pebble

DEVELOPMENTAL BIOLOGY

Embryonic

Analysis of the subcellular distribution of Pbl has demonstrated that during mitosis, PBL accumulates at the cleavage furrow at the anaphase to telophase transition, when assembly of a contractile ring is initiated. In addition, levels of PBL protein cycle during each round of cell division with the highest levels of PBL found in telophase and interphase nuclei. PBL RNA and Pbl protein have a similar tissue distribution and are expressed in a highly dynamic pattern throughout embryogenesis. Pbl is strongly enriched in dividing nuclei of syncytial embryos and in pole cells as well as in nuclei of dividing cells in postblastoderm embryos. Pebble transcripts (4.0 and 5.5 kb) are abundant in adult flies and in embryos, but are not detected in third instar larvae. Pebble has lower levels of expression in late embryos (12-15 h after egg deposition, or stages 15-16), at a time when mitotic divisions are restricted mainly to the central nervous system. Western analysis using anti-Pbl immune sera shows that Pbl protein (115-120 kDa) is also abundant in wild-type embryos. The protein is expressed at elevated levels in embryos ectopically expressing Pbl (Prokopenko, 2000).

In preblastoderm embryos, both PBL RNA and Pbl protein are much enriched at the posterior pole where the pole cells form during telophase of mitotic cycle 10. PBL RNA and Pbl protein are deposited in the cytoplasm and nuclei, respectively, of budding pole cells. PBL RNA is expressed ubiquitously in preblastoderm and syncytial blastoderm embryos, but its levels decrease significantly upon cellularization. Pbl protein is expressed in all dividing nuclei in syncytial embryos beginning from the single nucleus stage. Levels of Pbl protein cycle during each round of nuclear divisions in the syncytium, similar to the cycling of Pbl during mitoses in the postblastoderm embryo. Pbl is also found in the interphase nuclei of all cells at cellular blastoderm. The dividing nuclei in syncytial embryos form dumbbell-shaped structures with PBL-positive late telophase nuclei being connected by a thin bridge of PBL staining surrounded by a nuclear envelope. Nuclear Pbl is not distributed homogeneously, but forms an intricate, speckled pattern (Prokopenko, 2000).

During gastrulation and germ band extension when many cells undergo mitotic cycles 14-16, Pbl is strongly enriched in the nuclei of most cells, including the pole cells. The highest levels of PBL are found in late telophase nuclei. This staining persists in early interphase nuclei in the following mitotic cycle, resulting in pairs of daughter cells being strongly labeled with nuclear Pbl. No PBL protein is observed during late prophase, metaphase, and early anaphase, suggesting that the protein levels undergo cyclic changes, during each cycle of cell division. During maximal germ band extension PBL RNA is expressed ubiquitously in the ectoderm and mesoderm with higher levels of expression in the brain and pole cells. At the end of germ band retraction (stage 12) PBL RNA is strongly expressed in the brain, pole cells, ventral nerve cord, and in many ectodermal and mesodermal cells. At this time Pbl protein is found ubiquitously with higher levels of expression in the brain. By stage 16 when most of the embryonic cells have stopped dividing, PBL RNA and especially Pbl protein expression is much lower in most tissues than at earlier embryonic stages. However, both RNA and protein continue to be expressed at elevated levels in neuroblasts in the brain, ventral nerve cord, and in gonads. In late embryos Pbl protein is also observed in the hindgut, and low levels of PBL RNA are observed in ectoderm, cells of the peripheral nervous system, pharynx, and gut (Prokopenko, 2000).

In conclusion, these observations demonstrate that there are high levels of maternally provided PBL RNA in preblastoderm and syncytial blastoderm embryos. The persistence of maternal RNA prior to cellularization allows cycling of levels of Pbl protein during the fast mitotic divisions 1-13. Furthermore, the reduction in the levels of Pbl RNA at the time of cellularization and the apparent cycling of the protein presumably account for the precise cycle 14 cytokinetic arrest phenotype in pbl mutants. The dramatic changes in the levels of Pbl during each round of nuclear division, together with the finding that Pbl is expressed in all nuclei in preblastoderm embryos suggest that prior to cellular blastoderm formation Pbl may play other roles, unrelated to its role in cytokinesis in late embryogenesis. To examine the function of pbl prior to cellularization, mitotic clones were generated in the germline. Interestingly, removal of maternally provided Pbl results in female sterility, suggesting a requirement for pbl in cell division during oogenesis. This observation precludes assessment the role of pbl in preblastoderm embryos (Prokopenko, 2000).

Pbl is expressed in pole cells and in gonads throughout embryogenesis. Pole cells are the first mononuclear cells formed in a syncytial embryo in a process mechanistically resembling cytokinesis. Both PBL RNA and Pbl protein are localized to the posterior pole prior to the formation of the first pole cells and are deposited, respectively, in their cytoplasm and nuclei. Interestingly, mutations in pbl affect proliferation of pole cells resulting in formation of few multinucleate cells (Lehner, 1992). This suggests that a 50% reduction in the levels of maternally provided PBL RNA causes the pole cell phenotype (Prokopenko, 2000).

These results also demonstrate that throughout embryogenesis Pbl protein has the highest levels of expression in the nuclei of dividing cells or in young postmitotic cells. Pbl expression seems to be specific for proliferating tissues or tissues with proliferative potential (e.g. embryonic neuroblasts). Furthermore, cessation of proliferation and terminal differentiation of cells correlate with the downregulation of pbl expression as observed in late embryos. However, pbl continues to be expressed in tissues which resume proliferation during larval development, that is, neuroblasts of the CNS and gonads (Prokopenko, 2000).

Pebble/ECT2 RhoGEF negatively regulates the Wingless/Wnt signaling

Effects of Mutation or Deletion

Mutations in pbl lead to a complete block of cytokinesis in mitotic cycle 14 (Hime, 1992 and Lehner 1992). Later rounds of nuclear divisions without cytokinesis result in the formation of polyploid multinucleate cells. Other events of the cell cycle (including nuclear envelope breakdown, chromosome condensation, and assembly and function of mitotic spindle) are not affected (Hime, 1992; Lehner 1992); during cycle 15 two mitotic figures are formed that independently enter anaphase. Affected cells fail to initiate a cleavage furrow, suggesting a defect in contractile ring function. To further characterize the defects in cytokinesis, the localization of actin, Pnut, and anillin, components of the contractile ring, were examined in pbl mutant cells. In wild-type cells, actin is associated with the cell cortex throughout mitosis and accumulates in the equatorial region of the cell, in which the contractile ring is assembled. Anillin, an actin-binding protein required for cytokinesis, is thought to play a role in organizing contractile domains of the actin cytoskeleton. In wild-type cells, anillin accumulates at the cleavage furrow at the onset of anaphase and is restricted to the contractile ring during anaphase and telophase. Pnut, a member of the septin family of proteins, accumulates at the cleavage furrow in late anaphase and is associated with the contractile ring during telophase. During cycle 14 in pbl mutant cells, actin, anillin, and Pnut fail to relocalize from the cell cortex to the cleavage furrow. Subsequently, the cleavage furrow is not initiated and cytokinesis fails. Similarly, in later rounds of cell division, actin, anillin, and Pnut fail to accumulate at the equatorial region of the cell, and there are no signs of a cleavage furrow. These results indicate that in pbl mutant cells the contractile ring does not assemble, leading to a failure of cytokinesis (Prokopenko, 1999).

In an effort to identify genes involved in the Pbl signaling pathway, a genetic screen was designed to isolate second-site mutations interacting with pbl. The GMR-GAL4 driver, which drives expression in all cells posterior to the morphogenetic furrow, was used to express Pbl in the adult fly eye. Overexpression of Pbl (with the UAS-Pbl^3.2 line) results in a dominant rough eye phenotype characterized by misshaped ommatidia, disrupted bristle distribution, misshapen rhabdomeres, and a significant increase in the number of pigment cells. In contrast, the external eye morphology of UAS-Pbl^3.2flies is indistinguishable from wild type. Thus, overexpression of Pbl in the eye perturbs normal retinal development. Furthermore, the developing eye seems to be more sensitive than the embryo to overexpression of Pbl, in which it does not cause a phenotype when expressed in ectodermal cells using the prd-GAL4 driver (Prokopenko, 1999).

To identify dominant second-site modifiers of the rough eye phenotype caused by overexpression of Pbl, the GMR-Pbl flies were crossed to males that carry deficiencies covering most of the II chromosome. The resulting F₁ progeny were examined for dominant enhancement or suppression of the rough eye phenotype. Df(2R)Jp8 deficiency, identified as the only suppressor in the modifier screen, shows an almost complete suppression of the rough eye phenotype. One of the genes uncovered by this deficiency is Rho1. To investigate the possibility that a mutation in Rho1 causes the suppression, the phenotype of the progeny from a cross between the GMR-Pbl flies and null alleles of Rho1 was analyzed. Removal of a single copy of Rho1 dominantly suppresses the rough eye phenotype caused by overexpression of Pbl. Transverse sections show rhabdomeres of more regular shape, number, and arrangement. In addition, the number and density of pigment cells is significantly decreased compared with the GMR-Pbl flies. Hypomorphic alleles of Rho1 cause a similar, but much weaker suppression than Rho1 null mutations. In contrast, alleles of Cdc42 or a deficiency removing Rac1 do not suppress or enhance this phenotype. These observations suggest that pbl interacts genetically with Rho1 and that the phenotypic suppression is specific for mutations in Rho1, but not for genes encoding other Rho family proteins, i.e., Cdc42 andRac1 (Prokopenko, 1999).

Overexpression of Rho1 in the eye results in disruption of both external and internal eye morphology. Mutations in pbl (pbl¹, pbl², pbl³, pbl⁵, pbl^P81, pbl^S23, and pbl^V58 alleles) cause a strong dominant suppression of the GMR-Rho1-induced rough eye phenotype. Because pbl encodes a putative RhoGEF, these results suggest that a reduction in exchange factor activity in pbl alleles is responsible for the suppression of the GMR-Rho1-induced eye phenotype. Similarly, it is proposed that decreased levels of Rho1, a putative effector of Pbl, account for suppression of the GMR-Pbl-induced eye phenotype by Rho1 mutations (Prokopenko, 1999).

To further characterize the genetic interaction between pbl and Rho1, a new deletion allele of pbl, PblDeltaDH497-549, was generated that affects the DH domain only. Site-directed mutagenesis in a number of RhoGEFs reveals that point mutations and deletions within the DH domain completely abolishes exchange factor activity. Hence, to create an inactive form of Pbl that behaves as a dominant-negative protein, a short deletion (amino acids 497-549) was introduced removing the most highly conserved CR3 region within the DH domain. Mutations within this region dramatically diminish the DH domain function, suggesting that this domain is responsible for GTPase binding and nucleotide exchange activity. Expression of PblDeltaDH497-549 in the eye with the GMR-GAL4 driver results in a rough eye phenotype, with pronounced fusion of some ommatidia and a significant reduction in the number of bristles. Transverse sections show a variation in the number of rhabdomeres per ommatidium and, unlike overexpression of the full-length Pbl, a decrease in the number of pigment cells. This phenotype is significantly enhanced in a dominant fashion by mutations in pbl. Similarly, mutations in Rho1 act as dominant enhancersof this phenotype, with a dramatic increase in the number of fused ommatidia and loss of many bristles. Transverse sections show a severe disorganization of the internal architecture of the eye, a complete absence of rhabdomeres in some regions of the eye, and a further decrease in the number of pigment cells. Because the PblDeltaDH497-549 protein is likely to be inactive, it is suggested that this protein may mimic the phenotype of hypomorphic alleles of pbl by sequestering proteins (other than Rho1) that normally bind to or interact with endogenous Pbl. As expected, mutations in Rho1 that interact genetically with pbl enhance the phenotype of PblDeltaDH497-549. In summary, pbl shows strong genetic interaction with Rho1, but not with Rac1 or Cdc42, indicating that pbl and Rho1 are in the same genetic pathway in vivo (Prokopenko, 1999).

Cytokinesis is developmentally controlled during Drosophila embryogenesis. It is omitted during the initial nuclear division cycles. The nuclei of the resulting syncytium are then cellularized at a defined stage, and cytokinesis starts in somatic cells with mitosis 14. However, cytokinesis never occurs in somatic cells of embryos homozygous or transheterozygous for mutations in the pebble gene. Interestingly, the process of cellularization, which involves steps mechanistically similar to cytokinesis, is not affected. Moreover, all the nuclear aspects of mitosis (nuclear envelope breakdown, chromosome condensation, spindle assembly and function) proceed normally in pebble mutant embryos, indicating that pebble is specifically required for the coordination of mitotic spindle and contractile ring functions. The pebble phenotype is also observed, but only with very low penetrance, during the early divisions of the germ line progenitors (the pole cells). alpha-Amanitin injection experiments indicate that these early pole cell divisions, the first cell divisions during embryogenesis, do not require zygotic gene expression. These divisions might therefore rely on maternally contributed pebble function. The maternal contribution from heterozygous mothers might be insufficient in rare cases for all the pole cell divisions (Lehner, 1992).

Mutations at the pebble locus of Drosophila result in embryonic lethality. Examination of homozygous mutant embryos at the end of embryogenesis reveal the presence of fewer and larger cells that contain enlarged nuclei. Characterization of the embryonic cell cycles using DAPI, propidium iodide, anti-tubulin and anti-spectrin staining shows that the first thirteen rapid syncytial nuclear divisions proceed normally in pebble mutant embryos. Following cellularization, the postblastoderm nuclear divisions occur (mitoses 14, 15 and 16), but cytokinesis was never observed. Multinucleate cells and duplicate mitotic figures are seen within single cells at the time of the cycle 15 mitoses. It is concluded that zygotic expression of the pebble gene is required for cytokinesis following cellularization during Drosophila embryogenesis. It is postulated that developmental regulation of zygotic transcription of the pebble gene is a consequence of the transition from syncytial to cellular mitoses during cycle 14 of embryogenesis (Hime, 1992).

The proper localization of Numb depends on its interaction with the adapter protein Partner of numb (Pon). In pon mutant embryos, the formation of Numb crescent is delayed in neuroblasts and is disrupted in muscle progenitor cells (Lu, 1998). Pon was isolated on the basis of its physical interaction with Numb. Pon is asymmetrically localized during mitosis and colocalizes with Numb. Ectopically expressed Pon responds to the apical-basal polarity of epithelial cells and is sufficient to localize Numb basally. It is proposed that PON is one component of a multimolecular machinery that localizes Numb by responding to polarity cues conserved in neural precursors and epithelial cells (Lu, 1998 and Lu, 1999).

In principle, the asymmetric localization of Numb/Pon can be accomplished by one or a combination of the following mechanisms: localization and local translation of their mRNAs; active transport of the proteins by motor molecules along the cytoplasmic or cortical cytoskeleton; passive diffusion (3D in the cytosol or 2D along the cortex) and trapping of the proteins by basally localized anchor molecules, or protein targeting to the membrane followed by selective degradation at one side of the cortex. The available method to detect protein localization by immunostaining of fixed embryos only provides static images of the proteins in different cells and is inadequate to distinguish among the above possibilities (Lu, 1999).

The mechanism of asymmetric Pon localization has been shown to operate at the protein level. The asymmetric localization domain of Pon has been mapped to its C-terminal region. Using a fusion between this localization domain and GFP, the entire process of Pon localization was monitored in neuroblasts of living embryos. This in vivo analysis reveals that the asymmetric localization of Pon is a dynamic, multistep process. The protein is first recruited from the cytosol to the cell cortex, a step that requires cell cycle progression into mitosis. Cortically recruited Pon then moves on the cortex and is later restricted to the basal side to form a crescent. The crescent disintegrates upon exit from mitosis. Photobleaching experiments reveal both apical and basal movements of Pon on the cortex. These movements can still occur when myosin motor activity is inhibited by drug treatment. Genetic and pharmacological analyses further reveal that the formation and anchoring of the Pon crescent at the basal cortex require actomyosin and Inscuteable (Lu, 1999).

The gradual recruitment of Pon from the cytosol to the cell cortex at early stages of the cell cycle appears to be coupled to cell cycle progression. To test whether entry into mitosis is a prerequisite for this cortical recruitment, Pon-GFP was introduced into cell cycle mutant backgrounds. In string mutants, postblastoderm cells arrest at the G2 phase of the cell cycle. In neuroblasts of string mutants, the GFP signal is diffuse in the cytoplasm, with some uniform cortical staining. Even after 1 hr of recording, the cytoplasmic signal is not cleared, although the uniform cortical signal seems to increase slightly over time. In contrast, in wild-type neuroblasts, the cytoplasmic signal is cleared within 5-6 min after its appearance. Thus, cortical recruitment of Pon-GFP depends on entry into mitosis (Lu, 1999).

In pebble mutants, cytokinesis of postblastoderm cell divisions is blocked, but other cell cycle events including the asymmetric localization of Numb and Prospero still occur. The initial cortical recruitment and formation of a basal Pon-GFP crescent are normal in pebble mutant neuroblasts. However, by continuously monitoring the Pon-GFP crescent, it was observed that within 10-15 min of its formation, the crescent starts to disintegrate and the protein is dispersed uniformly on the cortex. Gradually, the cortical signal is decreased to background levels, presumably due to degradation or release from the cortex. By comparing the time interval between crescent formation and disintegration in pebble mutants (10-15 minutes) with the interval between crescent formation and the later stages of the neuroblast cell cycle in wild-type embryos, it was determined that the timing of Pon-GFP crescent disintegration in pebble mutants coincides with the end of a normal neuroblast division. This result suggests that at the exit from mitosis certain cell cycle events disassemble or inactivate the localization machinery and that this can occur in the absence of cytokinesis (Lu, 1999).

The defect in the cortical recruitment of Pon-GFP in string mutants suggests that the assembly or proper functioning of the machinery that recruits Pon to the cortex depends on cell cycle progression into mitosis. It is also possible that the cortical recruitment of Pon may depend on its posttranslational modification such as phosphorylation by the p34cdc2 kinase, which is inactive in string mutants. Further biochemical characterization of Pon protein, such as analyzing its posttranslational modification during the cell cycle, should provide more insight into the mechanistic aspects of this regulation (Lu, 1999).

The disintegration of Pon-GFP crescent at the exit from mitosis in pebble mutants implicates a role for the cell cycle machinery in disabling the protein localization machinery. The absence of cytokinesis in pebble mutants allows this step to be observed in more detail. In wild-type neuroblasts, the cleavage furrow coincides with the border of the Pon-GFP crescent at cytokinesis, therefore the GFP signal is distributed all around the GMC cell membrane as soon as the GMC is formed. This makes the crescent disintegration step not observable in wild-type embryos. However, in both wild-type GMC cells and pebble mutant neuroblasts, the uniform cortical GFP signal is gradually decreased to background levels as the cell cycle progresses, suggesting that Pon-GFP is eventually released from the cortex and becomes degraded or delocalized in both wild-type and pebble mutant embryos. In this regard, it will be interesting to test whether the anaphase-promoting complex/cyclosome ubiquitin ligase or components of the mitosis exit signaling pathway are involved in the disintegration of Pon-GFP crescent and the subsequent release of the protein from the cortex (Lu, 1999).

Neurons and glia are produced in stereotyped patterns after neuroblast cell division during development of the Drosophila central nervous system. The first cell division of thoracic neuroblast 6-4 (NB6-4T) is asymmetric, giving rise to a glial precursor cell and a neuronal precursor cell. In contrast, abdominal NB6-4 (NB6-4A) divides symmetrically to produce two glial cells. To understand the relationship between cell division and glia-neuron cell fate determination, the effects of known cell division mutations on the NB6-4T and NB6-4A lineages were examined and compared. Based on observation of expression of glial fate determination and early glial differentiation genes, the onset of glial differentiation occurs in NB6-4A but not in NB6-4T when both cell cycle progression and cytokinesis are genetically arrested. In contrast, glial differentiation starts in both lineages when cytokinesis is blocked with intact cell cycle progression. These results show that NB6-4T, but not NB6-4A, requires cell cycle progression for acquisition of glial fate, suggesting that distinct mechanisms trigger glial differentiation in the different lineages (Akiyama-Oda, 2000).

Cell division mutants stg, cycA, and pbl were used to investigate the relationship between cell division and glia- neuron cell fate determination in NB6-4. To determine the effects of cell cycle arrest on cell fate of NB6-4, expression of a glial fate determination protein, Gcm, and an early glial marker protein, Repo, were examined. In pbl mutant embryos, which lack cytokinesis, all the nuclei of NB6-4T express the glial proteins, suggesting that cytokinesis is not required for the onset of glial differentiation in the NB6-4T lineage. It has been suggested that cytokinesis may be required for negative regulation of glial differentiation, since more than three nuclei in the pbl mutant NB6-4T, in contrast to three glial cells in the wild-type, express Repo (Akiyama-Oda, 2000).

In the NB6-4T lineage, the first cell division is a critical step for triggering glial differentiation. Coincident with the onset of glial differentiation is the occurence of cell fate bifurcation. In the cell division rescue experiments using eg-GAL4, Gcm-positive and Gcm-negative cells appear after the first cell division, although surrounding cells are still stg mutant. The cell fate bifurcation is probably regulated cell intrinsically and coupled to cell division. In contrast, all the nuclei of NB6-4T in the pbl mutant express Gcm and Repo. This may be because these proteins contain the nuclear localization signal that enables them to enter the nuclei within the single cell after translation, even if asymmetry might initially appear within the cell (Akiyama-Oda, 2000).

During Drosophila embryogenesis the Malpighian tubules evaginate from the hindgut anlage and in a series of morphogenetic events form two pairs of long narrow tubes, each pair emptying into the hindgut through a single ureter. Some of the genes that are involved in specifying the cell type of the tubules have been described. Mutations of previously described genes were surveyed and ten were identified that are required for morphogenesis of the Malpighian tubules. Of those ten, four block tubule development at early stages; four block later stages of development, and two, rib and raw, alter the shape of the tubules without arresting specific morphogenetic events. Three of the genes, sna, twi, and trh, are known to encode transcription factors and are therefore likely to be part of the network of genes that dictate the Malpighian tubule pattern of gene expression (Jack, 1999).

Another group of genes that alter the morphology of the tubules are those that are required for normal cell proliferation. However, mutations of these genes affect not only the number of cells in the tubules but also their shape. Tubules mutant for these genes are generally defective in cell rearrangement. pimples (pim), whose mutations arrest development when the tubules are relatively small, encodes a protein required for sister chromatid separation, and mutants are defective in cytokinesis. The small tubule size in pim mutants is undoubtedly caused by the failure of tubule cells to divide normally. Mutations of pbl and three rows (thr), which are defective in cytokinesis and mitosis, respectively, also cause a reduction in the size of the tubules. Mutants of all three fail to undergo elongation to the final two cell circumference. crooked neck (crn) encodes a protein of unknown function that is required for normal proliferation of neuroblasts and has been suggested to function in progression through the cell cycle. In crn mutations only a few cells in the distal part of the tubules rearrange, while the more proximal cells fail to rearrange at all. Since crn is required for normal cell proliferation, the tubules may have a reduced number of cells. crn tubes are closer to the wild-type cell number than mutants of other genes that are defective for cell proliferation, and a small segment of the distal tubules elongate. Nevertheless, the association between defective cell proliferation and failure of elongation is maintained in crn mutants. One possibility is that the attainment of the proper cell number in the tubules triggers both the termination of cell proliferation and cell rearrangement (Jack, 1999).

Molecular dissection of cytokinesis by RNA interference in Drosophila cultured cells

Double-stranded RNA-mediated interference (RNAi) was used to study Drosophila cytokinesis. Double-stranded RNAs for anillin, RacGAP50C, pavarotti, rho1, pebble, spaghetti squash, syntaxin1A, and twinstar all disrupt cytokinesis in S2 tissue culture cells, causing gene-specific phenotypes. The phenotypic analyses identify genes required for different aspects of cytokinesis, such as central spindle formation, actin accumulation at the cell equator, contractile ring assembly or disassembly, and membrane behavior. Moreover, the cytological phenotypes elicited by RNAi reveal simultaneous disruption of multiple aspects of cytokinesis. These phenotypes suggest interactions between central spindle microtubules, the actin-based contractile ring, and the plasma membrane, and led to a proposal that the central spindle and the contractile ring are interdependent structures. Finally, these results indicate that RNAi in S2 cells is a highly efficient method to detect cytokinetic genes, and predict that genome-wide studies using this method will permit identification of the majority of genes involved in Drosophila mitotic cytokinesis (Somma, 2002).

The finding that chicadee, four wheel drive (fwd), and Kinesin-like protein at 3A (klp3A) are not required for cytokinesis in S2 cells is not surprising, because previous studies pointed toward a specific involvement of these genes in meiotic cytokinesis of males. Null mutations in klp3A, a gene encoding a kinesin-like protein expressed both in testes and somatic tissues, disrupt meiotic cytokinesis but have no effect on larval neuroblast division. Similarly, flies homozygous for null mutations in fwd, which encodes a phosphatidyl-inositol kinase, are viable but male sterile, and are specifically defective in male meiotic cytokinesis. In contrast with fwd and klp3A that are not required for viability, chic is an essential gene that specifies a Drosophila homolog of profilin. However, both male sterile chic mutants and heteroallelic chic combinations resulting in lethality, display severe disruptions in meiotic cytokinesis but have no defects in neuroblast cytokinesis (Somma, 2002).

The phenotypical analyses of RNAi-induced mutants in the RacGAP50C, rho1, and sqh genes provide the first description of the cytological defects that lead to cytokinesis failures when the function of these genes is ablated. Previous studies have shown that mutations in rho1 and sqh disrupt mitotic cytokinesis but have not defined the cytological phenotypes elicited by these mutations. In addition, pav and pbl (RNAi) cells have been characterized; the phenotypes of these (RNAi) cells are consistent with those previously observed in animals homozygous for mutations in these genes (Somma, 2002 and references therein).

Cells in which the RacGAP50C, pav, pbl, rho1, and sqh genes are ablated by RNAi normally undergo anaphase A, but they then fail to elongate and to undergo anaphase B. After anaphase A, mutant cells proceed toward telophase and decondense their chromosomes, forming typical telophase nuclei. However, these cells fail to develop a central spindle, to assemble an actomyosin contractile ring and to concentrate anillin in the cleavage furrow. This results in the formation of short, aberrant telophases that are unable to undergo cytokinesis and will thus give rise to binucleated cells (Somma, 2002).

The functional ablation of genes influencing either the actin or the microtubule cytoskeleton have similar effects on cytokinesis. The genes pbl, rho1, and sqh likely play primary roles in controlling the actin cytoskeleton. The sqh gene encodes a regulatory light chain of myosin II. Rho1 is a member of the Rho family GTPases that cycle from an inactive GDP-bound state to an active GTP-bound state under the regulation of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs enhance the exchange of bound GDP for GTP, whereas GAPs increase the GTPase activity of Rho. Rho proteins and Rho GEFs, such as Drosophila Pbl and human ECT2, localize to the cleavage furrow and are required for contractile ring assembly. In contrast, the activities of RacGAP50C and pav are likely to primarily influence the function of the central spindle. The Pav kinesin-like protein, a homolog of the C. elegans ZEN-4, is localized in the central spindle, and is thought to mediate microtubule cross-linking at the central spindle midzone. The RacGAP50C gene encodes a Rho GAP, and it is orthologous to the cyk-4 gene of C. elegans. CYK-4 interacts with ZEN-4, and the two proteins are mutually dependent for their localization to the central spindle. The complete absence of Pav immunostaining in RacGAP50C (RNAi) telophases suggests a similar interaction between RacGAP50C and Pav, pointing to a role of RacGAP50C in central spindle assembly. In summary, the cytological phenotypes of pbl, rho1, and sqh (RNAi) cells indicate that a primary defect in acto-myosin ring formation results in a secondary defect in central spindle assembly. The phenotypes of RacGAP50C- and Pav-depleted cells suggest the converse: that a primary defect in the central spindle can secondarily disrupt contractile ring formation. Thus, taken together, these data indicate that the central spindle and the actomyosin ring are interrelated structures. Although the molecular mechanisms underlying the cross talk between these structures is not conpletely understood, two possibilities can be envisioned. The formation and maintenance of both the central spindle and the actomyosin ring could be mediated by physical interactions between interzonal microtubules and components of the contractile ring. Alternatively, the central spindle and the contractile ring could be coupled by a checkpoint-like regulatory mechanism, which would inhibit the formation of either of these structures when the other is not properly assembled (Somma, 2002).

Although RacGAP50C, pav, pbl, rho1, and sqh (RNAi) cells display similar terminal phenotypes, the aberrant telophases observed in these cultures differ in both actin and anillin distribution. In rho1 telophases these proteins are excluded from the cell equator, in pbl they are uniformly distributed, and in RacGAP50C, pav, and sqh they concentrate in a wide equatorial band. This suggests that rho1 and pbl are required for actin and anillin accumulation in the equatorial region of the dividing cell. In contrast RacGAP50C, pav, and sqh seem to be required for the assembly of the contractile machinery from proteins already concentrated at the cell equator. In sqh (RNAi) cells the failure to assemble an actomyosin ring is likely to be a direct consequence of the depletion of an essential component of the ring. In RacGAP50C and pav cells this failure is instead likely to be a secondary effect of problems in central spindle assembly (Somma, 2002).

An interplay between the central spindle and the contractile ring has been suggested by studies on Drosophila male meiosis. Mutant spermatocytes in the chic, and dia loci, which encode products thought to be involved in contractile ring formation, and mutants in the kinesin-encoding gene klp3A, all display severe defects in both structures. Although all the extant results on Drosophila cells strongly suggest an interdependence of the central spindle and the contractile ring, it is currently unclear whether this is true in all animal cells. Studies on mammalian cells have shown that central spindle plays an essential role during cytokinesis. However, these experiments have provided limited information on whether perturbations in the actomyosin ring assembly disrupt the central spindle. The best evidence of an interplay between the central spindle and the contractile ring has been in rat kidney cells. By puncturing these cells with a blunt needle a physical barrier is created between the central spindle and the equatorial cortex. This barrier not only abrogates actomyosin ring assembly on the side of perforation facing the cortex, but also disrupts the organization of central spindle microtubules on the opposite side (Somma, 2002 and references therein).

In contrast, studies on C. elegans embryos indicate that, at least in the early stages of cytokinesis, the actomyosin ring and the central spindle can assemble independently. Why do Drosophila, and possibly mammalian cells, differ from C. elegans in the interactions between the central spindle and the contractile ring? It is believed that the answer to this question reflects differences in the distance between the central spindle and the equatorial cortex. In Drosophila and mammalian cells during central spindle assembly the equatorial cortex is very close to the interzonal microtubules. In contrast, in C. elegans embryos the central spindle assembles in the center of the cell when the cleavage furrow has just began to ingress, so that during their assembly the actomyosin ring and the central spindle lie a considerable distance apart. Only later in cell division, after substantial furrow ingression, can the actomyosin ring and the central spindle come into contact. It is thus hypothesized that in embryonic cells of C. elegans the cytokinetic process consists of two steps: an early step, where the central spindle and the contractile ring assemble independently in distant cellular regions, and a late step that begins when the central spindle and the contractile ring have come into contact. The early stage might be mediated by interactions between astral (rather than central spindle) microtubules and the contractile ring. The late step of C. elegans cytokinesis may then require that the contractile ring and the central spindle interact cooperatively to complete cytokinesis successfully. This two-step hypothesis also applies to other large cells, such as echinoderm eggs, where the central spindle and the cortex are separated by large masses of cytoplasmic material and seem to assemble independently (Somma, 2002 and references therein).

The RhoGEF Pebble is required for cell shape changes during cell migration triggered by the Drosophila FGF receptor Heartless

The FGF receptor Heartless (HTL) is required for mesodermal cell migration in the Drosophila gastrula. Mesoderm cells undergo different phases of specific cell shape changes during mesoderm migration. During the migratory phase, the cells adhere to the basal surface of the ectoderm and exhibit extensive protrusive activity. HTL is required for the protrusive activity of the mesoderm cells. Moreover, the early phenotype of htl mutants suggests that HTL is required for the adhesion of mesoderm cells to the ectoderm. In a genetic screen pebble was identfied as a novel gene required for mesoderm migration. pbl encodes a guanyl nucleotide exchange factor (GEF) for RHO1 and is known as an essential regulator of cytokinesis. The function of Pbl in cell migration is shown to be independent of the function of Pbl in cytokinesis. Although the small GTPase Rho1 acts as a substrate for Pbl in cytokinesis, compromising Rho1 function in the mesoderm does not block cell migration. These data suggest that the function of Pbl in cell migration might be mediated through a pathway distinct from Rho1. This idea is supported by allele-specific differences in the expressivity of the cytokinesis and cell migration phenotypes of different pbl mutants. Pbl is shown to be autonomously required in the mesoderm for cell migration. Like Htl, Pbl is required for early cell shape changes during mesoderm migration. Expression of a constitutively active form of Htl is unable to rescue the early cellular defects in pbl mutants, suggesting that Pbl is required for the ability of Htl to trigger these cell shape changes. These results provide evidence for a novel function of the Rho-GEF Pbl in Htl-dependent mesodermal cell migration (Schumacher, 2004).

In Drosophila the mesoderm originates from a ventral population of cells in the monolayered blastoderm epithelium. At the onset of gastrulation these cells are first internalized through an invagination of the epithelium. After internalization, the cells undergo mitosis, lose their epithelial characteristics, and start to spread as an aggregate between the central yolk sac and the basal cell surfaces of the ectoderm (Schumacher, 2004).

To follow cell shape changes of mesoderm cells, a transgene was used driving expression of the transmembrane protein CD2 from rat under the control of the twist (twi) promotor (twi::CD2). twi::CD2 is already expressed during invagination and represents a cell-surface marker specific for the mesoderm. Mesoderm migration can be divided into three phases with characteristic cell shape changes. After invagination, the mesoderm initially forms an epithelial tube. At phase 1 of migration, the surface of the mesoderm cells appears relatively smooth. After disassembly of the epithelial tube and mitosis, phase 2 begins, in which the mesodermal aggregate migrates out in dorsolateral direction. Cells at the leading edge of the aggregate are stretched along the dorsoventral axis and extend multiple cellular protrusions. The longest cellular protrusions often measure half to two-thirds the size of a cell diameter (to a length of 10-15 µm in fixed samples). Cross-sections reveal that not only the leading edge cells exhibit this polarized morphology, but that the cells immediately following the leading edge cells frequently also extend in dorsolateral direction. The term 'protrusive activity' is used to describe the formation and/or the dynamics of the filoform and lamelliform protrusions that are observed in fixed preparations. The protrusive activity is specific for the migratory phase, because when the cells have reached their final positions (phase 3) and form a coherent monolayer, large extensions are absent and only few filoform protrusions are observed (Schumacher, 2004).

Examination of embryos homozygously mutant for htl reveal that Htl is required for cell shape changes during phase 1 and 2 of mesoderm migration. In phase 1, the mesodermal epithelial tube extends further into the interior of the embryo when compared with wild type. During phase 2, the leading edge cells do not extend dorsolaterally. This phenotype is not simply explained by the possibility that htl mutant mesoderm cells were not able to contact the ectoderm, because cells directly apposed to the ectoderm also fail to extend. In phase 3, mesoderm cells of htl mutant embryos do not establish a monolayer configuration. Interestingly, during and after phase 3, htl mutant mesoderm cells exhibit directional protrusions suggesting that some migration might occur at these stages. This result indicates that Htl is not generally required for protrusive activity of the mesoderm cells. This late migration in htl mutants is never able to rescue the defects in mesoderm differentiation, most probably because of a second requirement of Htl for mesoderm differentiation (Schumacher, 2004).

These observations suggest that Htl is required for the early interaction of the mesoderm with the ectoderm. By analyzing sections, wild-type cells at the base of the mesodermal tube are observed to be attached to the basal surfaces of the ectoderm. By contrast, htl mutant mesoderm cells at the respective stage and position fail to establish contact to the ectoderm. This phenotype correlates well with a misalignment of the mesodermal tube in htl mutants. It is concluded that Htl is required for the effective attachment of mesoderm cells to the ectoderm, which might promote the protrusive activity of mesoderm cells during migration (Schumacher, 2004).

htl and dof represent the only zygotically expressed genes that have thus far been described to be essential for mesoderm migration. To obtain a better insight into the genetic control of mesoderm migration, a genetic screen was performed to identify zygotically expressed genes involved in mesoderm migration. Three loci mapped to the third chromosome and were characterized using chromosomal deletions and available point mutations. Two loci corresponded to the genes htl and dof, respectively. Embryos lacking the chromosomal interval 61A to 68 (based on breakpoints of the chromosomal translocation T(2;3)C309) displayed defects in mesoderm migration. Genetic mapping revealed that small overlapping chromosomal deletions, which exhibited the phenotype, all removed the gene pbl. Analysis of a strong loss-of-function point mutation in pbl, pbl³, indicated that pbl is required for mesoderm morphogenesis. Embryos homo- or hemizygously mutant for pbl³ show a dramatic reduction in the number of Eve-positive mesoderm cells at the extended germband stage. These results demonstrate a thus far unrecognized function for pbl in mesoderm differentiation (Schumacher, 2004).

pbl encodes a RHO1-GEF most similar to the vertebrate ect2 proto-oncogene. Both pbl and ect2 are required for the assembly of the contractile actin ring during cytokinesis. Interfering with the function of PBL or ECT2 results in a failure of cytokinesis and the generation of multinucleate cells. Because mutations in pbl affect cell shape changes before mitoses in the mesoderm occur, it was suspected that the requirement of pbl for mesoderm migration might be independent from its cytokinesis function. To determine, whether the defects in mesoderm migration in pbl mutants are direct rather than a secondary consequence of the failure in cytokinesis, the pbl phenotype was examined in division-defective embryos (Schumacher, 2004).

Postblastoderm mitotic divisions are controlled by zygotic expression of the cell cycle regulator String. Since mesoderm migration and specification of Eve-positive mesoderm cells occur normally in stg mutant embryos, this mutation provides a genetic condition to assay cytokinesis-independent functions of pbl. The cytokinesis defect of pbl is completely blocked by stg. In pbl stg double mutant embryos, migration of the mesoderm and specification of dorsal mesodermal derivatives are impaired similar to pbl single mutants. Moreover, cell shape changes in phase 2 occur normally in stg mutant embryos, but protrusive activity of the mesoderm cells is blocked in pbl stg double mutants. These results indicate that the activity of pbl is required for mesoderm migration even in the absence of mitosis and thus in the absence of cytokinesis defects. It is therefore concluded that Pbl has independent functions in cytokinesis and cell migration (Schumacher, 2004).

Thus Htl is required for protrusive activity only during phase 1 and 2 of mesoderm migration. However, Htl activity is not essential for the protrusive activity of the cells per se, because cells do extend dorsolaterally during phase 3 in htl mutant embryos. These data demonstrate that Htl activation is unlikely to provide the only directional cue in mesoderm migration. The results presented in this paper suggest that Htl signaling provides temporal information for protrusion formation during phases 1 and 2, and might be therefore acting as a permissive factor during mesoderm migration (Schumacher, 2004).

Pbl is required for protrusive activity of mesoderm cells also in phase 3 and later. It is therefore possible that Pbl function might be required in a more general way for the cell to extend protrusions. The specificity of Pbl for protrusive activity is also supported by the fact that loss of epithelial characteristics is unaffected in pbl mutant embryos. Although the specific mechanism of Pbl function in cell migration is currently unknown, it is important to note that not all morphogenetic movements are compromised in pbl mutants. For example, cephalic furrow formation, invagination of the ventral furrow and germband extension movements, which all depend on a functional cytoskeleton are normal in pbl mutant embryos. It is therefore proposed that Pbl might constitute an important component for cytoskeletal changes, which are triggered by FGFR signaling events (Schumacher, 2004).

Of the multiple responses generated downstream of FGFR activation, only little is known of the molecular pathways by which FGFRs trigger cell shape changes in vivo. The Rho GEF Pbl represents a good candidate for mediating cell shape changes triggered by Htl signaling. Importantly, the early phenotypes of htl and pbl mutants are almost identical, indicating that both gene products are required in a narrow time window for early cell shape changes after invagination of the mesoderm. Furthermore, in both mutants this phenotype is completely penetrant, indicating that the gene products do not act in a redundant fashion (Schumacher, 2004).

The function of Pbl for mesoderm migration is specific for mesoderm cells. Because htl is expressed only in the mesoderm at this stage of development, Pbl might be involved in the presentation of the receptor or its ligand and thus acting upstream, or Pbl might be involved in downstream events triggered by the Htl signaling cascade. If Pbl was acting upstream of Htl, signaling events downstream of Htl should be blocked in such mutants. By contrast, Pbl is shown to be dispensable for activation of MAP kinase in the early mesoderm cells. These results suggest that Pbl does not act upstream of Htl, and a model is favored in which Pbl acts downstream of the Htl signaling cascade (Schumacher, 2004).

The present results render it unlikely that Pbl is directly involved in a signaling pathway downstream of Htl FGFR. In contrast to htl mutants, no cell shape changes and no protrusive activity was observed in pbl mutant mesoderm cells in phase 3. In addition, the pbl null mutant phenotype still allows a few cells to undergo eve expression, probably owing to the larger cells and abnormal cytoarchitecture in the division defective embryos. This is in contrast to htl loss of function mutants where EVE-positive mesoderm cells are never observed. If pbl is essential for signaling downstream of the Htl receptor, the phenotype of htl and pbl mutants should be more similar with respect to mesoderm differentiation: in fact, the phenotypes of htl and dof mutant embryos are identical. It is therefore proposed that Pbl might represent a regulator of the cytoskeleton or adhesive mechanisms of the cell, which provide targets of the Htl signaling cascade to trigger cell shape changes (Schumacher, 2004).

Although the activation of MAP kinase in the mesoderm depends on Htl, it is not known whether this is a direct response or whether it is indirect, i.e. MAP kinase may not be directly activated by Htl itself, but through interactions of the mesoderm with the ectoderm. In this case, activation of MAP kinase would be a response rather than a cause of the cell shape changes. The phenotype of pbl mutants, however, argues against the latter possibility, because it shows that in the absence of cell shape changes, MAP kinase can still be activated. This result also suggests that activation of MAP kinase alone cannot account for the cell shape changes that occur. This idea is supported by the fact that activated forms of RAS1 are unable to completely rescue the defects in mesoderm migration of htl or dof mutant embryos, including the defects in cell shape changes in phase 1 and 2. These results suggest the presence of a signaling pathway acting in parallel to the Ras/Raf MAP kinase pathway to be involved in mesoderm migration (Schumacher, 2004).

The pbl gene was originally identified and characterized as an essential factor for cytokinesis. Two lines of evidence indicate that the function of Pbl in cell migration is mediated through a pathway different from the cytokinesis pathway. (1) Expression of a dominant-negative form of the small GTPase Rho1, which blocks cytokinesis in the mesoderm, has no effect on mesoderm migration, cell shape changes associated with it or expression of differentiation markers specific for dorsal mesoderm derivatives. (2) A mutation in pbl, pbl^11D exhibits significantly weaker defects in mesoderm differentiation compared to the strong loss of function mutation pbl³. These allele-specific differences indicate distinct requirements of the Pbl protein for its two functions, because both alleles exhibit identical cytokinesis defects and only differ in mesoderm differentiation defects significantly. It is therefore proposed that the function of Pbl for cell migration might not involve Rho1 and might therefore be using another mechanism (Schumacher, 2004).

How does Pbl act in cell migration and which GTPase represents its substrate? Both Pbl and its mammalian orthologs belong to the Dbl family of Rho-GEFs, which promote activation of Rho GTPases through a conserved Dbl-homology (DH) domain. The DH domain is required for both functions of Pbl, because a missense mutation in pbl, called pbl⁵, in which an amino acid exchange renders the DH domain inactive, exhibits equally strong defects in cell migration and cytokinesis. Data from yeast two-hybrid assays, as well as genetic interactions indicate that Pbl binds to and interacts with RHO1. During cytokinesis, Pbl is proposed to locally activate RHO1, which then interacts with its effector Diaphanous, a Drosophila homolog of the Formin family of actin regulators. Although Pbl appears to interact with RHO1, but not with CDC42 or RAC1, mammalian homologs of Pbl promote GTP/GDP exchange of the GTPases RHO1, RAC1 and CDC42. Because these discrepancies might reflect differences in the sensitivity of the assays applied, it remains to be determined which substrate Pbl uses for its function in cell migration (Schumacher, 2004).

Although a role of Pbl in FGFR triggered cell migration has been detected, it is currently unclear how general the requirement of Pbl is for the protrusive activity of migrating cells. Interestingly, mutations in pbl have been discovered in a screen for genes required for the development of the peripheral nervous system. These mutants affect the correct migration of the axons in the PNS without obvious defects in cytokinesis. It will therefore be interesting to assess the function of Pbl in a variety of migrating cells to further characterize its potential role as a mediator of cell shape changes triggered by extracellular signals (Schumacher, 2004).

The epithelial-mesenchymal transition of the Drosophila mesoderm requires the Rho GTP exchange factor Pebble

Drosophila pebble encodes a Rho-family GTP exchange factor (GEF) required for cytokinesis. The accumulation of high levels of Pbl protein during interphase and the developmentally regulated expression of pbl in mesodermal tissues suggests that the primary cytokinetic mutant phenotype might be masking other roles. Using various muscle differentiation markers, it was found that Even skipped (Eve) expression in the dorsal mesoderm is greatly reduced in pbl mutant embryos. Eve expression in the dorsalmost mesodermal cells is induced in response to Dpp secreted by the dorsal epidermal cells. Further analysis has revealed that this phenotype is likely to be a consequence of an earlier defect. pbl mutant mesodermal cells fail to undergo the normal epithelial-mesenchymal transition (EMT) and dorsal migration that follows ventral furrow formation. This phenotype is not a secondary consequence of failed cytokinesis, since it is rescued by a mutant form of pbl that does not rescue the cytokinetic defect. In wild-type embryos, newly invaginated cells at the lateral edges of the mesoderm extend numerous protrusions. In pbl mutant embryos, however, cells appear more tightly adhered to their neighbours and extend very few protrusions. Consistent with the dependence of the mesoderm EMT and cytokinesis on actin organisation, the GTP exchange function of the PBL RhoGEF is required for both processes. By contrast, the N-terminal BRCT domains of Pbl are required only for the cytokinetic function of Pbl. These studies reveal that a novel Pbl-mediated intracellular signalling pathway operates in mesodermal cells during the transition from an epithelial to migratory mesenchymal morphology during gastrulation (Smallhorn, 2004).

pbl mRNA is found at high levels in pole cells at cellularisation and zygotic expression is induced during interphase of cycle 14. However, induction of zygotic expression is not uniform throughout the embryo. Specifically, expression is lower in the ventral region of the blastoderm epithelium than in other parts of the embryo. pbl expression in the presumptive mesoderm is first observed immediately prior to invagination, the expression pattern becoming more pronounced and discrete as stage 6 progresses. After mesoderm invagination, pbl is strongly expressed in the invaginated tissue (Smallhorn, 2004).

To address whether PBL functions as a RhoGEF in mesodermal cell migration, two experiments were carried out. In the first, a GEF mutated form of PBL, PBL^DeltaDH, in which amino acids 497-549 within the DH domain are removed, was expressed using a prd-GAL4 driver in a pbl mutant background. Expression of UAS-pblDeltaDH with prd-GAL4 in a pbl mutant background failed to rescue the Eve-positive mesodermal cell formation phenotype. The number of Eve-positive hemisegments in UAS-pblDelta^DH; pbl³/prd-GAL4, pbl² embryos ranged from 0-18, with a mean of 8.81±0.56. This number is similar to the number observed in pbl²/pbl³ mutant embryos. It is concluded that the DH domain, and therefore the GEF activity, is required for EVE-positive mesodermal cell formation (Smallhorn, 2004).

The second approach used the pbl⁵ allele that contains a single missense mutation in the most highly conserved region (CR3) of the DH domain. This point mutation (valine to an aspartate at amino acid 531) has been shown in other systems to significantly reduce the nucleotide exchange activity of RhoGEFs. Consistent with this observation, pbl⁵ homozygous mutant embryos exhibit a strong cytokinetic phenotype. pbl⁵ homozygotes have few Eve-positive hemisegments. The number of EVE-positive hemisegments in pbl⁵/pbl⁵ mutant embryos ranges from 0-14, with a mean of 4.5±0.37. This number is even fewer than the number observed in pbl²/pbl³ embryos (Smallhorn, 2004).

The morphology of pbl⁵ mutant cells was examined using F-actin staining and mesodermal expression of GFP-Actin. The results were comparable with pbl²/pbl³ embryos, with mesodermal cells showing a similar range of defects in spreading, morphology and the extent of rounding/dissociation in the body of the mesoderm. These results show that the GEF activity of PBL is required for the normal epithelial-mesenchymal transition, migratory morphology and subsequent formation of EVE-positive mesodermal cells (Smallhorn, 2004).

Heartless (HTL), a receptor tyrosine kinsase (RTK) of the fibroblast growth factor receptor (FGFR) subfamily is required for the mesoderm EMT, where it is known to activate the conserved Ras/MAP kinase pathway. In htl mutant embryos, mesodermal cells fail to dissociate from each other following invagination and fail to migrate dorsally. Mesoderm migration also fails in embryos mutant for three other genes: Downstream-of-FGFR (Dof), Sugarless and Sulphateless. In each case, the failure in mesoderm migration is accompanied by a failure in the activation of the Ras1/MAPK pathway (Smallhorn, 2004).

To investigate whether the pbl mutant phenotype is also due to a failure in the activation of the HTL/MAPK pathway, pbl mutant embryos were stained with an antibody directed towards the dual phosphorylated form of MAP kinase (dp-ERK). In wild-type embryos following gastrulation, dp-ERK is expressed in the dorsalmost mesodermal cell rows on each lateral surface of the embryo, a staining pattern that is Htl dependent. In pbl mutant embryos, dp-ERK staining is seen in the dorsalmost mesodermal cell rows similar to wild type. This result shows that Pbl function is not required for Htl-dependent activation of the MAP kinase signalling pathway, and that the mesoderm migration defect in pbl mutants is not due to a failure in the activation of the MAPK pathway (Smallhorn, 2004).

Pebble/ECT2 RhoGEF negatively regulates the Wingless/Wnt signaling pathway

Wingless (Wg)/Wnt signaling is essential for patterning invertebrate and vertebrate embryos, and inappropriate Wnt activity is associated with a variety of human cancers. Despite intensive study, Wnt pathway mechanisms are not fully understood. A new mechanism has been discovered for regulating the Wnt pathway: activity of a Rho guanine nucleotide exchange factor (GEF) encoded by pebble (pbl) in Drosophila and ECT2 in humans. This RhoGEF has an essential role in cytokinesis, but also plays an unexpected, conserved role in inhibiting Wg/Wnt activity. Loss and gain of pbl function in Drosophila embryos cause pattern defects that indicate altered Wg activity. Both Pbl and ECT2 repress Wg/Wnt target gene expression in cultured Drosophila and human cells. The GEF activity is required for Wnt regulation, whereas other protein domains important for cytokinesis are not. Unlike most negative regulators of Wnt activity, Pbl/ECT2 functions downstream of Armadillo (Arm)/beta-catenin stabilization. These results indicate GTPase regulation at a novel point in Wg/Wnt signal transduction, and provide new insight into the categorization of ECT2 as a human proto-oncogene (Greer, 2013).

Pbl was originally investigated because of its interaction with the Wnt regulators Tumbleweed (Tum) and Pav during cytokinesis. The data show that although Pbl does regulate Wnt signaling, it does so independently of Tum and Pav. First, Pbl regulation of Wg signaling does not require the Tum-binding BRCT1 domain. Indeed, some constructs lacking this region were more effective than full-length Pbl in repressing DROPflash, a reporter containing six tandem TCF binding sites. Second, Tum and Pav require nuclear localization to regulate Wg signaling (Jones, 2010). By contrast, no nuclear role for Pbl or ECT2 was found in Wg/Wnt regulation. Third, Pbl regulation of Wg/Wnt signaling correlates with its GEF activity, whereas Tum does not require GAP activity to regulate Wg signaling. Together, these results suggest that although Tum, Pav and Pbl act together in a complex during cytokinesis, they play distinctly different roles in their influence on the Wg/Wnt pathway. Analysis of Pbl and ECT2 domain requirements further established distinctions between cytokinesis and signaling roles: for example, the BRCT domains of Pbl or ECT2 are essential for cytokinesis but are not required for Wg/Wnt inhibition (Greer, 2013).

Most negative regulators of Wnt signaling alter Arm/beta-cat stabilization; this is true of Axin, which consistently repressed the pathway more effectively than did Tum, Pav or Pbl. However, Pbl/ECT2 RhoGEF can repress Wg/Wnt activity induced by stabilized forms of Arm/beta-cat, which Axin cannot do. Thus Pbl/ECT2 functions downstream of the destruction complex to modulate expression of target genes. Although the mild changes in endogenous Arm stability observed in pbl loss- and gain-of-function conditions are consistent with mutant phenotypes, they may instead be a secondary effect of RhoGEF on Arm localization. The data suggest that Pbl/ECT2 acts on Arm/beta-cat at a point between its stabilization and its translocation into the nucleus. Several ways in which this could occur can be imagined. First, Wg/Wnt repression could result from a general effect on the actin cytoskeleton. Rho family GTPases regulate formins and other effector molecules that control actin dynamics. Both Arm/beta-cat and Apc are known to interact with the actin cytoskeleton and to assume a cortical localization in polarized epithelial cells. Changes in the actin network might alter subcellular localization or activity of Arm. Alternatively, GTPase activation of an effector molecule might have a direct effect on some component of the Wnt pathway or of a parallel signaling pathway that antagonizes Wnt signaling. For instance, Diaphanous, a Rho1 effector, interacts genetically and physically with Apc2. All GTPases tested had some capacity to repress Wg/Wnt signaling, raising the possibility that any change in the actin cytoskeleton can negatively influence signaling and/or that multiple effectors are involved. Alternatively, these GTPases may cross-activate Rho1, and only Rho1 activation mediates the Pbl regulation of Wnt signaling. These possibilities are currently being explored (Greer, 2013).

The role of Rho GTPases in planar cell polarity, a non-canonical Wnt signaling pathway, has been well-studied, but growing evidence suggests that Rho GTPases modulate canonical Wnt signaling in mammalian cells. Several GEFs potentiate Wnt signaling by activating Rac1: the GEF DOCK4 promotes beta-cat degradation, whereas the GEF TiamI is recruited to Wnt-responsive promoters where it activates Rac1 and promotes transcription. Rac1 also appears to be activated by Wnt signaling, leading to activation of JNK2 (MAPK9) kinase and nuclear import of beta-catenin. By contrast, this study found that Pbl antagonizes Wnt signaling through Rho1 activation. Thus, Rho GTPases can have either positive or negative effects on Wnt signaling. The challenge now is to identify the effector molecules with which these G proteins interact in their activated, GTP-bound forms to modulate Wnt pathway output (Greer, 2013).

The finding that ECT2 negatively regulates Wnt signaling in human cells suggests that it would be protective against oncogenesis. Prior work categorizing ECT2 as a proto-oncogene was based on clones that lacked the N-terminus or nuclear localization signals. Although full-length ECT2 cannot transform cells, full-length ECT2 is overexpressed in many human tumors. It is unclear if the upregulation observed in tumors is a cause versus a consequence of oncogenesis; it may simply reflect the essential role of ECT2 in the cell cycle. No upregulation of ECT2 is observed in colorectal cancer, the cancer most strongly associated with aberrations in Wnt signaling. The full role that ECT2 plays in human tumorigenesis is just beginning to be explored and the current work demonstrates that any proposed role for ECT2 in cancer must consider its conserved function in inhibiting Wnt signaling (Greer, 2013).