A Statistically Representative Atlas for Mapping Neuronal Circuits in the Drosophila Adult Brain

Published: 23 March 2018The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fninf.2018.00013/full#supplementary-material Supplementary Figure 1. 3D renderings of the 14 regions used for quantitative evaluation of atlas performances in segmentation and registration tasks. The 14 regions shown here were extracted from the atlas of Ito et al. (2014) that has been registered onto the group-wise inter-sex atlas (available from http://fruitfly.tefor.net). Supplementary Figure 2. Selected lines from the Janelia Farm collection showing an overlap value with the search pattern ranking among the first 50 for at least three of the five PDF profiles. (Left) GAL4-driven GFP profile registered on the standard brain. (Right) overlap between the first PDF profile and the GAL4-driven GFP profile. Numbers refer to Janelia Farm lines with associated gene names. Scale bar: 20 μm. Supplementary Table 1. Results of the 3D space query for each of the five PDF profiles. Overlap values are indicated for each Janelia Farm line and the corresponding gene name (FlyBase nomenclature) is indicated for the overlap values ranking among the first 50 for at least three of the five PDF profiles (blue). Bold names correspond to the three lines shown in Figure 10. Supplementary Movie 1. Animated rendering of the group-wise inter-sex atlas. Successively: nc82 template image (2D sections then 3D volume rendering, opaque then transparent); label image (3D surface rendering of anatomical regions, defined following Ito et al. 2014); six registered patterns of GAL4-GFP expression (3D surface rendering of intensity-thresholded pattern images); same patterns (left half of the brain) with the anatomical regions (right half of the brain).Imaging the expression patterns of reporter constructs is a powerful tool to dissect the neuronal circuits of perception and behavior in the adult brain of Drosophila, one of the major models for studying brain functions. To date, several Drosophila brain templates and digital atlases have been built to automatically analyze and compare collections of expression pattern images. However, there has been no systematic comparison of performances between alternative atlasing strategies and registration algorithms. Here, we objectively evaluated the performance of different strategies for building adult Drosophila brain templates and atlases. In addition, we used state-of-the-art registration algorithms to generate a new group-wise inter-sex atlas. Our results highlight the benefit of statistical atlases over individual ones and show that the newly proposed inter-sex atlas outperformed existing solutions for automated registration and annotation of expression patterns. Over 3,000 images from the Janelia Farm FlyLight collection were registered using the proposed strategy. These registered expression patterns can be searched and compared with a new version of the BrainBaseWeb system and BrainGazer software. We illustrate the validity of our methodology and brain atlas with registration-based predictions of expression patterns in a subset of clock neurons. The described registration framework should benefit to brain studies in Drosophila and other insect species.IA-C, TM, NM, FS, and AJ were funded by the Tefor Infrastructure under the Investments for the Future program of the French National Research Agency (Grant #ANR-11-INBS-0014). FR was supported by INSERM. Work at Institut des Neurosciences Paris-Saclay was supported by ANR Infrastructure Tefor and by ANR ClockEye(#ANR-14-CE13-0034-01). JI was supported by the Spanish Ministry of Economy and Competitiveness (TEC2014-51882-P), the European Union's Horizon 2020 research and innovation programme (Marie Sklodowska-Curie grant 654911, project THALAMODEL), and the European Research Council (ERC Starting Grant no. 677697 BUNGEE-TOOLS). VRVis (KB, FS) is funded by BMVIT, BMWFW, Styria, SFG and Vienna Business Agency in the scope of COMET - Competence Centers for Excellent Technologies (854174) which is managed by FFG. The Institut Jean-Pierre Bourgin benefits from the support of the LabEx Saclay Plant Sciences-SPS (#ANR-10-LABX-0040-SPS)

Quality assessment in light microscopy for routine use through simple tools and robust metrics

International audienceAlthough there is a need to demonstrate reproducibility in light microscopy acquisitions, the lack of standardized guidelines monitoring microscope health status over time has so far impaired the widespread use of quality control (QC) measurements. As scientists from 10 imaging core facilities who encounter various types of projects, we provide affordable hardware and open source software tools, rigorous protocols, and define reference values to assess QC metrics for the most common fluorescence light microscopy modalities. Seven protocols specify metrics on the microscope resolution, field illumination flatness, chromatic aberrations, illumination power stability, stage drift, positioning repeatability, and spatial-temporal noise of camera sensors. We designed the MetroloJ_QC ImageJ/Fiji Java plugin to incorporate the metrics and automate analysis. Measurements allow us to propose an extensive characterization of the QC procedures that can be used by any seasoned microscope user, from research biologists with a specialized interest in fluorescence light microscopy through to core facility staff, to ensure reproducible and quantifiable microscopy results

Image1.PDF

<p>Imaging the expression patterns of reporter constructs is a powerful tool to dissect the neuronal circuits of perception and behavior in the adult brain of Drosophila, one of the major models for studying brain functions. To date, several Drosophila brain templates and digital atlases have been built to automatically analyze and compare collections of expression pattern images. However, there has been no systematic comparison of performances between alternative atlasing strategies and registration algorithms. Here, we objectively evaluated the performance of different strategies for building adult Drosophila brain templates and atlases. In addition, we used state-of-the-art registration algorithms to generate a new group-wise inter-sex atlas. Our results highlight the benefit of statistical atlases over individual ones and show that the newly proposed inter-sex atlas outperformed existing solutions for automated registration and annotation of expression patterns. Over 3,000 images from the Janelia Farm FlyLight collection were registered using the proposed strategy. These registered expression patterns can be searched and compared with a new version of the BrainBaseWeb system and BrainGazer software. We illustrate the validity of our methodology and brain atlas with registration-based predictions of expression patterns in a subset of clock neurons. The described registration framework should benefit to brain studies in Drosophila and other insect species.</p

Image2.PDF

<p>Imaging the expression patterns of reporter constructs is a powerful tool to dissect the neuronal circuits of perception and behavior in the adult brain of Drosophila, one of the major models for studying brain functions. To date, several Drosophila brain templates and digital atlases have been built to automatically analyze and compare collections of expression pattern images. However, there has been no systematic comparison of performances between alternative atlasing strategies and registration algorithms. Here, we objectively evaluated the performance of different strategies for building adult Drosophila brain templates and atlases. In addition, we used state-of-the-art registration algorithms to generate a new group-wise inter-sex atlas. Our results highlight the benefit of statistical atlases over individual ones and show that the newly proposed inter-sex atlas outperformed existing solutions for automated registration and annotation of expression patterns. Over 3,000 images from the Janelia Farm FlyLight collection were registered using the proposed strategy. These registered expression patterns can be searched and compared with a new version of the BrainBaseWeb system and BrainGazer software. We illustrate the validity of our methodology and brain atlas with registration-based predictions of expression patterns in a subset of clock neurons. The described registration framework should benefit to brain studies in Drosophila and other insect species.</p

Video1.mp4

<p>Imaging the expression patterns of reporter constructs is a powerful tool to dissect the neuronal circuits of perception and behavior in the adult brain of Drosophila, one of the major models for studying brain functions. To date, several Drosophila brain templates and digital atlases have been built to automatically analyze and compare collections of expression pattern images. However, there has been no systematic comparison of performances between alternative atlasing strategies and registration algorithms. Here, we objectively evaluated the performance of different strategies for building adult Drosophila brain templates and atlases. In addition, we used state-of-the-art registration algorithms to generate a new group-wise inter-sex atlas. Our results highlight the benefit of statistical atlases over individual ones and show that the newly proposed inter-sex atlas outperformed existing solutions for automated registration and annotation of expression patterns. Over 3,000 images from the Janelia Farm FlyLight collection were registered using the proposed strategy. These registered expression patterns can be searched and compared with a new version of the BrainBaseWeb system and BrainGazer software. We illustrate the validity of our methodology and brain atlas with registration-based predictions of expression patterns in a subset of clock neurons. The described registration framework should benefit to brain studies in Drosophila and other insect species.</p

Table1.XLS

<p>Imaging the expression patterns of reporter constructs is a powerful tool to dissect the neuronal circuits of perception and behavior in the adult brain of Drosophila, one of the major models for studying brain functions. To date, several Drosophila brain templates and digital atlases have been built to automatically analyze and compare collections of expression pattern images. However, there has been no systematic comparison of performances between alternative atlasing strategies and registration algorithms. Here, we objectively evaluated the performance of different strategies for building adult Drosophila brain templates and atlases. In addition, we used state-of-the-art registration algorithms to generate a new group-wise inter-sex atlas. Our results highlight the benefit of statistical atlases over individual ones and show that the newly proposed inter-sex atlas outperformed existing solutions for automated registration and annotation of expression patterns. Over 3,000 images from the Janelia Farm FlyLight collection were registered using the proposed strategy. These registered expression patterns can be searched and compared with a new version of the BrainBaseWeb system and BrainGazer software. We illustrate the validity of our methodology and brain atlas with registration-based predictions of expression patterns in a subset of clock neurons. The described registration framework should benefit to brain studies in Drosophila and other insect species.</p

Endothelial and hematopoietic hPSCs differentiation via a hematoendothelial progenitor

International audienceAbstract Background hPSC-derived endothelial and hematopoietic cells (ECs and HCs) are an interesting source of cells for tissue engineering. Despite their close spatial and temporal embryonic development, current hPSC differentiation protocols are specialized in only one of these lineages. In this study, we generated a hematoendothelial population that could be further differentiated in vitro to both lineages. Methods Two hESCs and one hiPSC lines were differentiated into a hematoendothelial population, hPSC-ECs and blast colonies (hPSC-BCs) via CD144 + -embryoid bodies (hPSC-EBs). hPSC-ECs were characterized by endothelial colony-forming assay, LDL uptake assay, endothelial activation by TNF-α, nitric oxide detection and Matrigel-based tube formation. Hematopoietic colony-forming cell assay was performed from hPSC-BCs. Interestingly, we identified a hPSC-BC population characterized by the expression of both CD144 and CD45. hPSC-ECs and hPSC-BCs were analyzed by flow cytometry and RT-qPCR; in vivo experiments have been realized by ischemic tissue injury model on a mouse dorsal skinfold chamber and hematopoietic reconstitution in irradiated immunosuppressed mouse from hPSC-ECs and hPSC-EB-CD144 + , respectively. Transcriptomic analyses were performed to confirm the endothelial and hematopoietic identity of hESC-derived cell populations by comparing them against undifferentiated hESC, among each other’s ( e.g. hPSC-ECs vs. hPSC-EB-CD144 + ) and against human embryonic liver (EL) endothelial, hematoendothelial and hematopoietic cell subpopulations. Results A hematoendothelial population was obtained after 84 h of hPSC-EBs formation under serum-free conditions and isolated based on CD144 expression. Intrafemorally injection of hPSC-EB-CD144 + contributed to the generation of CD45 + human cells in immunodeficient mice suggesting the existence of hemogenic ECs within hPSC-EB-CD144 + . Endothelial differentiation of hPSC-EB-CD144 + yields a population of > 95% functional ECs in vitro. hPSC-ECs derived through this protocol participated at the formation of new vessels in vivo in a mouse ischemia model. In vitro, hematopoietic differentiation of hPSC-EB-CD144 + generated an intermediate population of > 90% CD43 + hPSC-BCs capable to generate myeloid and erythroid colonies. Finally, the transcriptomic analyses confirmed the hematoendothelial, endothelial and hematopoietic identity of hPSC-EB-CD144 + , hPSC-ECs and hPSC-BCs, respectively, and the similarities between hPSC-BC-CD144 + CD45 + , a subpopulation of hPSC-BCs, and human EL hematopoietic stem cells/hematopoietic progenitors. Conclusion The present work reports a hPSC differentiation protocol into functional hematopoietic and endothelial cells through a hematoendothelial population. Both lineages were proven to display characteristics of physiological human cells, and therefore, they represent an interesting rapid source of cells for future cell therapy and tissue engineering