Image analysis platforms for exploring genetic and neuronal mechanisms regulating animal behavior

An important aim of neuroscience is to understand how gene interactions and neuronal networks regulate animal behavior. The larvae of the marine annelid Platynereis dumerilii provide a convenient system for such integrative studies. These larvae exhibit a wide range of behaviors, including phototaxis, chemotaxis and gravitaxis and at the same time exhibit relatively simple nervous system organization. Due to its small size and transparent body, the Platynereis larva is compatible with whole-body light microscopic imaging following tissue staining protocols. It is also suitable for serial electron microscopic imaging and subsequent neuronal connectome reconstruction. Despite advances in imaging techniques, automated computational tools for large data analysis are not well-established in Platynereis. In the current work, I developed image analysis software for exploring genetic and nervous system mechanisms modulating Platynereis behavior. Exploring gene expression patterns Current labeling and imaging techniques restrict the number of gene expression patterns that can be labelled and visualized in a single specimen, which hinders the study of behaviors driven by multi-molecular interactions. To address this problem, I employed image registration to generate a gene expression atlas that integrates gene expression information from multiple specimens in a common reference space. The gene expression atlas was used to investigate mechanisms regulating larval locomotion, settlement and phototaxis in Platynereis. The atlas can assist in the identification of inter-individual and inter-species variations in gene expression. To provide a representation convenient for exploring gene expression patterns, I created a model of the atlas using 3D graphics software, which enabled convenient data visualization and efficient data storage and sharing. Exploring neuronal networks regulating behavior Neuronal circuitry can be reconstructed from the images obtained from electron microscopy, which resolves very fine structures such as neuron morphology or synapses. The amount of data resulting from electron microscopy and the complexity of neuronal networks represent a significant challenge for manual analysis. To solve this problem, I developed the NeuroDetective software, which models a neuronal circuitry and analyzes the information flow within it. The software combines the advantages of 3D visualization and graph analysis software by integrating neuron morphology and spatial distribution together with synaptic connectivity. NeuroDetective allowed studying the neuronal circuitry responsible for phototaxis in Platynereis larvae, revealing the connections and the neurons important for the network functionality. NeuroDetective facilitated the establishment of a relationship between the function and the structure of the neuronal circuitry in Platynereis phototaxis. Integrating gene expression patterns with neuronal connectivity Neuronal circuitry and its associated modulating biomolecules, such as neurotransmitters and neuropeptides, are thought to be the main factors regulating animal behavior. Therefore it was important to integrate both genetic and neuronal information in order to fully understand how biomolecules in conjunction with neuronal anatomy elicit certain animal behavior. To resolve the difference in specimen preparation for gene expression versus electron microscopy preparations, I developed an image registration procedure to match the signals from these two different datasets. This method enabled the integration the spatial distribution of specific modulators into the analysis of neuronal networks, leading to an improved understanding of the genetic and neuronal mechanisms modulating behavior in Platynereis

Object-based representation and analysis of light and electron microscopic volume data using Blender

This is the final version of the article. Available from the publisher via the DOI in this record.BACKGROUND: Rapid improvements in light and electron microscopy imaging techniques and the development of 3D anatomical atlases necessitate new approaches for the visualization and analysis of image data. Pixel-based representations of raw light microscopy data suffer from limitations in the number of channels that can be visualized simultaneously. Complex electron microscopic reconstructions from large tissue volumes are also challenging to visualize and analyze. RESULTS: Here we exploit the advanced visualization capabilities and flexibility of the open-source platform Blender to visualize and analyze anatomical atlases. We use light-microscopy-based gene expression atlases and electron microscopy connectome volume data from larval stages of the marine annelid Platynereis dumerilii. We build object-based larval gene expression atlases in Blender and develop tools for annotation and coexpression analysis. We also represent and analyze connectome data including neuronal reconstructions and underlying synaptic connectivity. CONCLUSIONS: We demonstrate the power and flexibility of Blender for visualizing and exploring complex anatomical atlases. The resources we have developed for Platynereis will facilitate data sharing and the standardization of anatomical atlases for this species. The flexibility of Blender, particularly its embedded Python application programming interface, means that our methods can be easily extended to other organisms.The research leading to these results received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/European Research Council Grant Agreement 260821

Neuronal connectome of a sensory-motor circuit for visual navigation.

This is the final version of the article. Available from eLife Sciences Publications via the DOI in this record.Animals use spatial differences in environmental light levels for visual navigation; however, how light inputs are translated into coordinated motor outputs remains poorly understood. Here we reconstruct the neuronal connectome of a four-eye visual circuit in the larva of the annelid Platynereis using serial-section transmission electron microscopy. In this 71-neuron circuit, photoreceptors connect via three layers of interneurons to motorneurons, which innervate trunk muscles. By combining eye ablations with behavioral experiments, we show that the circuit compares light on either side of the body and stimulates body bending upon left-right light imbalance during visual phototaxis. We also identified an interneuron motif that enhances sensitivity to different light intensity contrasts. The Platynereis eye circuit has the hallmarks of a visual system, including spatial light detection and contrast modulation, illustrating how image-forming eyes may have evolved via intermediate stages contrasting only a light and a dark field during a simple visual task.The research leading to these results received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013)/European Research Council Grant Agreement 260821

A serial multiplex immunogold labeling method for identifying peptidergic neurons in connectomes.

This is the final version of the article.Available from eLife Sciences Publications via the DOI in this record.Electron microscopy-based connectomics aims to comprehensively map synaptic connections in neural tissue. However, current approaches are limited in their capacity to directly assign molecular identities to neurons. Here, we use serial multiplex immunogold labeling (siGOLD) and serial-section transmission electron microscopy (ssTEM) to identify multiple peptidergic neurons in a connectome. The high immunogenicity of neuropeptides and their broad distribution along axons, allowed us to identify distinct neurons by immunolabeling small subsets of sections within larger series. We demonstrate the scalability of siGOLD by using 11 neuropeptide antibodies on a full-body larval ssTEM dataset of the annelid Platynereis. We also reconstruct a peptidergic circuitry comprising the sensory nuchal organs, found by siGOLD to express pigment-dispersing factor, a circadian neuropeptide. Our approach enables the direct overlaying of chemical neuromodulatory maps onto synaptic connectomic maps in the study of nervous systems.The research leading to these results received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/European Research Council Grant Agreement 260821. This project is supported by the Marie Curie ITN "Neptune", GA 317172, funded under the FP7, PEOPLE Work Programme of the European Commission

Synaptic and peptidergic connectome of a neurosecretory centre in the annelid brain

This is the author accepted manuscript. The final version is available from eLife Sciences Publications via the DOI in this record.Neurosecretory centers in animal brains use peptidergic signaling to influence physiology and behavior. Understanding neurosecretory center function requires mapping cell types, synapses, and peptidergic networks. Here we use transmission electron microscopy and gene expression mapping to analyze the synaptic and peptidergic connectome of an entire neurosecretory center. We reconstructed 78 neurosecretory neurons and mapped their synaptic connectivity in the brain of larval Platynereis dumerilii, a marine annelid. These neurons form an anterior neurosecretory center expressing many neuropeptides, including hypothalamic peptide orthologs and their receptors. Analysis of peptide-receptor pairs in spatially mapped single-cell transcriptome data revealed sparsely connected networks linking specific neuronal subsets. We experimentally analyzed one peptide-receptor pair and found that a neuropeptide can couple neurosecretory and synaptic brain signaling. Our study uncovered extensive networks of peptidergic signaling within a neurosecretory center and its connection to the synaptic brain.The research leading to these results received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ European Research Council Grant Agreement 260821. The research was supported by a grant from the DFG - Deutsche Forschungsgemeinschaft (Reference no. JE 777/1)

КЛИНИЧЕСКИЙ СЛУЧАЙ ГРИБОВИДНОГО МИКОЗА

Mucosis fungoides (Sezary syndrome) is a rare disease. Because of polymorphism of clinical manifestations that mimic benign diseases of the skin, it often takes several years before the disease is diagnosed. The paper presents a clinical case of progressive mucosis fungoides with unfavorable outcome. The diagnosis of mycosis fungoides was first suspected on the basis of laboratory studies of peripheral blood.Грибовидный микоз/синдром Сезари относится к редким заболеваниям. Из-за полиморфизма клинических проявлений, имитирующих доброкачественные заболевания кожи, нередко от момента появления первых симптомов до постановки диагноза проходит несколько лет. Представлен клинический случай прогрессирующего течения грибовидного микоза с неблагоприятным исходом. Диагноз грибовидного микоза впервые был заподозрен на основании лабораторного исследования периферической крови

High diversity in neuropeptide immunoreactivity patterns among three closely related species of Dinophilidae (Annelida).

This is the author accepted manuscript.The final version is available from Wiley via the DOI in this record.Neuropeptides are conserved metazoan signaling molecules, and represent useful markers for comparative investigations on the morphology and function of the nervous system. However, little is known about the variation of neuropeptide expression patterns across closely related species in invertebrate groups other than insects. In this study, we compare the immunoreactivity patterns of 14 neuropeptides in three closely related microscopic dinophilid annelids (Dinophilus gyrociliatus, D. taeniatus and Trilobodrilus axi). The brains of all three species were found to consist of around 700 somata, surrounding a central neuropil with 3-5 ventral and 2-5 dorsal commissures. Neuropeptide immunoreactivity was detected in the brain, the ventral cords, stomatogastric nervous system, and additional nerves. Different neuropeptides are expressed in specific, non-overlapping cells in the brain in all three species. FMRFamide, MLD/pedal peptide, allatotropin, RNamide, excitatory peptide, and FVRIamide showed a broad localization within the brain, while calcitonin, SIFamide, vasotocin, RGWamide, DLamide, FLamide, FVamide, MIP, and serotonin were present in fewer cells in demarcated regions. The different markers did not reveal ganglionic subdivisions or physical compartmentalization in any of these microscopic brains. The non-overlapping expression of different neuropeptides may indicate that the regionalization in these uniform, small brains is realized by individual cells, rather than cell clusters, representing an alternative to the lobular organization observed in several macroscopic annelids. Furthermore, despite the similar gross brain morphology, we found an unexpectedly high variation in the expression patterns of neuropeptides across species. This suggests that neuropeptide expression evolves faster than morphology, representing a possible mechanism for the evolutionary divergence of behaviors.Villum Fonde

The evolutionary origin of bilaterian smooth and striated myocytes

The dichotomy between smooth and striated myocytes is fundamental for bilaterian musculature, but its evolutionary origin is unsolved. In particular, interrelationships of visceral smooth muscles remain unclear. Absent in fly and nematode, they have not yet been characterized molecularly outside vertebrates. Here, we characterize expression profile, ultrastructure, contractility and innervation of the musculature in the marine annelid Platynereis dumerilii and identify smooth muscles around the midgut, hindgut and heart that resemble their vertebrate counterparts in molecular fingerprint, contraction speed and nervous control. Our data suggest that both visceral smooth and somatic striated myocytes were present in the protostome-deuterostome ancestor and that smooth myocytes later co-opted the striated contractile module repeatedly for example, in vertebrate heart evolution. During these smooth-to-striated myocyte conversions, the core regulatory complex of transcription factors conveying myocyte identity remained unchanged, reflecting a general principle in cell type evolutio