A locomotor assay reveals deficits in heterozygous Parkinson's disease model and proprioceptive mutants in adult Drosophila.

Severe locomotor impairment is a common phenotype of neurodegenerative disorders such as Parkinson's disease (PD). Drosophila models of PD, studied for more than a decade, have helped in understanding the interaction between various genetic factors, such as parkin and PINK1, in this disease. To characterize locomotor behavioral phenotypes for these genes, fly climbing assays have been widely used. While these simple current assays for locomotor defects in Drosophila mutants measure some locomotor phenotypes well, it is possible that detection of subtle changes in behavior is important to understand the manifestation of locomotor disorders. We introduce a climbing behavior assay which provides such fine-scale behavioral data and tests this proposition for the Drosophila model. We use this inexpensive, fully automated assay to quantitatively characterize the climbing behavior at high parametric resolution in 3 contexts. First, we characterize wild-type flies and uncover a hitherto unknown sexual dimorphism in climbing behavior. Second, we study climbing behavior of heterozygous mutants of genes implicated in the fly PD model and reveal previously unreported prominent locomotor defects in some of these heterozygous fly lines. Finally, we study locomotor defects in a homozygous proprioceptory mutation (Trp-γ1 ) known to affect fine motor control in Drosophila Moreover, we identify aberrant geotactic behavior in Trp-γ1 mutants, thereby opening up a finer assay for geotaxis and its genetic basis. Our assay is therefore a cost-effective, general tool for measuring locomotor behaviors of wild-type and mutant flies in fine detail and can reveal subtle motor defects.

An Emerging System to Study Photosymbiosis, Brain Regeneration, Chronobiology, and Behavior: The Marine Acoel Symsagittifera roscoffensis.

The acoel worm Symsagittifera roscoffensis, an early offshoot of the Bilateria and the only well-studied marine acoel that lives in a photosymbiotic relationship, exhibits a centralized nervous system, brain regeneration, and a wide repertoire of complex behaviors such as circatidal rhythmicity, photo/geotaxis, and social interactions. While this animal can be collected by the thousands and is studied historically, significant progress is made over the last decade to develop it as an emerging marine model. The authors here present the feasibility of culturing it in the laboratory and describe the progress made on different areas, including genomic and tissue architectures, highlighting the associated challenges. In light of these developments, and on the ability to access abundant synchronized embryos, the authors put forward S. roscoffensis as a marine system to revisit questions in the areas of photosymbiosis, regeneration, chronobiology, and the study of complex behaviors from a molecular and evolutionary perspective.

The asymmetrically segregating lncRNA cherub is required for transforming stem cells into malignant cells.

Tumor cells display features that are not found in healthy cells. How they become immortal and how their specific features can be exploited to combat tumorigenesis are key questions in tumor biology. Here we describe the long non-coding RNA cherub that is critically required for the development of brain tumors in Drosophila but is dispensable for normal development. In mitotic Drosophila neural stem cells, cherub localizes to the cell periphery and segregates into the differentiating daughter cell. During tumorigenesis, de-differentiation of cherub-high cells leads to the formation of tumorigenic stem cells that accumulate abnormally high cherub levels. We show that cherub establishes a molecular link between the RNA-binding proteins Staufen and Syncrip. As Syncrip is part of the molecular machinery specifying temporal identity in neural stem cells, we propose that tumor cells proliferate indefinitely, because cherub accumulation no longer allows them to complete their temporal neurogenesis program.

Identification of a Single Pair of Interneurons for Bitter Taste Processing in the Drosophila Brain.

Drosophila has become an excellent model system for investigating the organization and function of the gustatory system due to the relatively simple neuroanatomical organization of its brain and the availability of powerful genetic and transgenic technology. Thus, at the molecular and cellular levels, a great deal of insight into the peripheral detection and coding of gustatory information has already been attained. In contrast, much less is known about the central neural circuits that process this information and induce behaviorally appropriate motor output. Here, we combine functional behavioral tests with targeted transgene expression through specific driver lines to identify a single bilaterally homologous pair of bitter-sensitive interneurons that are located in the subesophageal zone of the brain. Anatomical and functional data indicate that these interneurons receive specific synaptic input from bitter-sensitive gustatory receptor neurons. Targeted transgenic activation and inactivation experiments show that these bitter-sensitive interneurons can largely suppress the proboscis extension reflex to appetitive stimuli, such as sugar and water. These functional experiments, together with calcium-imaging studies and calcium-modulated photoactivatable ratiometric integrator (CaMPARI) labeling, indicate that these first-order local interneurons play an important role in the inhibition of the proboscis extension reflex that occurs in response to bitter tastants. Taken together, our studies present a cellular identification and functional characterization of a key gustatory interneuron in the bitter-sensitive gustatory circuitry of the adult fly.

GABAergic inhibition of leg motoneurons is required for normal walking behavior in freely moving Drosophila.

Walking is a complex rhythmic locomotor behavior generated by sequential and periodical contraction of muscles essential for coordinated control of movements of legs and leg joints. Studies of walking in vertebrates and invertebrates have revealed that premotor neural circuitry generates a basic rhythmic pattern that is sculpted by sensory feedback and ultimately controls the amplitude and phase of the motor output to leg muscles. However, the identity and functional roles of the premotor interneurons that directly control leg motoneuron activity are poorly understood. Here we take advantage of the powerful genetic methodology available in Drosophila to investigate the role of premotor inhibition in walking by genetically suppressing inhibitory input to leg motoneurons. For this, we have developed an algorithm for automated analysis of leg motion to characterize the walking parameters of wild-type flies from high-speed video recordings. Further, we use genetic reagents for targeted RNAi knockdown of inhibitory neurotransmitter receptors in leg motoneurons together with quantitative analysis of resulting changes in leg movement parameters in freely walking Drosophila Our findings indicate that targeted down-regulation of the GABAA receptor Rdl (Resistance to Dieldrin) in leg motoneurons results in a dramatic reduction of walking speed and step length without the loss of general leg coordination during locomotion. Genetically restricting the knockdown to the adult stage and subsets of motoneurons yields qualitatively identical results. Taken together, these findings identify GABAergic premotor inhibition of motoneurons as an important determinant of correctly coordinated leg movements and speed of walking in freely behaving Drosophila.

Structure and development of the subesophageal zone of the Drosophila brain. II. Sensory compartments.

The subesophageal zone (SEZ) of the Drosophila brain processes mechanosensory and gustatory sensory input from sensilla located on the head, mouth cavity and trunk. Motor output from the SEZ directly controls the movements involved in feeding behavior. In an accompanying paper (Hartenstein et al., ), we analyzed the systems of fiber tracts and secondary lineages to establish reliable criteria for defining boundaries between the four neuromeres of the SEZ, as well as discrete longitudinal neuropil domains within each SEZ neuromere. Here we use this anatomical framework to systematically map the sensory projections entering the SEZ throughout development. Our findings show continuity between larval and adult sensory neuropils. Gustatory axons from internal and external taste sensilla of the larva and adult form two closely related sensory projections, (a) the anterior central sensory center located deep in the ventromedial neuropil of the tritocerebrum and mandibular neuromere, and (b) the anterior ventral sensory center (AVSC), occupying a superficial layer within the ventromedial tritocerebrum. Additional, presumed mechanosensory terminal axons entering via the labial nerve define the ventromedial sensory center (VMSC) in the maxilla and labium. Mechanosensory afferents of the massive array of chordotonal organs (Johnston's organ) of the adult antenna project into the centrolateral neuropil column of the anterior SEZ, creating the antenno-mechanosensory and motor center (AMMC). Dendritic projections of dye back-filled motor neurons extend throughout a ventral layer of the SEZ, overlapping widely with the AVSC and VMSC. Our findings elucidate fundamental structural aspects of the developing sensory systems in Drosophila.

Structure and development of the subesophageal zone of the Drosophila brain. I. Segmental architecture, compartmentalization, and lineage anatomy.

The subesophageal zone (SEZ) of the Drosophila brain houses the circuitry underlying feeding behavior and is involved in many other aspects of sensory processing and locomotor control. Formed by the merging of four neuromeres, the internal architecture of the SEZ can be best understood by identifying segmentally reiterated landmarks emerging in the embryo and larva, and following the gradual changes by which these landmarks become integrated into the mature SEZ during metamorphosis. In previous works, the system of longitudinal fibers (connectives) and transverse axons (commissures) has been used as a scaffold that provides internal landmarks for the neuromeres of the larval ventral nerve cord. We have extended the analysis of this scaffold to the SEZ and, in addition, reconstructed the tracts formed by lineages and nerves in relationship to the connectives and commissures. As a result, we establish reliable criteria that define boundaries between the four neuromeres (tritocerebrum, mandibular neuromere, maxillary neuromere, labial neuromere) of the SEZ at all stages of development. Fascicles and lineage tracts also demarcate seven columnar neuropil domains (ventromedial, ventro-lateral, centromedial, central, centrolateral, dorsomedial, dorsolateral) identifiable throughout development. These anatomical subdivisions, presented in the form of an atlas including confocal sections and 3D digital models for the larval, pupal and adult stage, allowed us to describe the morphogenetic changes shaping the adult SEZ. Finally, we mapped MARCM-labeled clones of all secondary lineages of the SEZ to the newly established neuropil subdivisions. Our work will facilitate future studies of function and comparative anatomy of the SEZ.

Drosophila adult muscle development and regeneration.

Myogenesis is a highly orchestrated, complex developmental process by which cell lineages that are mesodermal in origin generate differentiated multinucleate muscle cells as a final product. Considerable insight into the process of myogenesis has been obtained for the embryonic development of the larval muscles of Drosophila. More recently, the postembryonic development of the muscles of the adult fly has become a focus of experimental investigation of myogenesis since specific flight muscles of the fly manifest remarkable similarities to vertebrate muscles in their development and organization. In this review, we catalog some of the milestones in the study of myogenesis in the large adult-specific flight muscles of Drosophila. The identification of mesoderm-derived muscle stem cell lineages, the characterization of the symmetric and asymmetric divisions through which they produce adult-specific myoblasts, the multifaceted processes of myoblast fusion, and the unexpected discovery of quiescent satellite cells that can be activated by injury are discussed. Moreover, the finding that all of these processes incorporate a plethora of signaling interactions with other myogenic cells and with niche-like neighboring tissue is considered. Finally, we briefly point out possible future developments in the area of Drosophila myogenesis that may lead to of new avenues of genetic research into the roles of muscle stem cells in development, disease and aging.

Identification and functional characterization of muscle satellite cells in Drosophila.

Work on genetic model systems such as Drosophila and mouse has shown that the fundamental mechanisms of myogenesis are remarkably similar in vertebrates and invertebrates. Strikingly, however, satellite cells, the adult muscle stem cells that are essential for the regeneration of damaged muscles in vertebrates, have not been reported in invertebrates. In this study, we show that lineal descendants of muscle stem cells are present in adult muscle of Drosophila as small, unfused cells observed at the surface and in close proximity to the mature muscle fibers. Normally quiescent, following muscle fiber injury, we show that these cells express Zfh1 and engage in Notch-Delta-dependent proliferative activity and generate lineal descendant populations, which fuse with the injured muscle fiber. In view of strikingly similar morphological and functional features, we consider these novel cells to be the Drosophila equivalent of vertebrate muscle satellite cells.

The splicing co-factor Barricade/Tat-SF1 is required for cell cycle and lineage progression in Drosophila neural stem cells.

Stem cells need to balance self-renewal and differentiation for correct tissue development and homeostasis. Defects in this balance can lead to developmental defects or tumor formation. In recent years, mRNA splicing has emerged as an important mechanism regulating cell fate decisions. Here we address the role of the evolutionarily conserved splicing co-factor Barricade (Barc)/Tat-SF1/CUS2 in Drosophila neural stem cell (neuroblast) lineage formation. We show that Barc is required for the generation of neurons during Drosophila brain development by ensuring correct neural progenitor proliferation and differentiation. Barc associates with components of the U2 small nuclear ribonucleoprotein (snRNP) complex, and its depletion causes alternative splicing in the form of intron retention in a subset of genes. Using bioinformatics analysis and a cell culture-based splicing assay, we found that Barc-dependent introns share three major traits: they are short, GC rich and have weak 3' splice sites. Our results show that Barc, together with the U2 snRNP complex, plays an important role in regulating neural stem cell lineage progression during brain development and facilitates correct splicing of a subset of introns.

How the humble insect brain became a powerful experimental model system.

In the 21st century, neurobiological studies focused on the insect brain are revealing unprecedented insight into the molecular, cellular, developmental, and circuit aspects of brain organization and function, notably in the genetic model system of Drosophila melanogaster. Underlying this accelerating progress in understanding the insect brain is a century-long history of ground breaking experimental investigation, methodological advance, and conceptual insight catalyzed by the integration of two emerging research fields, neuroscience and genetics. This review traces some of the key early steps in this remarkable historical scientific adventure of exploring the brain of "these apparently humble representatives of life".

Motor control of Drosophila feeding behavior.

The precise coordination of body parts is essential for survival and behavior of higher organisms. While progress has been made towards the identification of central mechanisms coordinating limb movement, only limited knowledge exists regarding the generation and execution of sequential motor action patterns at the level of individual motoneurons. Here we use Drosophila proboscis extension as a model system for a reaching-like behavior. We first provide a neuroanatomical description of the motoneurons and muscles contributing to proboscis motion. Using genetic targeting in combination with artificial activation and silencing assays we identify the individual motoneurons controlling the five major sequential steps of proboscis extension and retraction. Activity-manipulations during naturally evoked proboscis extension show that orchestration of serial motoneuron activation does not rely on feed-forward mechanisms. Our data support a model in which central command circuits recruit individual motoneurons to generate task-specific proboscis extension sequences.

Glial and neuronal Semaphorin signaling instruct the development of a functional myotopic map for Drosophila walking.

Motoneurons developmentally acquire appropriate cellular architectures that ensure connections with postsynaptic muscles and presynaptic neurons. In Drosophila, leg motoneurons are organized as a myotopic map, where their dendritic domains represent the muscle field. Here, we investigate mechanisms underlying development of aspects of this myotopic map, required for walking. A behavioral screen identified roles for Semaphorins (Sema) and Plexins (Plex) in walking behavior. Deciphering this phenotype, we show that PlexA/Sema1a mediates motoneuron axon branching in ways that differ in the proximal femur and distal tibia, based on motoneuronal birth order. Importantly, we show a novel role for glia in positioning dendrites of specific motoneurons; PlexB/Sema2a is required for dendritic positioning of late-born motoneurons but not early-born motoneurons. These findings indicate that communication within motoneurons and between glia and motoneurons, mediated by the combined action of different Plexin/Semaphorin signaling systems, are required for the formation of a functional myotopic map.

Functional brain regeneration in the acoel worm Symsagittifera roscoffensis.

The ability of some animals to regrow their head and brain after decapitation provides a striking example of the regenerative capacity within the animal kingdom. The acoel worm Symsagittifera roscoffensis can regrow its head, brain and sensory head organs within only a few weeks after decapitation. How rapidly and to what degree it also reacquires its functionality to control behavior however remains unknown. We provide here a neuroanatomical map of the brain neuropils of the adult S. roscoffensis and show that after decapitation a normal neuroanatomical organization of the brain is restored in the majority of animals. By testing different behaviors we further show that functionality of both sensory perception and the underlying brain architecture are restored within weeks after decapitation. Interestingly not all behaviors are restored at the same speed and to the same extent. While we find that phototaxis recovered rapidly, geotaxis is not restored within 7 weeks. Our findings show that regeneration of the head, sensory organs and brain result in the restoration of directed navigation behavior, suggesting a tight coordination in the regeneration of certain sensory organs with that of their underlying neural circuits. Thus, at least in S. roscoffensis, the regenerative capacity of different sensory modalities follows distinct paths.

Microanatomy and development of the dwarf male of Symbion pandora (Phylum Cycliophora): new insights from ultrastructural investigation based on serial section electron microscopy.

Cycliophorans have a complex life cycle that involves several sexual and asexual stages. One of the sexual stages is the 40 µm-long dwarf male, which is among the smallest free-living metazoans. Although the dwarf male has a highly complex body plan, this minute organism is composed of a very low number of somatic cells (~50). The developmental processes that give rise to this unique phenotype are largely unknown. Here we use high resolution serial block face-scanning electron microscopy to analyze the anatomy and morphogenesis of three cycliophoran dwarf males at different developmental stages ranging from internal bud to mature male. The anatomical and morphological features of the mature dwarf male stage reported here largely correspond to those reported in earlier studies. Interestingly, the organs that typically characterize the anatomy of the mature dwarf male, e.g., muscles, brain, testis and glands, are already formed in the young male. However, there are striking differences between the mature male and young male stages at the level of cellular architecture. Thus, while the young male stage, like the internal bud stage, possesses approximately 200 nucleated cells, the mature male stage comprises only around 50 nucleated cells; muscle and epidermal cells of the mature male lack nuclei. Moreover, the total body volume of the mature male is only 63% of the body of the young male implying that the maturation of the young male into a mature male involves a marked reduction of internal body volume, mainly by massive nuclei loss. Our comparative analysis of these dwarf male specimens reveals unprecedented insight into the striking morphological and developmental differences that characterize these highly miniaturized male stages both at the level of body organization and at the level of cellular ultrastructure.

Control of neural stem cell self-renewal and differentiation in Drosophila.

The neural stem cells of Drosophila, called neuroblasts, have the ability to self-renew and at the same time produce many different types of neurons and glial cells. In the central brain and ventral ganglia, neuroblasts are specified and delaminate from the neuroectoderm during embryonic development under the control of proneural and neurogenic genes. In contrast, in the optic lobes, neuroepithelial cells are transformed into neuroblasts postembryonically by a spatial wave of proneural gene expression. Central brain and ventral nerve cord neuroblasts manifest a short embryonic proliferation period followed by a stage of quiescence and then undergo a prolonged postembryonic proliferation period during which most of the differentiated neurons of the adult CNS are generated. While most neuroblasts belong to a type I class that produces neuronal lineages through non-self-renewing ganglion mother cells, a small subset of type II neuroblasts generates exceptionally large neuronal lineages through self-renewing intermediate progenitor cells that have a transit amplifying function. All neuroblasts in the CNS generate their neural progeny through an asymmetric cell division mode in which the interplay of apical complex and basal complex molecules in the mitotically active progenitor results in the segregation of cell fate determinants into the smaller more differentiated daughter cell. Defects in this molecular control of asymmetric cell division in neuroblasts can result in brain tumor formation. Proliferating neuroblast lineages in the developing CNS utilize transcription factor cascades as a generic mechanism for temporal patterning and birth order-dependent determination of differential neural cell fate. This contributes to the generation of a remarkable diversity of cell types in the developing CNS from a surprisingly small set of neural stem cell-like precursors.

Genetic transformation of structural and functional circuitry rewires the Drosophila brain.

Acquisition of distinct neuronal identities during development is critical for the assembly of diverse functional neural circuits in the brain. In both vertebrates and invertebrates, intrinsic determinants are thought to act in neural progenitors to specify their identity and the identity of their neuronal progeny. However, the extent to which individual factors can contribute to this is poorly understood. We investigate the role of orthodenticle in the specification of an identified neuroblast (neuronal progenitor) lineage in the Drosophila brain. Loss of orthodenticle from this neuroblast affects molecular properties, neuroanatomical features, and functional inputs of progeny neurons, such that an entire central complex lineage transforms into a functional olfactory projection neuron lineage. This ability to change functional macrocircuitry of the brain through changes in gene expression in a single neuroblast reveals a surprising capacity for novel circuit formation in the brain and provides a paradigm for large-scale evolutionary modification of circuitry.

Maintaining neural stem cell identity in the brain.

In the developing fruit fly brain, a protein called Trithorax increases the number of neural cells produced from a single stem cell, in part by regulating the transcription of the target genes buttonhead and pointed.

The chimerical and multifaceted marine acoel Symsagittifera roscoffensis: from photosymbiosis to brain regeneration.

A remarkable example of biological engineering is the capability of some marine animals to take advantage of photosynthesis by hosting symbiotic algae. This capacity, referred to as photosymbiosis, is based on structural and functional complexes that involve two distantly unrelated organisms. These stable photosymbiotic associations between metazoans and photosynthetic protists play fundamental roles in marine ecology as exemplified by reef communities and their vulnerability to global changes threats. Here we introduce a photosymbiotic tidal acoel flatworm, Symsagittifera roscoffensis, and its obligatory green algal photosymbiont, Tetraselmis convolutae (Lack of the algal partner invariably results in acoel lethality emphasizing the mandatory nature of the photosymbiotic algae for the animal's survival). Together they form a composite photosymbiotic unit, which can be reared in controlled conditions that provide easy access to key life-cycle events ranging from early embryogenesis through the induction of photosymbiosis in aposymbiotic juveniles to the emergence of a functional "solar-powered" mature stage. Since it is possible to grow both algae and host under precisely controlled culture conditions, it is now possible to design a range of new experimental protocols that address the mechanisms and evolution of photosymbiosis. S. roscoffensis thus represents an emerging model system with experimental advantages that complement those of other photosymbiotic species, in particular corals. The basal taxonomic position of S. roscoffensis (and acoels in general) also makes it a relevant model for evolutionary studies of development, stem cell biology and regeneration. Finally, it's autotrophic lifestyle and lack of calcification make S. roscoffensis a favorable system to study the role of symbiosis in the response of marine organisms to climate change (e.g., ocean warming and acidification). In this article we summarize the state of knowledge of the biology of S. roscoffensis and its algal partner from studies dating back over a century, and provide an overview of ongoing research efforts that take advantage of this unique system.