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Neural representations of head direction have been discovered in many different species. A large body of theoretical work has proposed that the dynamics associated with these representations might be generated, maintained, and updated by recurrent network structures called ring attractors. Ring attractor models rely on specific assumptions about the structure of excitatory and inhibitory connectivity. These assumptions have been difficult to test directly. We therefore performed electron-microscopy-based circuit reconstruction and RNA profiling of identified cell types in the heading direction system of Drosophila melanogaster to directly examine excitatory and inhibitory synaptic connectivity, generating a dataset that should serve as a reference for future functional studies of the network. Consistent with several theoretical models, we identified network motifs that have been hypothesized to maintain the heading representation in darkness, update it when the animal turns, and tether it to visual cues. Genetically targeted two-photon calcium imaging and thermogenetic perturbation of the constituent neuron types during behavior provided additional support for these functional roles. However, we also discovered network motifs absent in current models, including a surprising degree of recurrence between arbors of different neurons with mixed pre- and post-synaptic specializations. Overall, our results confirm that the Drosophila heading direction network contains the core components of a ring attractor while also revealing unpredicted structural features that might enable the heading system to accurately track the animal9s heading with a small number of neurons.

SummaryAnimals employ multiple and distributed neuronal networks with diverse learning rules and synaptic plasticity dynamics to record distinct temporal and statistical information about the world. However, the molecular mechanisms underlying this diversity are poorly understood. The anatomically defined compartments of the insect mushroom body function as parallel units of associative learning, with different learning rates, memory decay dynamics and flexibility (Aso & Rubin 2016). Here we show that nitric oxide (NO) acts as a neurotransmitter in a subset of dopaminergic neurons in Drosophila. NO’s effects develop more slowly than those of dopamine and depend on soluble guanylate cyclase in postsynaptic Kenyon cells. NO acts antagonistically to dopamine; it shortens memory retention and facilitates the rapid updating of memories. The interplay of NO and dopamine enables memories stored in local domains along Kenyon cell axons to be specialized for predicting the value of odors based only on recent events. Our results provide key mechanistic insights into how diverse memory dynamics are established in parallel memory systems.

Animals employ diverse learning rules and synaptic plasticity dynamics to record temporal and statistical information about the world. However, the molecular mechanisms underlying this diversity are poorly understood. The anatomically defined compartments of the insect mushroom body function as parallel units of associative learning, with different learning rates, memory decay dynamics and flexibility (Aso and Rubin, 2016). Here, we show that nitric oxide (NO) acts as a neurotransmitter in a subset of dopaminergic neurons in Drosophila. NO’s effects develop more slowly than those of dopamine and depend on soluble guanylate cyclase in postsynaptic Kenyon cells. NO acts antagonistically to dopamine; it shortens memory retention and facilitates the rapid updating of memories. The interplay of NO and dopamine enables memories stored in local domains along Kenyon cell axons to be specialized for predicting the value of odors based only on recent events. Our results provide key mechanistic insights into how diverse memory dynamics are established in parallel memory systems.

Insect wing shapes are remarkably diverse and the combination of shape and kinematics determines both aerial capabilities and power requirements. However, the contribution of any specific morphological feature to performance is not known. Using targeted RNA interference to modify wing shape far beyond the natural variation found within the population of a single species, we show a direct effect on flight performance that can be explained by physical modelling of the novel wing geometry. Our data show that altering the expression of a single gene can significantly enhance aerial agility and that the Drosophila wing shape is not, therefore, optimized for certain flight performance characteristics that are known to be important. Our technique points in a new direction for experiments on the evolution of performance specialities in animals., Insect wings are under multiple competing selection pressures, but which are important in natural populations is not clear. Using RNAi to modify wing shape, Ray et al. show that aerial agility can be significantly enhanced in Drosophila, suggesting that natural variation does not reflect an optimization solely for flight agility.

Summary Epithelial tissues can elongate in two dimensions by polarized cell intercalation, oriented cell division, or cell shape change, owing to local or global actomyosin contractile forces acting in the plane of the tissue. In addition, epithelia can undergo morphogenetic change in three dimensions. We show that elongation of the wings and legs of Drosophila involves a columnar-to-cuboidal cell shape change that reduces cell height and expands cell width. Remodeling of the apical extracellular matrix by the Stubble protease and basal matrix by MMP1/2 proteases induces wing and leg elongation. Matrix remodeling does not occur in the haltere, a limb that fails to elongate. Limb elongation is made anisotropic by planar polarized Myosin-II, which drives convergent extension along the proximal-distal axis. Subsequently, Myosin-II relocalizes to lateral membranes to accelerate columnar-to-cuboidal transition and isotropic tissue expansion. Thus, matrix remodeling induces dynamic changes in actomyosin contractility to drive epithelial morphogenesis in three dimensions., Graphical Abstract, Highlights • Apical and basal extracellular matrices are degraded to elongate Drosophila limbs • Apical matrix is degraded by the Stubble protease and basal matrix by MMPs • Limbs elongate via convergent extension and cell flattening, driven by Myosin-II • In the haltere, Ultrabithorax prevents matrix remodeling and tissue elongation, Diaz-de-la-Loza et al. show that morphogenetic elongation of Drosophila limbs occurs via both convergent extension and columnar-to-cuboidal cell shape change. These processes are spatially organized by Myosin-II and temporally organized by remodeling of the extracellular matrix, including both apical (ZP-domain-containing) and basal (Collagen IV/Laminin/Perlecan-containing) matrices.

Background: Bone morphogenetic proteins require proteolytic processing to generate the mature ligand. Results: The BMPs Gbb and Scw have three cleavage sites, and BMP7 has one, which are differentially required to produce the functional ligand. Conclusion: Gbb, Scw, and BMP7 have distinct processing requirements despite being closely related. Significance: Rapid evolution of cleavage sites is a general mechanism for fine-tuning BMP ligand activity to function., Bone morphogenetic proteins (BMPs) are synthesized as proproteins that undergo proteolytic processing by furin/subtilisin proprotein convertases to release the active ligand. Here we study processing of BMP5/6/7/8 proteins, including the Drosophila orthologs Glass Bottom Boat (Gbb) and Screw (Scw) and human BMP7. Gbb and Scw have three functional furin/subtilisin proprotein convertase cleavage sites; two between the prodomain and ligand domain, which we call the Main and Shadow sites, and one within the prodomain, which we call the Pro site. In Gbb each site can be cleaved independently, although efficient cleavage at the Shadow site requires cleavage at the Main site, and remarkably, none of the sites is essential for Gbb function. Rather, Gbb must be processed at either the Pro or Main site to produce a functional ligand. Like Gbb, the Pro and Main sites in Scw can be cleaved independently, but cleavage at the Shadow site is dependent on cleavage at the Main site. However, both Pro and Main sites are essential for Scw function. Thus, Gbb and Scw have different processing requirements. The BMP7 ligand rescues gbb mutants in Drosophila, but full-length BMP7 cannot, showing that functional differences in the prodomain limit the BMP7 activity in flies. Furthermore, unlike Gbb, cleavage-resistant BMP7, although non-functional in rescue assays, activates the downstream signaling cascade and thus retains some functionality. Our data show that cleavage requirements evolve rapidly, supporting the notion that changes in post-translational processing are used to create functional diversity between BMPs within and between species.

Gene duplication and divergence is widely considered to be a fundamental mechanism for generating evolutionary novelties. The Bone Morphogenetic Proteins (BMPs) are a diverse family of signalling molecules found in all metazoan genomes that have evolved by duplication and divergence from a small number of ancestral types. In the fruit fly Drosophila, there are three BMPs: Decapentaplegic (Dpp) and Glass bottom boat (Gbb), which are the orthologues of vertebrate BMP2/4 and BMP5/6/7/8, respectively, and Screw (Scw), which, at the sequence level, is equally divergent from Dpp and Gbb. It has recently been shown that Scw has arisen from a duplication of Gbb in the lineage leading to higher Diptera. We show that since this duplication event, Gbb has maintained the ancestral BMP5/6/7/8 functionality while Scw has rapidly diverged. The evolution of Scw was accompanied by duplication and divergence of a suite of extracellular regulators that continue to diverge together in the higher Diptera. In addition, Scw has become restricted in its receptor specificity: Gbb proteins can signal through the Type I receptors Thick veins (Tkv) and Saxophone (Sax), while Scw signals through Sax. Thus, in a relatively short span of evolutionary time, the duplication event that gave rise to Scw produced not only a novel ligand but also a novel signalling mode that is functionally distinct from the ancestral Gbb mode. Our results demonstrate the plasticity of the BMP pathway not only in evolving new family members and new functions but also new signalling modes by redeploying key regulators in the pathway.

In following up the experiments reported in this paper, we have discovered that the polarity phenotypes of Dystroglycan and perlecan mutant clones under starvation conditions shown in Figures 4, 5, 6F, 6H, and 6J are the result of an artifact. We now believe that these panels do not represent real clones with a polarity phenotype, but rather false clones caused by a damage-induced artifact that creates patches of GFP-negative cells that mimic the appearance of a mutant clone with apical-basal polarity defects. Bona fide Dystroglycan and perlecan mutant clones marked using a different system do not show this phenotype, and thus our conclusion that they are required for the apical-basal polarity of the follicle cells under conditions of energetic stress is incorrect. We have described this damage-induced artifact in an article in Biology Open (Haack, T., Bergstralh, D., and St Johnston, D. (2013). Damage to the Drosophila follicle cell epithelium produces “false clones” with apparent polarity phenotypes. http://dx.doi.org/10.1242/bio.20134671). We apologize for any inconvenience that this erroneous conclusion may have caused. The results pertaining to the characterization of null Dg alleles and their role in basal planar cell polarity and egg shape remain valid. In light of our new data, the results from the paper lead to the conclusion that neither Dg nor Dys has an essential role in follicle cell polarity.

Summary How tissues acquire their characteristic shape is a fundamental unresolved question in biology. While genes have been characterized that control local mechanical forces to elongate epithelial tissues, genes controlling global forces in epithelia have yet to be identified. Here, we describe a genetic pathway that shapes appendages in Drosophila by defining the pattern of global tensile forces in the tissue. In the appendages, shape arises from tension generated by cell constriction and localized anchorage of the epithelium to the cuticle via the apical extracellular-matrix protein Dumpy (Dp). Altering Dp expression in the developing wing results in predictable changes in wing shape that can be simulated by a computational model that incorporates only tissue contraction and localized anchorage. Three other wing shape genes, narrow, tapered, and lanceolate, encode components of a pathway that modulates Dp distribution in the wing to refine the global force pattern and thus wing shape., Graphical Abstract, Highlights • The apical extracellular matrix protein Dumpy (Dp) is required for appendage shape • Dp anchors the epidermis to the cuticle, generating tension during tissue contraction • Alteration of the pattern of Dp gives rise to predictable changes in appendage shape • Narrow (Nw), Tapered (Ta), and Lanceolate (Ll) affect shape by modulating Dp, Regulation of global tensile forces in epithelia is one mechanism of determining tissue shape. Ray and Matamoro-Vidal et al. show that tissue contraction, in combination with localized anchorage to the cuticle by the apical extracellular matrix protein Dumpy, gives rise to anisotropic tensions that shape the appendages in the Drosophila pupa.

The Drosophila BMP5/6/7/8 homolog, glass bottom boat (gbb), has been shown to be involved in proliferation and vein patterning in the wing disk. To better understand the roles for gbb in wing development, as well as its relationship with the Drosophila BMP2/4 homolog decapentaplegic (dpp), we have used clonal analysis to define the functional foci of gbb during wing development. Our results show that gbb has both local and long-range functions in the disk that coincide both spatially and functionally with the established functions of dpp, suggesting that both BMPs contribute to the same processes during wing development. Indeed, comparison of the mutant phenotypes of dpp and gbb hypomorphs and null clones shows that both BMPs act locally along the longitudinal and cross veins to affect the process of vein promotion during pupal development, and long-range from a single focus along the A/P compartment boundary to affect the processes of disk proliferation and vein specification during larval development. Moreover, we show that duplications of dpp are able to rescue many of the phenotypes associated with gbb mutants and clones, indicating that the functions of gbb are at least partially redundant with those of dpp. While this relationship is similar to that described for dpp and the BMP screw (scw) in the embryo, we show that the mechanisms underlying both local and long-range functions of gbb and dpp in the wing are different. For the local foci, gbb function is confined to the regions of the veins that require the highest levels of dpp signaling, suggesting that gbb acts to augment dpp signaling in the same way as scw is proposed to do in the embryo. However, unlike scw-dependent signals in the embryo, these gbb signals are not transduced by the Type I receptor saxophone (sax), thus, the cooperativity between gbb and dpp is not achieved by signaling through distinct receptor complexes. For the long-range focus along the A/P compartment boundary, gbb function does not appear to affect the high point of the dpp gradient, but, rather, appears to be required for low points, which is the reciprocal of the relationship between dpp and scw in the embryo. Moreover, these functions of gbb also do not require the Type I receptor sax. Given these results, we conclude that the relationships between gbb and dpp in the wing disk represent novel paradigms for how multiple BMP ligands signal during development, and that signaling by multiple BMPs involves a variety of different inter-ligand relationships that depend on the developmental context in which they act.

The pattern of the large sensory bristles on the notum ofDrosophila arises as a consequence of the expression of theachaete and scute genes. The gene u-shapedencodes a novel zinc finger that acts as a transregulator ofachaete and scute in the dorsal region of the notum. Viable hypomorphic u-shaped mutants display additional dorsocentral and scutellar bristles that result from overexpression ofachaete and scute. In contrast, overexpression ofu-shaped causes a loss of achaete–scute expression and consequently a loss of dorsal bristles. The effects on the dorsocentral bristles appear to be mediated through the enhancer sequences that regulate achaete and scute at this site. The effects ofu-shaped mutants are similar to those of a class of dominant alleles of the gene pannier with which they display allele-specific interactions, suggesting that the products of both genes cooperate in the regulation of achaete and scute.A study of the sites at which the dorsocentral bristles arise in mosaicu-shaped nota, suggests that the levels of the u-shapedprotein are crucial for the precise positioning of the precursors of these bristles.

The anterior-posterior and dorsal-ventral axes of the Diosophila egg and embryo are established during oogen­ esis as the egg is being formed. The mechanisms under­ lying this process involve intercellular signaling events, including bidirectional communication between germline and somatic cells, and local cell-cell interactions in the soma. On the molecular level, these interactions ap­ pear to be mediated by a small number of intercellular signaling systems, including the Epidermal growth fac­ tor receptor [Egfr] and Notch systems, that are used mul­ tiple times during oogenesis to trigger different develop­ mental switches. The precisely regulated interplay be­ tween these various signaling systems forms a network of interdependencies that leads to the establishment of both the anterior-posterior and dorsal-ventral polarity in the egg chamber and embryo. Because both the Egfr and Notch signaling systems are widely conserved, the mechanisms involved in the regulation of these pro­ cesses in Drosophila oogenesis may serve as a paradigm for understanding intercellular signaling events in other organisms.

decapentaplegic (dpp) is a zygotically expressed gene encoding a TGF-β-related ligand that is necessary for dorsal-ventral patterning in the Drosophila embryo. We show here that dpp is an integral part of a gradient that specifies many different cell fates via intercellular signalling. There is a graded requirement for dpp activity in the early embryo: high levels of dpp activity specify the amnioserosa, while progressively lower levels specify dorsal and lateral ectoderm. This potential for dpp to specify cell fate is highly dosage sensitive. In the wildtype embryo, increasing the gene dosage of dpp can shift cell fates along the dorsal-ventral axis. Furthermore, in mutant embryos, in which only a subset of the dorsalventral pattern elements are represented, increasing the gene dosage of dpp can specifically transform those pattern elements into more dorsal ones. We present evidence that the zygotic dpp gradient and the maternal dorsal gradient specify distinct, non-overlapping domains of the dorsal-ventral pattern.

In mammals, the Transforming Growth Factor-s (TGF-s) superfamily controls a variety of developmental processes. In Drosophila, by contrast, a single member of the superfamily, decapentaplegic (dpp) performs most TGF-s developmental functions. The complexity of dpp functions is reflected in the complex cis-regulatory sequences that flank the gene. Dpp is divided into three regions: Hin, including the protein-coding exons; disk, including 3' cis-regulatory sequences; and shortvein (shv), including noncoding exons and 5' cis-regulatory sequences. We analyzed the cis-regulatory structure of the shortvein region using a nested series of rearrangement breakpoints and rescue constructs. We delimit the molecular regions responsible for three mutant phenotypes: larval lethality, wing venation defects, and head capsule defects. Multiple overlapping elements are responsible for larval lethality and wing venation defects. However, the area regulating head capsule formation is distinct, and resides 5' to these elements. We have demonstrated this by isolating and describing two novel dpp alleles, which affect only the adult head capsule. genesis 42:181?192, 2005. © 2005 Wiley-Liss, Inc.

Members of the Rho family of small GTPases are required for many of the morphogenetic processes required to shape the animal body. The activity of this family is regulated in part by a class of proteins known as RhoGTPase Activating Proteins (RhoGAPs) that catalyse the conversion of RhoGTPases to their inactive state. In our search for genes that regulate Drosophila morphogenesis, we have isolated several lethal alleles of crossveinless-c (cv-c). Molecular characterisation reveals that cv-c encodes the RhoGAP protein RhoGAP88C. During embryonic development, cv-c is expressed in tissues undergoing morphogenetic movements; phenotypic analysis of the mutants reveals defects in the morphogenesis of these tissues. Genetic interactions between cv-c and RhoGTPase mutants indicate that Rho1, Rac1 and Rac2 are substrates for Cv-c, and suggest that the substrate specificity might be regulated in a tissue-dependent manner. In the absence of cv-c activity, tubulogenesis in the renal or Malpighian tubules fails and they collapse into a cyst-like sack. Further analysis of the role of cv-c in the Malpighian tubules demonstrates that its activity is required to regulate the re-organisation of the actin cytoskeleton during the process of convergent extension. In addition, overexpression of cv-c in the developing tubules gives rise to actin-associated membrane extensions. Thus, Cv-c function is required in tissues actively undergoing morphogenesis, and we propose that its role is to regulate RhoGTPase activity to promote the coordinated organisation of the actin cytoskeleton, possibly by stabilising plasma membrane/actin cytoskeleton interactions., This work was supported by the Wellcome Trust (H.S., J.C.-G.H., B.D. and S.B.); The Royal Society (J.C.-G.H. and R.P.R.); a grant from the Dirección General de Investigación, Ministerio de Ciencia y Tecnología (BMC2003-05056); and an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa (M.R.-G.).

okra (okr), spindle-B (spnB), andspindle-D (spnD) are three members of a group of female sterile loci that produce defects in oocyte and egg morphology, including variable dorsal–ventral defects in the eggshell and embryo, anterior–posterior defects in the follicle cell epithelium and in the oocyte, and abnormalities in oocyte nuclear morphology. Many of these phenotypes reflect defects in grk-Egfr signaling processes, and can be accounted for by a failure to accumulate wild-type levels of Gurken and Fs(1)K10. We have cloned okr and spnB, and show that okr encodes the Drosophila homolog of the yeast DNA-repair protein Rad54, and spnB encodes a Rad51-like protein related to the meiosis-specific DMC1 gene. In functional tests of their role in DNA repair, we find that okrbehaves like its yeast homolog in that it is required in both mitotic and meiotic cells. In contrast, spnB and spnD appear to be required only in meiosis. The fact that genes involved in meiotic DNA metabolism have specific effects on oocyte patterning implies that the progression of the meiotic cell cycle is coordinated with the regulation of certain developmental events during oogenesis.

View Large Image | Download PowerPoint SlideWhen Darwin proposed the idea of natural selection during the 19th century, the major limitation to his theory was how to explain the notion of ‘heritable variation’. During the 20th century, one half of this problem (the explanation of the inheritance of genetic variation) was resolved through the work of Fisher, Haldane, and Wright, which culminated with the ‘modern synthesis’. The other half of the problem (how genetic variation gives rise to morphological differences between species) has only begun to be addressed more recently through advances in the field of evolutionary developmental biology, or evo-devo, and this will undoubtedly be a major contribution to evolutionary theory, ushering in a ‘new modern synthesis’ for the 21st century.In this light, Wallace Arthur's Evolution: A Developmental Approach looks forward to this new synthesis, covering the major themes of evo-devo in a brief and focussed text. The book begins with a rather lengthy introductory section covering the fundamentals of genetics, molecular, cell, and developmental biology (a necessary evil for any introductory development text), and this is followed by two main sections that the author has entitled ‘Developmental Repatterning’ and ‘The Direction of Evolution’. The first of these deals with the different ways in which development can evolve, providing examples of heterochrony, heterotopy, heterometry, and heterotypy and how these different forms of change are integrated. The second section deals with the mechanisms that steer the course of evolution, including co-adaptation, exaptation, developmental bias and constraint, plasticity, and the evolution of complexity. The book ends with a short section that offers a summary of the main conclusions and prospects for the future of evo-devo.In the preface to Evolution: A Developmental Approach, Arthur explains that he has written the text for students taking a course in evolution at university or college, yet it is not clearly focussed on this audience. It is unquestionably an introductory text: it is too technical for popular science, but not comprehensive or detailed enough for an upper division or graduate-level course, despite the references provided in the bibliography. The book is written in a casual, conversational style (for which it has been praised) and, although this will no doubt be appealing to an undergraduate audience, it is less appropriate for a more advanced one. By contrast, the text does more than simply recount the various experiments and conclusions that have formed the foundations of the evo-devo field, as a typical introductory text would. Arthur takes a step back from the data and tries to reassess the field as a whole, redefining terms, proposing new ones, and challenging some of the long-standing dogmas in the field. This is a very thought-provoking perspective; however, because it has not been vetted out in the wider scientific community, it is questionable whether it is the best approach for a naive audience. One of the strengths of the text is its candid approach to the limitations to the interpretation of scientific experiments, which is important in a field where the temptation to propose ‘just-so’ stories is all too strong. However, although this is important for developing critical thinking in more advanced students, it is so prevalent in the book that one is sometimes left wondering whether definitive conclusions can be made from evo-devo studies. This may put off the young biology students at whom the book is aimed. It is also not clear how this text would fit into a typical undergraduate curriculum. Although it is an excellent overview of evo-devo, Arthur has purposely left out most of the basic material on evolutionary theory and focussed his text on developmental evolution. As forward thinking as this may be, a typical undergraduate course will still need to cover classical evolutionary theory and, thus, this text would be, at best, a companion alongside a more conventional evolution text.These issues aside, the text is a first step in the right direction and is looking forward to the new synthesis that developmental biology is going to bring to evolutionary theory during the 21st century.

We have identified the molecular lesions associated with six point mutations in the Drosophila TGF-β homologue decapentaplegic (dpp). The sites of these mutations define residues within both the pro and ligand regions that are essential for dpp function in vivo. While all of these mutations affect residues that are highly conserved among TGF-β superfamily members, the phenotypic consequences of the different alleles are quite distinct. Through an analysis of these mutant phenotypes, both in cuticle preparations and with molecular probes, we have assessed the functional significance of specific residues that are conserved among the different members of the superfamily. In addition, we have tested for conditional genetic interactions between the different alleles. We show that two of the alleles are temperature sensitive for the embyronic functions of dpp, such that these alleles are not only embryonic viable as homozygotes but also partially complement other dpp hypomorphs at low temperatures. Our results are discussed with regard to in vitro mutagenesis data on other TGF-β-like molecules, as well as with regard to the regulation of dpp cell signaling in Drosophila.

Dystrophin and Dystroglycan are the two central components of the multimeric Dystrophin Associated Protein Complex, or DAPC, that is thought to provide a mechanical link between the extracellular matrix and the actin cytoskeleton, disruption of which leads to muscular dystrophy in humans. We present the characterization of the Drosophila ‘crossveinless’ mutation detached (det), and show that the gene encodes the fly ortholog of Dystrophin. Our genetic analysis shows that, in flies, Dystrophin is a non-essential gene, and the sole overt morphological defect associated with null mutations in the locus is the variable loss of the posterior crossvein that has been described for alleles of det. Null mutations in Drosophila Dystroglycan (Dg) are similarly viable and exhibit this crossvein defect, indicating that both of the central DAPC components have been co-opted for this atypical function of the complex. In the developing wing, the Drosophila DAPC affects the intercellular signalling pathways involved in vein specification. In det and Dg mutant wings, the early BMP signalling that initiates crossvein specification is not maintained, particularly in the pro-vein territories adjacent to the longitudinal veins, and this results in the production of a crossvein fragment in the intervein between the two longitudinal veins. Genetic interaction studies suggest that the DAPC may exert this effect indirectly by down-regulating Notch signalling in pro-vein territories, leading to enhanced BMP signalling in the intervein by diffusion of BMP ligands from the longitudinal veins.