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Wnt signaling plays key roles in embryonic development and adult stem cell homeostasis and is altered in human cancer. We explore β-catenin transfer from the destruction complex to the E3 ligase, and test models suggesting Dishevelled and APC2 compete for association with Axin.

Forty years ago, Horace Judson’s The Eighth Day of Creationwas published, a book vividly recounting the foundations of modern biology, the molecular biology revolution. This book inspired many in my generation. The anniversary provides a chance for a new generation to take a look back, to see how science has and hasn’t changed. Many central players in the book, including Sydney Brenner, Seymour Benzer, and François Jacob, would go on to be among the founders of modern cell biology, developmental biology, and neurobiology. These players come alive via their own words, as complex individuals, both heroes and anti-heroes. The technologies and experimental approaches they pioneered, ranging from cell fractionation to immunoprecipitation to structural biology, and the multidisciplinary approaches they took continue to power and inspire our work today. In the process, Judson brings out of the shadows the central roles played by women in many of the era’s discoveries. He provides us with a vision of how science and scientists have changed, of how many things about our endeavor never change, and of how some new ideas are perhaps not as new as we would like to think.

Small adapter proteins of the Crk family play important roles in signal amplification and transduction. Here we describe the roles of Crk in embryonic development in Drosophila. Crk regulates diverse events in embryogenesis that require orchestrated cytoskeletal dynamics—these include the dynamic events of syncytial development during which Crk regulates actin dynamics via SCAR and the Arp 2/3 complex.

During morphogenesis, cells must change shape and move without disrupting tissue integrity. This requires cell–cell junctions to allow dynamic remodeling while resisting force generated by the actomyosin cytoskeleton. Canoe and Polychaetoid, the DrosophilaAfadin and ZO-1 homologues, act in parallel during this process.

The United States has been a leader in biomedical science for decades, in large part because of the strategy used by the National Institutes of Health (NIH) to invest its budgetary portfolio. They identified talented young scientists from each generation and gave them the resources they needed to initiate and maintain strong research programs. However, recently this investment has become less diversified, with a larger fraction of grant dollars in the hands of a smaller fraction of researchers. This threatens the future of our field, as many productive early and midcareer scientists are facing having to close their labs. NIH and others have studied this problem, gathering data that suggest that over a certain level of funding to an individual investigator, there are diminishing returns in scientific output. Here I review these data and examine the issues that led NIH to propose and then reverse a cap on funding to individual investigators, the Grant Support Index. I consider other proposed solutions, and call on all in the field to examine whether the status quo is acceptable, and if not, urge them to propose and advocate for concrete alternatives.

APC and Axin are key negative regulators of Wnt signaling in development and oncogenesis. They form a multiprotein complex targeting the key Wnt effector β-catenin for destruction. Essential components of APC and Axin required for their cooperative function are identified, and the data are used to design a minimal β-catenin–destruction machine.

A PhD in biomedical science and the critical thinking skills that it provides can open the door to many different careers. The current popular scientific press and blogosphere too often portray the job of a research-intensive faculty member and principal investigator (PI) as both unattainable and undesirable. We want to make sure our trainees include our own career path among their options, as for each of us it has been a fantastic, family-friendly, and highly impactful career.

During embryonic development dramatic cell shape changes and movements re-shape the embryonic body plan. These require robust but dynamic linkage between the cell-cell adherens junctions and the force-generating actomyosin cytoskeleton. Our view of this linkage has evolved, and we now realize linkage is mediated by mechanosensitive multiprotein complexes assembled via multivalent connections. Here we combine genetic, cell biological and modeling approaches to define the mechanism of action and functions of an important player, DrosophilaPolychaetoid, homolog of mammalian ZO-1. Our data reveal that Pyd reinforces cell junctions under elevated tension, and facilitates cell rearrangements. Pyd is important to maintain junctional contractility and in its absence cell rearrangements stall. We next use structured illumination microscopy to define the molecular architecture of cell-cell junctions during these events. The cadherin-catenin complex and Cno both localize to puncta along the junctional membrane, but are differentially enriched in different puncta. Pyd, in contrast, exhibits a distinct localization to strands that extend out from the region occupied by core junction proteins. We then discuss the implications for the protein network at the junction-cytoskeletal interface, suggesting different proteins localize and function in distinct ways, perhaps in distinct subcomplexes, but combine to produce robust connections.Movie S1Movie S1Video of the wildtype simulation for germband extension. It includes 400 timesteps, equating to roughly 20 simulated minutes of germband extension (∼0.5 min of germband extension per timestep). This simulation includes supercontractilty', modelled by a condition where junction shrinkage rate increases as length decreases.Movie S2Movie S2Video in which ‘supercontractilty’ was removed to mimic the reduction in contractility seen in pydmutants.

The importance of Abl kinase activity, the F-actin–binding site, and scaffolding ability in Abl’s many cell biological roles during Drosophilamorphogenesis is examined. Abl is a robust multidomain scaffold with different protein motifs and activities contributing differentially to diverse cellular behaviors.

Among the defining features of the animal kingdom is the ability of cells to change shape and move. This underlies embryonic and postembryonic development, tissue homeostasis, regeneration, and wound healing. Cell shape change and motility require linkage of the cell’s force-generating machinery to the plasma membrane at cell–cell and cell–extracellular matrix junctions. Connections of the actomyosin cytoskeleton to cell–cell adherens junctions need to be both resilient and dynamic, preventing tissue disruption during the dramatic events of embryonic morphogenesis. In the past decade, new insights radically altered the earlier simple paradigm that suggested simple linear linkage via the cadherin–catenin complex as the molecular mechanism of junction–cytoskeleton interaction. In this Perspective we provide a brief overview of our current state of knowledge and then focus on selected examples highlighting what we view as the major unanswered questions in our field and the approaches that offer exciting new insights at multiple scales from atomic structure to tissue mechanics.

Actin-based protrusions are important for signaling and migration during development and homeostasis. Gain- and loss-of-function and quantitative approaches are used to define differential roles for the actin elongation factors Diaphanous and Enabled in regulating distinct protrusive behaviors in different tissues during Drosophilamorphogenesis.

The small GTPase Rap1 and the actin-junctional linker protein Canoe/afadin are essential for the initial establishment of polarity in Drosophila, acting upstream of Bazooka/Par3 and the adherens junctions. However, feedback and cross-regulation occur, so polarity establishment is regulated by a network of proteins rather than a linear pathway.

In this study, we test two current models for the function of the tumor suppressor adenomatous polyposis coli (APC). We find that APC can regulate Wnt signaling from diverse cytoplasmic locations, suggesting that its roles in the nucleus or in localizing the β-catenin destruction complex are not essential.

Coordination of adhesion and the actin cytoskeleton is critical in morphogenesis. Drosophilagermband extension is a model for convergent extension. Canoe/afadin is found to have a novel role in this process. It helps to coordinate a contractile apical actomyosin network with cell shape change and regulates apical polarity protein localization.

Integrating individual cell movements to create tissue-level shape change is essential to building an animal. We explored mechanisms of adherens junction (AJ):cytoskeleton linkage and roles of the linkage regulator Canoe/afadin during Drosophila germband extension (GBE), a convergent-extension process elongating the body axis. We found surprising parallels between GBE and a quite different morphogenetic movement, mesoderm apical constriction. Germband cells have an apical actomyosin network undergoing cyclical contractions. These coincide with a novel cell shape change—cell extension along the anterior–posterior (AP) axis. In Canoe's absence, GBE is disrupted. The apical actomyosin network detaches from AJs at AP cell borders, reducing coordination of actomyosin contractility and cell shape change. Normal GBE requires planar polarization of AJs and the cytoskeleton. Canoe loss subtly enhances AJ planar polarity and dramatically increases planar polarity of the apical polarity proteins Bazooka/Par3 and atypical protein kinase C. Changes in Bazooka localization parallel retraction of the actomyosin network. Globally reducing AJ function does not mimic Canoe loss, but many effects are replicated by global actin disruption. Strong dose-sensitive genetic interactions between canoe and bazooka are consistent with them affecting a common process. We propose a model in which an actomyosin network linked at AP AJs by Canoe and coupled to apical polarity proteins regulates convergent extension.

Adherens and tight junctions play key roles in assembling epithelia and maintaining barriers. In cell culture zonula occludens (ZO)–family proteins are important for assembly/maturation of both tight and adherens junctions (AJs). Genetic studies suggest that ZO proteins are important during normal development, but interpretation of mouse and fly studies is limited by genetic redundancy and/or a lack of null alleles. We generated null alleles of the single Drosophila ZO protein Polychaetoid (Pyd). Most embryos lacking Pyd die with striking defects in morphogenesis of embryonic epithelia including the epidermis, segmental grooves, and tracheal system. Pyd loss does not dramatically affect AJ protein localization or initial localization of actin and myosin during dorsal closure. However, Pyd loss does affect several cell behaviors that drive dorsal closure. The defects, which include segmental grooves that fail to retract, a disrupted leading edge actin cable, and reduced zippering as leading edges meet, closely resemble defects in canoe zygotic null mutants and in embryos lacking the actin regulator Enabled (Ena), suggesting that these proteins act together. Canoe (Cno) and Pyd are required for proper Ena localization during dorsal closure, and strong genetic interactions suggest that Cno, Pyd, and Ena act together in regulating or anchoring the actin cytoskeleton during dorsal closure.

Analysis of the function of the fly ZO-1 homologue Polychaetoid shows that it is not essential for junctional assembly or maintenance but does play an important role in embryonic morphogenesis. The data suggest that it works with Canoe/afadin and the actin regulator Enabled to regulate actin anchoring at junctions.

APC is a key tumor suppressor and Wnt signaling regulator, but its mechanism of action remains mysterious. We combined parallel assays in Drosophilaand cultured human colon cancer cell lines to test hypotheses regarding APC function and to develop novel hypotheses, using mutants altering its structure in specific ways.

Negatively regulating signaling by targeting key effectors for ubiquitina­tion/destruction is essential for development and oncogenesis. The tumor suppressor adenomatous polyposis coli (APC), an essential negative regulator of Wnt signaling, provides a paradigm. APC mutations occur in most colon cancers. Acting in the "destruction complex" with Axin, glycogen synthase kinase 3, and casein kinase, APC targets sscatenin (sscat) for phosphorylation and recognition by an E3 ubiquitin-ligase. Despite 20 years of work, the internal workings of the destruction complex and APC's role remain largely mysterious. We use both Drosophila and colon cancer cells to test hypotheses for APC's mechanism of action. Our data are inconsistent with current models suggesting that high-affinity sscat-binding sites on APC play key roles. Instead, they suggest that multiple sscat-binding sites act additively to fine-tune signaling via cytoplasmic retention. We identify essential roles for two putative binding sites for new partners—20-amino-acid repeat 2 and conserved sequence B—in destruction complex action. Finally, we demonstrate that APC interacts with Axin by two different modes and provide evidence that conserved sequence B helps ensure release of APC from Axin, with disassembly critical in regulating sscat levels. Using these data, we suggest a new model for destruction complex action in development, which also provides new insights into functions of truncated APC proteins in cancer.

During migration cell protrusions power cell extension and sample the environment. Different cells produce different protrusions, from keratocytes dominated by lamellipodia, to growth cones combining filopodia and lamellipodia, to dendritic spines. One key challenge is to determine how the toolkit of actin regulators are coordinated to generate these diverse protrusive arrays. Here we use Drosophilaleading-edge (LE) cells to explore how Diaphanous (Dia)-related formins and Ena/VASP proteins cooperate in this process. We first dissect the Dia regulatory region, revealing novel roles for the GTPase-binding and FH3 domains in cortical localization, filopodial initiation, and lengthening. Second, we provide evidence that activating Dia mobilizes Ena from storage places near the LE to act at the LE. Further, Dia and Ena coIP and can recruit one another to new locations, suggesting cooperation is key to their mechanisms of action. Third, we directly explore the functional relationship between Dia and Ena, varying their levels and activity separately in the same cell type. Surprisingly, although each is sufficient to induce filopodia, together they induce lamellipodia. Our data suggest they work together in a complex and nonadditive way, with the ratio between active Dia and Ena being one factor that modulates the balance between filopodia and lamellipodia.

During migration cell protrusions power cell extension and sample the environment. Different cells produce different protrusions, from keratocytes dominated by lamellipodia, to growth cones combining filopodia and lamellipodia, to dendritic spines. One key challenge is to determine how the toolkit of actin regulators are coordinated to generate these diverse protrusive arrays. Here we use Drosophila leading-edge (LE) cells to explore how Diaphanous (Dia)-related formins and Ena/VASP proteins cooperate in this process. We first dissect the Dia regulatory region, revealing novel roles for the GTPase-binding and FH3 domains in cortical localization, filopodial initiation, and lengthening. Second, we provide evidence that activating Dia mobilizes Ena from storage places near the LE to act at the LE. Further, Dia and Ena coIP and can recruit one another to new locations, suggesting cooperation is key to their mechanisms of action. Third, we directly explore the functional relationship between Dia and Ena, varying their levels and activity separately in the same cell type. Surprisingly, although each is sufficient to induce filopodia, together they induce lamellipodia. Our data suggest they work together in a complex and nonadditive way, with the ratio between active Dia and Ena being one factor that modulates the balance between filopodia and lamellipodia.

Embryonic morphogenesis is powered by dramatic changes in cell shape and arrangement, driven by the cytoskeleton and its connections to adherens junctions. This requires robust linkage, allowing morphogenesis without disrupting tissue integrity. The small GTPase Rap1 is a key regulator of cell adhesion, controlling both cadherin-mediated and integrin-mediated processes. We have defined multiple roles in morphogenesis for one Rap1 effector, Canoe/Afadin, which ensures robust junction-cytoskeletal linkage. We now ask what mechanisms regulate Canoe and other junction-cytoskeletal linkers during Drosophilamorphogenesis, defining roles for Rap1 and one of its guanine nucleotide exchange factor (GEF) regulators, Dizzy. Rap1 uses Canoe as one effector, regulating junctional planar polarity. However, Rap1 has additional roles in junctional protein localization and balanced apical constriction—in its absence, Bazooka/Par3 localization is fragmented, and cells next to mitotic cells apically constrict and invaginate, disrupting epidermal integrity. In contrast, the GEF Dizzy has phenotypes similar to but slightly less severe than Canoe loss, suggesting this GEF regulates Rap1 action via Canoe. Taken together, these data reveal that Rap1 is a crucial regulator of morphogenesis, likely acting in parallel via Canoe and other effectors, and that different Rap1 GEFs regulate distinct functions of Rap1.

In animal cells, centrosomes nucleate microtubules that form polarized arrays to organize the cytoplasm. Drosophilapresents an interesting paradox however, as centrosome-deficient mutant animals develop into viable adults. To understand this discrepancy, we analyzed behaviors of centrosomes and microtubules in Drosophilacells, in culture and in vivo, using a combination of live-cell imaging, electron microscopy, and RNAi. The canonical model of the cycle of centrosome function in animal cells states that centrosomes act as microtubule-organizing centers throughout the cell cycle. Unexpectedly, we found that many Drosophilacell-types display an altered cycle, in which functional centrosomes are only present during cell division. On mitotic exit, centrosomes disassemble producing interphase cells containing centrioles that lack microtubule-nucleating activity. Furthermore, steady-state interphase microtubule levels are not changed by codepleting both γ-tubulins. However, γ-tubulin RNAi delays microtubule regrowth after depolymerization, suggesting that it may function partially redundantly with another pathway. Therefore, we examined additional microtubule nucleating factors and found that Mini-spindles, CLIP-190, EB1, or dynein RNAi also delayed microtubule regrowth; surprisingly, this was not further prolonged when we codepleted γ-tubulins. Taken together, these results modify our view of the cycle of centrosome function and reveal a multi-component acentrosomal microtubule assembly pathway to establish interphase microtubule arrays in Drosophila.

In animal cells, centrosomes nucleate microtubules that form polarized arrays to organize the cytoplasm. Drosophila presents an interesting paradox however, as centrosome-deficient mutant animals develop into viable adults. To understand this discrepancy, we analyzed behaviors of centrosomes and microtubules in Drosophila cells, in culture and in vivo, using a combination of live-cell imaging, electron microscopy, and RNAi. The canonical model of the cycle of centrosome function in animal cells states that centrosomes act as microtubule-organizing centers throughout the cell cycle. Unexpectedly, we found that many Drosophila cell-types display an altered cycle, in which functional centrosomes are only present during cell division. On mitotic exit, centrosomes disassemble producing interphase cells containing centrioles that lack microtubule-nucleating activity. Furthermore, steady-state interphase microtubule levels are not changed by codepleting both γ-tubulins. However, γ-tubulin RNAi delays microtubule regrowth after depolymerization, suggesting that it may function partially redundantly with another pathway. Therefore, we examined additional microtubule nucleating factors and found that Mini-spindles, CLIP-190, EB1, or dynein RNAi also delayed microtubule regrowth; surprisingly, this was not further prolonged when we codepleted γ-tubulins. Taken together, these results modify our view of the cycle of centrosome function and reveal a multi-component acentrosomal microtubule assembly pathway to establish interphase microtubule arrays in DROSOPHILA:

Signaling by the nonreceptor tyrosine kinase Abelson (Abl) plays key roles in normal development, whereas its inappropriate activation helps trigger the development of several forms of leukemia. Abl is best known for its roles in axon guidance, but Abl and its relatives also help regulate embryonic morphogenesis in epithelial tissues. Here, we explore the role of regulation of Abl kinase activity during development. We first compare the subcellular localization of Abl protein and of active Abl, by using a phosphospecific antibody, providing a catalog of places where Abl is activated. Next, we explore the consequences for morphogenesis of overexpressing wild-type Abl or expressing the activated form found in leukemia, Bcr-Abl. We find dose-dependent effects of elevating Abl activity on morphogenetic movements such as head involution and dorsal closure, on cell shape changes, on cell protrusive behavior, and on the organization of the actin cytoskeleton. Most of the effects of Abl activation parallel those caused by reduction in function of its target Enabled. Abl activation leads to changes in Enabled phosphorylation and localization, suggesting a mechanism of action. These data provide new insight into how regulated Abl activity helps direct normal development and into possible biological functions of Bcr-Abl.

Signaling by the nonreceptor tyrosine kinase Abelson (Abl) plays key roles in normal development, whereas its inappropriate activation helps trigger the development of several forms of leukemia. Abl is best known for its roles in axon guidance, but Abl and its relatives also help regulate embryonic morphogenesis in epithelial tissues. Here, we explore the role of regulation of Abl kinase activity during development. We first compare the subcellular localization of Abl protein and of active Abl, by using a phosphospecific antibody, providing a catalog of places where Abl is activated. Next, we explore the consequences for morphogenesis of overexpressing wild-type Abl or expressing the activated form found in leukemia, Bcr-Abl. We find dose-dependent effects of elevating Abl activity on morphogenetic movements such as head involution and dorsal closure, on cell shape changes, on cell protrusive behavior, and on the organization of the actin cytoskeleton. Most of the effects of Abl activation parallel those caused by reduction in function of its target Enabled. Abl activation leads to changes in Enabled phosphorylation and localization, suggesting a mechanism of action. These data provide new insight into how regulated Abl activity helps direct normal development and into possible biological functions of Bcr-Abl.

The septins are a family of proteins involved in cytokinesis and other aspects of cell-cortex organization. In a two-hybrid screen designed to identify septin-interacting proteins in Drosophila, we isolated several genes, including homologues (Dmuba2 and Dmubc9) of yeast UBA2 and UBC9. Yeast Uba2p and Ubc9p are involved in the activation and conjugation, respectively, of the ubiquitin-like protein Smt3p/SUMO, which becomes conjugated to a variety of proteins through this pathway. Uba2p functions together with a second protein, Aos1p. We also cloned and characterized the Drosophila homologues of AOS1(Dmaos1) and SMT3 (Dmsmt3). Our biochemical data suggest that DmUba2/DmAos1 and DmUbc9 indeed act as activating and conjugating enzymes for DmSmt3, implying that this protein-conjugation pathway is well conserved in Drosophila. Immunofluorescence studies showed that DmUba2 shuttles between the embryonic cortex and nuclei during the syncytial blastoderm stage. In older embryos, DmUba2 and DmSmt3 are both concentrated in the nuclei during interphase but dispersed throughout the cells during mitosis, with DmSmt3 also enriched on the chromosomes during mitosis. These data suggest that DmSmt3 could modify target proteins both inside and outside the nuclei. We did not observe any concentration of DmUba2 at sites where the septins are concentrated, and we could not detect DmSmt3 modification of the three Drosophila septins tested. However, we did observe DmSmt3 localization to the midbody during cytokinesis both in tissue-culture cells and in embryonic mitotic domains, suggesting that DmSmt3 modification of septins and/or other midzone proteins occurs during cytokinesis in Drosophila.

Human β-catenin and its fly homolog Armadillo are best known for their roles in cadherin-based cell–cell adhesion and in transduction of Wingless/Wnt signals. It has been hypothesized that β-catenin may also regulate cell migration and cell shape changes, possibly by regulating the microtubule cytoskeleton via interactions with APC. This hypothesis was based on experiments in which a hyperstable mutant form of β-catenin was expressed in MDCK cells, where it altered their migratory properties and their ability to send out long cellular processes. We tested the generality of this hypothesis in vivoin Drosophila. We utilized three model systems in which cell migration and/or process extension are known to play key roles during development: the migration of the border cells during oogenesis, the extension of axons in the nervous system, and the migration and cell process extension of tracheal cells. In all cases, cells expressing activated Armadillo were able to migrate and extend cell processes essentially normally. The one alteration from normal involved an apparent cell fate change in certain tracheal cells. These results suggest that only certain cells are affected by activation of Armadillo/β-catenin, and that Armadillo/β-catenin does not play a general role in inhibiting cell migration or process extension.

DrosophilaArmadillo and its mammalian homologue β-catenin are scaffolding proteins involved in the assembly of multiprotein complexes with diverse biological roles. They mediate adherens junction assembly, thus determining tissue architecture, and also transduce Wnt/Wingless intercellular signals, which regulate embryonic cell fates and, if inappropriately activated, contribute to tumorigenesis. To learn more about Armadillo/β-catenin's scaffolding function, we examined in detail its interaction with one of its protein targets, cadherin. We utilized two assay systems: the yeast two-hybrid system to study cadherin binding in the absence of Armadillo/β-catenin's other protein partners, and mammalian cells where interactions were assessed in their presence. We found that segments of the cadherin cytoplasmic tail as small as 23 amino acids bind Armadillo or β-catenin in yeast, whereas a slightly longer region is required for binding in mammalian cells. We used mutagenesis to identify critical amino acids required for cadherin interaction with Armadillo/β-catenin. Expression of such short cadherin sequences in mammalian cells did not affect adherens junctions but effectively inhibited β-catenin–mediated signaling. This suggests that the interaction between β-catenin and T cell factor family transcription factors is a sensitive target for disruption, making the use of analogues of these cadherin derivatives a potentially useful means to suppress tumor progression.

The septins are a conserved family of proteins that are involved in cytokinesis and other aspects of cell-surface organization. InDrosophila melanogaster, null mutations in thepnutseptin gene are recessive lethal, but homozygouspnutmutants complete embryogenesis and survive until the pupal stage. Because the completion of cellularization and other aspects of early development seemed likely to be due to maternally contributed Pnut product, we attempted to generate embryos lacking the maternal contribution in order to explore the roles of Pnut in these processes. We used two methods, the production of germline clones homozygous for a pnutmutation and the rescue ofpnuthomozygous mutant flies by apnut+transgene under control of thehsp70promoter. Remarkably, the pnutgermline-clone females produced eggs, indicating that stem-cell and cystoblast divisions in the female germline do not require Pnut. Moreover, the Pnut-deficient embryos obtained by either method completed early syncytial development and began cellularization of the embryo normally. However, during the later stages of cellularization, the organization of the actin cytoskeleton at the leading edge of the invaginating furrows became progressively more abnormal, and the embryos displayed widespread defects in cell and embryo morphology beginning at gastrulation. Examination of two other septins showed that Sep1 was not detectable at the cellularization front in the Pnut-deficient embryos, whereas Sep2 was still present in normal levels. Thus, it is possible that Sep2 (perhaps in conjunction with other septins such as Sep4 and Sep5) fulfills an essential septin role during the organization and initial ingression of the cellularization furrow even in the absence of Pnut and Sep1. Together, the results suggest that some cell-division events in Drosophilado not require septin function, that there is functional differentiation among the Drosophilaseptins, or both.

Elaboration of the Drosophila body plan depends on a series of cell-identity decisions and morphogenetic movements regulated by intercellular signals. For example, Jun N-terminal kinase signaling regulates cell fate decisions and morphogenesis during dorsal closure, while Wingless signaling regulates segmental patterning of the larval cuticle via Armadillo. wingless or armadillo mutant embryos secrete a lawn of ventral denticles; armadillo mutants also exhibit dorsal closure defects. We found that mutations in puckered, a phosphatase that antagonizes Jun N-terminal kinase, suppress in a dose-sensitive manner both the dorsal and ventral armadillo cuticle defects. Furthermore, we found that activation of the Jun N-terminal kinase signaling pathway suppresses armadillo-associated defects. Jun N-terminal kinase signaling promotes dorsal closure, in part, by regulating decapentaplegic expression in the dorsal epidermis. We demonstrate that Wingless signaling is also required to activate decapentaplegic expression and to coordinate cell shape changes during dorsal closure. Together, these results demonstrate that MAP-Kinase and Wingless signaling cooperate in both the dorsal and ventral epidermis, and suggest that Wingless may activate both the Wingless and the Jun N-terminal kinase signaling cascades.

Drosophila Armadillo and its vertebrate homolog β-catenin are key effectors of Wingless/Wnt signaling. In the current model, Wingless/Wnt signal stabilizes Armadillo/β-catenin, which then accumulates in nuclei and binds TCF/LEF family proteins, forming bipartite transcription factors which activate transcription of Wingless/Wnt responsive genes. This model was recently challenged. Overexpression in Xenopus of membrane-tethered β-catenin or its paralog plakoglobin activates Wnt signaling, suggesting that nuclear localization of Armadillo/β-catenin is not essential for signaling. Tethered plakoglobin or β-catenin might signal on their own or might act indirectly by elevating levels of endogenous β-catenin. We tested these hypotheses in Drosophila by removing endogenous Armadillo. We generated a series of mutant Armadillo proteins with altered intracellular localizations, and expressed these in wild-type and armadillo mutant backgrounds. We found that membrane-tethered Armadillo cannot signal on its own; however it can function in adherens junctions. We also created mutant forms of Armadillo carrying heterologous nuclear localization or nuclear export signals. Although these signals alter the subcellular localization of Arm when overexpressed in Xenopus, in Drosophila they have little effect on localization and only subtle effects on signaling. This supports a model in which Armadillo’s nuclear localization is key for signaling, but in which Armadillo intracellular localization is controlled by the availability and affinity of its binding partners.

Cells must cooperate and communicate to form a multicellular animal. Information about the molecules required for these processes have come from a variety of sources; the convergence between the studies of particular molecules by vertebrate cell biologists and the genes identified by scientists investigating development in Drosophilahas been especially fruitful. We are interested in the connection between cadherin proteins that regulate cell-cell adhesion and the wingless/wnt-1cell-cell signaling molecules controlling pattern formation during development. The Drosophilasegment polarity gene armadillo,homolog of the vertebrate adherens junction protein-catenin, is required for both cell adhesion and wgsignaling. We review what is known about winglesssignaling in Drosophila,and discuss the role of cell-cell junctions in both cell adhesion and cell communication. We then describe the results of our preliminary structure-function analysis of Armadillo protein in both cell adhesion and winglesssignaling. Finally, we discuss evidence supporting a direct role for Armadillo and adherens junction in transduction of winglesssignal.

Recent results suggest that our current model of Wingless/Wnt signal transduction is over-simplified.

The epithelial sheet is a structural unit common to many tissues. Its organization appears to depend on the function of the multi-protein complexes that form adherens junctions. Elegant cell biological experiments have provided support for hypotheses explaining the function of adherens junctions and of their components. These systems, however, lack the ability to test function within an entire organism during development. The realization that the product of the Drosophila segment polarity gene armadillo is related to the vertebrate adhesive junction components plakoglobin and β-catenin led to the suggestion that armadillo might provide a genetic handle to study adhesive junction structure and function. An examination of the potential function of Armadillo in cell-cell adhesive junctions was initiated using the Drosophila ovary as the model system. We examined the distribution of Armadillo in the Drosophila ovary and demonstrated that this localization often parallels the location of cell-cell adhesive junctions. The consequences of removing armadillo function from the germ-line cells of the ovary were also examined. Germ-line armadillo mutations appear to disrupt processes requiring cell adhesion and integrity of the actin cytoskeleton, consistent with a role for Armadillo in cell-cell adhesive junctions. We have also used armadillo mutations to examine the effects on ovarian development of altering the stereotyped cell arrangements of the ovary. The implications of these results for the role of adhesive junctions during development are discussed.

wingless/wnt-1 signaling directs cell fate during development. Genetic analysis in Drosophila identified genes that may encode components of the wingless signal transduction system. Drosophila Armadillo, homolog of vertebrate β-catenin, is required for wingless signaling. Unlike armadillo RNA, Armadillo protein accumulates non-uniformly in different cells of each embryonic segment. We found that cells alter their intracellular distribution of Armadillo in response to Wingless signal, accumulating increased levels of cytoplasmic Armadillo relative to those of membrane-associated protein. Levels of cytoplasmic Armadillo are also regulated by Zeste-White 3 kinase. Analysis of double mutants demonstrates that Armadillo’s role in wingless signaling is direct, and that Armadillo functions down-stream of both wingless and zeste-white 3. We present a model for the role of Armadillo stripes in transduction of wingless signal.

Adherens junctions are multiprotein complexes mediating cell-cell adhesion and communication. They are organized around a transmembrane cadherin, which binds a set of cytoplasmic proteins required for adhesion and to link the complex to the actin cytoskeleton. Three components of Drosophilaadherens junctions, analogous to those in vertebrates, have been identified: Armadillo (homolog of β-catenin), DrosophilaE-cadherin (DE-cadherin), and α-catenin. We carried out the first analysis of the interactions between these proteins using in vitrobinding assays, the yeast two-hybrid system, and in vivoassays. We identified a 76-amino acid region of Armadillo that is necessary and sufficient for binding α-catenin and found that the N-terminal 258 amino acids of α-catenin interact with Armadillo. A large region of Armadillo, spanning six central Armadillo repeats, is required for DE-cadherin binding, whereas only 41 amino acids of the DE-cadherin cytoplasmic tail are sufficient for Armadillo binding. Our data complement and extend results obtained in studies of vertebrate adherens junctions, providing a foundation for understanding how junctional proteins assemble and a basis for interpreting existing mutations and creating new ones.

The Drosophilasegment polarity gene product Armadillo provides a link between two seemingly separate processes, regulation of segmental pattern by the Wingless intercellular signal and the function of cell-cell adherens junctions. armadillowas originally identified because of its segment polarity phenotype but subsequently was found to be the homolog of the vertebrate adherens junction protein β-catenin. We examined the nature of the post-translational modification of Armadillo and its possible role in regulating Armadillo function. Armadillo is a phosphoprotein. Its level of phosphorylation varies both during embryonic development and from tissue to tissue. Phosphorylation occurs on both serine or threonine and tyrosine residues. Finally, Wingless signal negatively regulates Armadillo phosphorylation, while the segment polarity gene product Zeste-white 3, a serine/threonine protein kinase, promotes Armadillo phosphorylation. We discuss the implications of these results for regulation of Wingless/Wnt-1 signaling and adherens junction function.

extradenticle (exd) and the homeotic selector proteins together establish segmental identities by coordinately regulating the expression of downstream target genes. The inappropriate expression of these targets in exd mutant embryos results in homeotic transformations and aberrant morphogenesis. Here we examine the role of exd in adult development by using genetic mosaics and a hypomorphic exd allele caused by a point mutation in the homeodomain. exd continues to be essential for the specification of segmental identities, consistent with a continuing requirement for exd as a cofactor of the homeotic selector proteins. Loss of exd results in the homeotic transformation of abdominal segments to an A5 or A6 segmental identity, the antenna and arista to leg, and the head capsule to dorsal thorax or notum. Proximal leg structures are particularly sensitive to the loss of exd, although exd does not affect the allocation of proximal positional values of the leg imaginal disc. Using heat-shocks to induce expression of a hsp70-exd fusion gene, we show that, in contrast to the homeotic selector genes, ubiquitously high levels of exd expression do not cause pattern abnormalities or segmental transformations.

Segmental pattern in Drosophila melanogaster is set up via a set of cell-cell interactions mediated by the products of the segment polarity genes. Among these is the armadillo gene, whose product seems to be required for the reception of an intercellular signal encoded by the wingless gene. As part of our effort to relate the structure of the armadillo protein to its function within the cell, we have examined the evolutionary conservation of the armadillo gene during insect evolution. We have cloned the armadillo gene from the housefly, Musca domestica, which diverged from Drosophila 100 million years ago. The Musca protein is 97.5% identical to that in Drosophila, while the noncoding sequences have diverged extensively. This remarkable degree of conservation at the protein level is mirrored in the expression pattern of the armadillo protein. Antibodies against the Drosophila protein cross-react with a Musca protein of the appropriate size. We have also used these antibodies to show that the Musca armadillo protein has a pattern of expression in larval and adult tissues similar to that of Drosophila armadillo. We discuss the implications of conservation of structure and expression for the cellular role of the armadillo protein and its mammalian homologs.

Drosophila Armadillo and its vertebrate homolog β-catenin play essential roles both in the transduction of Wingless/Wnt cell-cell signals and in the function of cell-cell adherens junctions. Wingless and Wnts direct numerous cell fate choices during development. We generated a mutant protein, ArmadilloS10, with a 54 amino acid deletion in its N-terminal domain. This mutant is con-stitutively active in Wingless signaling; its activity is inde-pendent of both Wingless signal and endogenous wild-type Armadillo. Armadillo’s role in signal transduction is normally negatively regulated by Zeste-white 3 kinase, which modulates Armadillo protein stability. ArmadilloS10 is more stable than wild-type Armadillo, suggesting that it is less rapidly targeted for degradation. We show that ArmadilloS10 has escaped from negative regulation by Zeste white-3 kinase, and thus accumulates outside junctions even in the absence of Wingless signal. Finally, we present data implicating kinases in addition to Zeste white-3 in Armadillo phosphorylation. We discuss two models for the negative regulation of Armadillo in normal development and discuss how escape from this regulation contributes to tumorigenesis.

Cells must cooperate and communicate to form a multicellular animal. Information about the molecules required for these processes have come from a variety of sources; the convergence between the studies of particular molecules by vertebrate cell biologists and the genes identified by scientists investigating development in Drosophila has been especially fruitful. We are interested in the connection between cadherin proteins that regulate cell-cell adhesion and the wingless/wnt-1 cell-cell signaling molecules controlling pattern formation during development. The Drosophila segment polarity gene armadillo, homolog of the vertebrate adherens junction protein |3-catenin, is required for both cell adhesion and wg signaling. We review what is known about wingless signaling in Drosophila, and discuss the role of cell-cell junctions in both cell adhesion and cell communication. We then describe the results of our preliminary structure-function analysis of Armadillo protein in both cell adhesion and wingless signaling. Finally, we discuss evidence supporting a direct role for Armadillo and adherens junction in transduction of wingless signal.

There are 10 known mammalian septin genes, some of which produce multiple splice variants. The current nomenclature for the genes and gene products is very confusing, with several different names having been given to the same gene product and distinct names given to splice variants of the same gene. Moreover, some names are based on those of yeast or Drosophilaseptins that are not the closest homologues. Therefore, we suggest that the mammalian septin field adopt a common nomenclature system, based on that adopted by the Mouse Genomic Nomenclature Committee and accepted by the Human Genome Organization Gene Nomenclature Committee. The human and mouse septin genes will be namedSEPT1–SEPT10and Sept1–Sept10, respectively. Splice variants will be designated by an underscore followed by a lowercase “v” and a number, e.g., SEPT4_v1.

The G protein–coupled receptor Mist is the receptor for Fog, a secreted factor that drives gastrulation in fruit fly embryos.

The GPCR Mist is required for Fog-induced contractility in cells and acts parallel to Fog in Drosophilaepithelial morphogenesis.

The protein sequence of a representative of the zeins, the major storage proteins of maize, has been derived from the nucleotide sequence of a zein cDNA clone. This cDNA was sequenced both by the Maxam and Gilbert and the M13-dideoxy techniques. The nucleotide sequence encompasses the non-translated 3′ terminus of the mRNA, the entire coding sequence specifying both the mature zein protein and a small signal peptide, and a portion of the non-translated 5′ region. The deduced amino acid composition and the amino-terminal amino acid sequence closely resemble those derived from chemical analysis of the zein protein fraction. The data presented represent the first complete amino acid sequence of a plant storage protein.