invagination defects, and 18W is a receptor protein that activates Rho signaling, whether 18W might be the FOG receptor was examined. This seems unlikely, however, because FOG overexpression within the salivary gland rescues the 18w mutant salivary gland defects. Since fog mutations do not completely eliminate apical constriction during ventral furrow and posterior midgut formation but RhoGEF2 mutations do, it has been argued that additional pathways must regulate apical constrictions via RhoGEF2. However, since 18w RhoGEF2 double mutants have more severe defects than either of the single mutants, 18W is not one of the additional upstream activators of RhoGEF2. Although neither present downstream of FOG nor upstream of RhoGEF2, 18W does appear to be positioned upstream of Sqh phosphorylation since the 18w salivary gland mutant phenotype can be rescued by introducing one copy of a phosphomimetic allele of sqh (Kolesnikov, 2007).
One possible way that 18W might activate Rho signaling is by negatively regulating RhoGAPs. Two RhoGAPs, RhoGAP5A and Cv-c, were identified that function partially redundantly during salivary gland morphogenesis. Embryos defective for both RhoGAPs exhibit invagination and migration defects similar to those observed when 18W is overexpressed within the salivary glands. Comparable defects are also seen upon expression of activated Rho, supporting the role of both RhoGAPs and 18W in Rho signaling (Kolesnikov, 2007).
Although overexpression and genetic interaction data demonstrate that 18W does indeed work in opposition to Cv-c activity, whether 18W actually negatively regulates RhoGAPs or if it controls Rho signaling through an alternate and unknown pathway has yet to to be deciphered. Another RhoGAP, RhoGAPp190, is regulated by the Src family of tyrosine kinases in both mammals and Drosophila. Depending on the site of phosphorylation, mammalian RhoGAPp190 can be either activated or inhibited by Src, while the Drosophila RhoGAPp190 appears to be only negatively regulated by the Drosophila Src homolog, Src64B. Genetic interactions and double mutant analysis with 18w and either Src64B or the other Drosophila Src gene, Src42A, however, suggest 18W does not regulate RhoGAPs via Src kinases in Drosophila (Kolesnikov, 2007).
Considering that 18W is a member of the Toll family of receptors, it might signal through the pathway used by Toll itself. Upon activation by its ligand, Spätzle, Toll signals via the cytoplasmic proteins MyD88, Tube, and Pelle to promote the degradation of the Cactus protein. This degradation releases the sequestered transcription factor Dorsal, allowing it to enter the nucleus and activate transcription. Although both 18w and Toll are expressed in the salivary gland, no evidence was found to suggest that 18W signals through the Toll-pathway or that it functions redundantly with Toll. Zygotic tube, pelle, MyD88, or Toll mutant embryos do not have salivary gland defects and MyD88 does not physically interact with any of the Toll-like receptors except for Toll itself. Similarly, there are no obvious genetic interactions between mutant alleles of 18w and Toll based both on lethality and salivary gland abnormalities (Kolesnikov, 2007).
Similar to the Toll family of receptors, many RhoGAPs and RhoGEFs are found in both mammals and flies. The Drosophila genome encodes 21 RhoGAPs and 20 RhoGEFs but only seven Rho-family GTPases. Since a specific RhoGTPase can be regulated by multiple RhoGAPs, there may be some redundancy in the function of the RhoGAPs. This appears to be the case during salivary gland development. Of the 17 RhoGAPs analyzed by RNAi, by available alleles, or by both, two resulted in distinct defects in the salivary glands. Mutant embryos that lack both of these RhoGAPs have more severe and penetrant gland defects than embryos that only lack one, indicating that the two have redundant roles during gland development (Kolesnikov, 2007).
Since 18W is expressed in several tissues undergoing morphogenesis, it will be interesting to establish whether it is important for the development of additional tissues other than the salivary gland. It will also be interesting to determine whether 18W functions in opposition to the particular RhoGAPs that are active within these other tissues. Overall, since very little is known about pathways controlling RhoGAP activity during apical constriction, identifying additional genes that interact with 18W may prove to be important not only in elucidating RhoGAP regulation but also in understanding the process of epithelial invagination (Kolesnikov, 2007).
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date revised: 25 August 2007
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