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.

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 GABA A 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 .

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.

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.

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.

Severe locomotor impairment is a common phenotype of neurodegenerative disorders such as Parkinson’s disease (PD). Drosophila models of PD, studied from 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 behavioural 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 behaviour is important to understand the early manifestation of locomotor disorders. We introduce a novel climbing behaviour assay which provides such fine-scale behavioural data and tests this proposition for the Drosophila model. We use this inexpensive, fully automated, high resolution assay to quantitatively characterize the parameters of climbing behaviour in three contexts. First, we characterize wild type flies and uncover a hitherto unknown sexual dimorphism in climbing behaviour. Second, we study climbing behaviour 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-γ 1 ) known to affect fine motor control in Drosophila . Moreover, we identify aberrant geotactic behaviour in Trp-γ 1 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 behaviours of wild type and mutant flies in fine detail and can reveal mild motor defects. Significance statement Fine control of neuronal activity is required for proper motor output. Severe locomotor impairment is a common result of neurodegenerative disorders such as Parkinson’s disease (PD). The fruitfly, Drosophila , has been widely used as a model system to study the genetics of these disorders and simple climbing assays have been used to study the behavioural phenotypes of mutations in these genes. Here we introduce a novel, fully automated, high resolution climbing behaviour assay and use this assay to characterize climbing behaviour in wild type flies and in various fly mutant lines related to PD and defects in proprioception. Our assay is a general tool for measuring locomotor behaviours of flies in fine detail and can reveal very mild motor defects.

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.

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.

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.

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.

Cycliophorans are characterized by a complex life cycle that involves an asexual generation and a sexual generation. The most prominent life cycle stage in both generations is the so-called feeding stage. Using RNA-Seq, we profiled differential gene expression between feeding stages from asexual and sexual generations to study this intergenerational shift. For this, we also generated a reference transcriptome for the cycliophoran Symbion pandora. We found that a total of 2660 contigs (more than 10% of the total transcriptome) correspond to genes that are expressed differentially in the feeding stages from the asexual generation as compared to the sexual feeding stages. Among these, 1236 genes are upregulated in the asexual stages as compared to the sexual stages. Conversely, 1424 genes are upregulated in the sexual stages as compared to the asexual stages. The asexual stages express genes predominantly related to RNA processing and splicing as well as protein folding, which suggests a high degree of regulation at the transcriptional and post-transcriptional levels. In marked contrast, the sexual stages highly express genes related to signal transduction and neurotransmission. This is the first time that a large whole-transcriptome RNA-Seq expression dataset has been generated for any cycliophoran. Moreover, this study provides important information for further studies on the molecular mechanisms that are involved in the shift from asexual to sexual generations in this still enigmatic group.

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.

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”.

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.

Meiofauna is the community of microscopically small, benthic animals living in and on soft aquatic substrates. Meiofaunal organisms, which have representatives of almost all invertebrate phyla, are smaller than macrofauna. The meiofauna of Singapore has never been explored. In the present study, we show results from the first exploration of the marine meiofauna of this geographical region. Several of the localities sampled yielded specimens of the meiofaunal groups Tardigrada, Kinorhyncha and/or meiofaunal Rhabditophora (Platyhelminthes). In the future, quantitative and qualitative studies would be required to assess the geographical distribution, as well as population density and taxon richness of the meiofaunal community in Singapore.

The origin of the first nervous system is an intriguing enigma. This chapter focuses on the evolution of complex nervous systems from simple neural origins and considers evidence from comparative, developmental, and molecular genetic studies that shed light on this fascinating evolutionary process. A phylogenetic assessment of the origin of nervous systems based on currently available paleontological data is both enlightening and disappointing. In its ontogeny, the bilaterian central nervous system is a complex three-dimensional structure that develops from a two-dimensional embryonic neuroepithelium. Cnidarian neurons are found in the ectodermal and the endodermal cell layers, and in terms of cell types, correspond to sensory cells, motor neurons and ganglionic interneurons. Many of the developmental control genes involved in axial patterning in insects and vertebrates are known to be conserved in cnidarians, implying that that a substantial number of these genes were already present in the last common ancestor of bilaterians and cnidarians.

Loricifera is a phylum of free-living, marine animals of microscopic size, which is distributed worldwide at an extensive depth range. After the description of Nanaloricus mysticus Kristensen, 1983, a total of 36 loriciferan species have now been described. However, additional new species already known await formal description. Indeed, new loriciferans are frequently found during oceanographic expeditions, though they are usually accounted as “undescribed species” amongst the meiofauna collected. This is because their taxonomic identification is not easy, especially at the genus and species level, and the number of researchers familiarized with the loriciferan anatomy is reduced. Aiming at contributing with new tools for the identification of Loricifera, we present here the first hierarchical single-access and multiple entry keys based on morphological characters of adults (or postlarva) and the most prominent larval stage, the so-called Higgins larva. The single-access keys facilitate the identification at the species level, while the multiple entry key summarizes the diagnostic features for each genus of Loricifera. The two keys may be used separately or supplementary to each other. In order to facilitate the use of the keys, we provide a detailed description of the loriciferan morphology, as well as an updated list of the terminology used. In addition, we include a short description of the intermediate life cycle stages and the hypothetical life cycles of all families. Finally, we hope to encourage more researchers to study the enigmatic phylum Loricifera.

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.

Abstract In this spotlight chapter the question of whether the urbilaterian ancestor already possessed a complex brain, or whether the various brains found in extant bilaterians arose by convergent evolution is considered. Since current palaeontological data gives little insight into the origin of brains during evolution earlier than the Cambrium, evidence from comparative, developmental, and molecular genetic studies that sheds light on the origin of the first brain is reviewed. Comparative neuroanatomical data reveal complex CNS throughout the protostomes and deuterostomes, but do not resolve the question of whether the diverse brains of extant bilaterians all had a common urbilaterian origin. Studies on regionalized expression of developmental patterning genes, such as Cephalic gap genes and Hox genes, show that they not only show similar expression patterns, but also have comparable developmental roles in vertebrate and invertebrate brain development. Moreover, even conserved organizational features of complex brain circuitry such as the visual and olfactory system have recently been reported in both protostomes and deuterostomes. Taken together, there is now increasing evidence for a brain with relatively sophisticated sensory and associative brain centres in the urbilaterian ancestor of vertebrates and invertebrates, implying that the first urbilaterian brain might already have been quite complex.