Precise segmentation of densely interweaving neuron clusters using G-Cut

脑是宇宙间最为复杂的系统之一，成人的脑中有约1000亿个神经元，单个神经元通常与其它神经元有成千上万个“突触”连接节点，形成拥有百万亿级连接的极其复杂的脑神经网络。当前多数神经元三维重建和分析工具仅适用于单个神经元的形态学重建，难以从神经元簇图像中正确追踪重建出多个神经元，而神经元的重建质量又影响到量化分析神经元的形态学特征及其功能。针对这一问题，课题组提出一种新的三维神经元簇重建工具G-Cut。具体地，为了度量神经元胞体与神经突起间的关联性，课题组从已有的带有标注的大规模神经元形态学数据集统计分析得到其规律和形态学信息。然后将神经元簇的重建问题转化为神经突起之间连接所形成的拓扑连接图的图分割问题，并结合神经元形态学规律和信息，在所有的神经突起与神经元胞体的关联性中寻找重建问题的最优解。通过在不同的合成数据集以及真实的脑组织图像数据集上测试，和已有的方法相比，G-Cut在不同密度和不同规模的神经元簇图像上均获得了更高的重建正确率。该项研究工作由厦门大学，南加州大学，加州大学洛杉矶分校等高校课题组合作完成，厦门大学信息学院智能科学与技术系为第一完成单位，厦门大学博士生李睿和USC博士生Muye Zhu为论文共同第一作者，张俊松博士和南加州大学的Hong-Wei Dong教授为论文共同通讯作者。厦门大学周昌乐教授和南加州大学的Arthur Toga教授为研究提供了大力支持。【Abstract】Characterizing the precise three-dimensional morphology and anatomical context of neurons is crucial for neuronal cell type classification and circuitry mapping. Recent advances in tissue clearing techniques and microscopy make it possible to obtain image stacks of intact, interweaving neuron clusters in brain tissues. As most current 3D neuronal morphology reconstruction methods are only applicable to single neurons, it remains challenging to reconstruct these clusters digitally. To advance the state of the art beyond these challenges, we propose a fast and robust method named G-Cut that is able to automatically segment individual neurons from an interweaving neuron cluster. Across various densely interconnected neuron clusters, G-Cut achieves significantly higher accuracies than other state-of-the-art algorithms. G-Cut is intended as a robust component in a high throughput informatics pipeline for large-scale brain mapping projects.This work was supported by NIH/NIMH MH094360-01A1 (H.W.D.), MH094360-06 (H.W.D.), NIH/NCI U01CA198932-01 (H.W.D.), NIH/NIMH MH106008 (X.W.Y. and H.W.D.), National Nature Science Foundation of China No. 61772440 (J.S.Z.), and National Basic Research Program of China 2013CB329502 (J.S.Z. and C.L.Z.). We thank a support of Graduate Student International Exchange Project of Xiamen University to R.L. and State Scholarship Fund of China Scholarship Council (No. 201406315023) to J.S.Z. 该项研究得到国家自然科学基金、国家重点基础研究发展计划973项目、国家留学基金、厦门大学研究生国际交流项目、美国脑计划和NIH等课题资助

Biochemical properties of Paracoccus denitrificans FnrP:Reactions with molecular oxygen and nitric oxide

In Paracoccus denitrificans, three CRP/FNR family regulatory proteins, NarR, NnrR and FnrP, control the switch between aerobic and anaerobic (denitrification) respiration. FnrP is a [4Fe-4S] cluster containing homologue of the archetypal O2 sensor FNR from E. coli and accordingly regulates genes encoding aerobic and anaerobic respiratory enzymes in response to O2, and also NO, availability. Here we show that FnrP undergoes O2-driven [4Fe-4S] to [2Fe-2S] cluster conversion that involves up to 2 O2 per cluster, with significant oxidation of released cluster sulfide to sulfane observed at higher O2 concentrations. The rate of the cluster reaction was found to be ~6-fold lower than that of E. coli FNR, suggesting that FnrP can remain transcriptionally active under microaerobic conditions. This is consistent with a role for FnrP in activating expression of the high O2 affinity cytochrome c oxidase under microaerobic conditions. Cluster conversion resulted in dissociation of the transcriptionally active FnrP dimer into monomers. Therefore, along with E. coli FNR, FnrP belongs to the subset of FNR proteins in which cluster type is correlated with association state. Interestingly, two key charged residues, Arg140 and Asp154, that have been shown to play key roles in the monomer-dimer equilibrium in E. coli FNR are not conserved in FnrP, indicating that different protomer interactions are important for this equilibrium. Finally, the FnrP [4Fe-4S] cluster is shown to undergo reaction with multiple NO molecules, resulting in iron nitrosyl species and dissociation into monomers

Natural climate solutions for the United States

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Science Advances 4 (2018): eaat1869, doi:10.1126/sciadv.aat1869.Limiting climate warming to <2°C requires increased mitigation efforts, including land stewardship, whose potential in the United States is poorly understood. We quantified the potential of natural climate solutions (NCS)—21 conservation, restoration, and improved land management interventions on natural and agricultural lands—to increase carbon storage and avoid greenhouse gas emissions in the United States. We found a maximum potential of 1.2 (0.9 to 1.6) Pg CO2e year−1, the equivalent of 21% of current net annual emissions of the United States. At current carbon market prices (USD 10 per Mg CO2e), 299 Tg CO2e year−1 could be achieved. NCS would also provide air and water filtration, flood control, soil health, wildlife habitat, and climate resilience benefits.This study was made possible by funding from the Doris Duke Charitable Foundation. C.A.W. and H.G. acknowledge financial support from NASA’s Carbon Monitoring System program (NNH14ZDA001N-CMS) under award NNX14AR39G. S.D.B. acknowledges support from the DOE’s Office of Biological and Environmental Research Program under the award DE-SC0014416. J.W.F. acknowledges financial support from the Florida Coastal Everglades Long-Term Ecological Research program under National Science Foundation grant no. DEB-1237517

Identification of Vascular and Hematopoietic Genes Downstream of etsrp by Deep Sequencing in Zebrafish

The transcription factor etsrp/Er71/Etv2 is a master control gene for vasculogenesis in all species studied to date. It is also required for hematopoiesis in zebrafish and mice. Several novel genes expressed in vasculature have been identified through transcriptional profiling of zebrafish embryos overexpressing etsrp by microarrays. Here we re-examined this transcriptional profile by Illumina RNA-sequencing technology, revealing a substantially increased number of candidate genes regulated by etsrp. Expression studies of 50 selected candidate genes from this dataset resulted in the identification of 39 new genes that are expressed in vascular cells. Regulation of these genes by etsrp was confirmed by their ectopic induction in etsrp overexpressing and decreased expression in etsrp deficient embryos. Our studies demonstrate the effectiveness of the RNA-sequencing technology to identify biologically relevant genes in zebrfish and produced a comprehensive profile of genes previously unexplored in vascular endothelial cell biology

Discovery and Characterization of Novel Vascular and Hematopoietic Genes Downstream of Etsrp in Zebrafish

The transcription factor Etsrp is required for vasculogenesis and primitive myelopoiesis in zebrafish. When ectopically expressed, etsrp is sufficient to induce the expression of many vascular and myeloid genes in zebrafish. The mammalian homolog of etsrp, ER71/Etv2, is also essential for vascular and hematopoietic development. To identify genes downstream of etsrp, gain-of-function experiments were performed for etsrp in zebrafish embryos followed by transcription profile analysis by microarray. Subsequent in vivo expression studies resulted in the identification of fourteen genes with blood and/or vascular expression, six of these being completely novel. Regulation of these genes by etsrp was confirmed by ectopic induction in etsrp overexpressing embryos and decreased expression in etsrp deficient embryos. Additional functional analysis of two newly discovered genes, hapln1b and sh3gl3, demonstrates their importance in embryonic vascular development. The results described here identify a group of genes downstream of etsrp likely to be critical for vascular and/or myeloid development

Mutations in Zebrafish lrp2 Result in Adult-Onset Ocular Pathogenesis That Models Myopia and Other Risk Factors for Glaucoma

The glaucomas comprise a genetically complex group of retinal neuropathies that typically occur late in life and are characterized by progressive pathology of the optic nerve head and degeneration of retinal ganglion cells. In addition to age and family history, other significant risk factors for glaucoma include elevated intraocular pressure (IOP) and myopia. The complexity of glaucoma has made it difficult to model in animals, but also challenging to identify responsible genes. We have used zebrafish to identify a genetically complex, recessive mutant that shows risk factors for glaucoma including adult onset severe myopia, elevated IOP, and progressive retinal ganglion cell pathology. Positional cloning and analysis of a non-complementing allele indicated that non-sense mutations in low density lipoprotein receptor-related protein 2 (lrp2) underlie the mutant phenotype. Lrp2, previously named Megalin, functions as an endocytic receptor for a wide-variety of bioactive molecules including Sonic hedgehog, Bone morphogenic protein 4, retinol-binding protein, vitamin D-binding protein, and apolipoprotein E, among others. Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals—but not all—develop high IOP and severe myopia with obviously enlarged eye globes. This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis. Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease

Morphological diversity of single neurons in molecularly defined cell types.

Dendritic and axonal morphology reflects the input and output of neurons and is a defining feature of neuronal types1,2, yet our knowledge of its diversity remains limited. Here, to systematically examine complete single-neuron morphologies on a brain-wide scale, we established a pipeline encompassing sparse labelling, whole-brain imaging, reconstruction, registration and analysis. We fully reconstructed 1,741 neurons from cortex, claustrum, thalamus, striatum and other brain regions in mice. We identified 11 major projection neuron types with distinct morphological features and corresponding transcriptomic identities. Extensive projectional diversity was found within each of these major types, on the basis of which some types were clustered into more refined subtypes. This diversity follows a set of generalizable principles that govern long-range axonal projections at different levels, including molecular correspondence, divergent or convergent projection, axon termination pattern, regional specificity, topography, and individual cell variability. Although clear concordance with transcriptomic profiles is evident at the level of major projection type, fine-grained morphological diversity often does not readily correlate with transcriptomic subtypes derived from unsupervised clustering, highlighting the need for single-cell cross-modality studies. Overall, our study demonstrates the crucial need for quantitative description of complete single-cell anatomy in cell-type classification, as single-cell morphological diversity reveals a plethora of ways in which different cell types and their individual members may contribute to the configuration and function of their respective circuits

A multimodal cell census and atlas of the mammalian primary motor cortex

ABSTRACT We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties

Mechanisms underlying optic nerve regeneration in zebrafish.

Following injury to the central nervous system (CNS), adult mammals are largely unable to regenerate damaged axons resulting in permanent loss of function. Zebrafish, on the other hand, can robustly regenerate damaged CNS axons and regain lost function. This model organism provides an opportunity to study the cellular and molecular mechanisms necessary for successful CNS regeneration. The identification of gene regulatory mechanisms active during successful nerve regeneration may identify targets for therapeutic enhancement of mammalian regeneration. Previously, our lab generated transgennc zebrafish harboring the alpha1 tubulin promoter driving green fluorescent protein expression. This transgene, like the endogenous alpha1 tubulin gene, is specifically expressed in the developing CNS, repressed in the adult CNS, and re-induced during nerve regeneration. Using this alpha1 tubulin promoter as a probe we have identified an enhancer that is necessary for alpha1 tubulin promoter activation following optic nerve injury. Transgenic zebrafish harboring deletions or mutations in the alpha1 tubulin promoter identified a G/C-rich element within this enhancer that is necessary, but not sufficient, for alpha1 tubulin promoter induction during optic nerve regeneration. Electrophoretic mobility supershift assays suggested Sp/KLF transcription factors could to bind the G/C-rich element. Zebrafish DNA microarrays were used to investigate the identity of specific Sp/KLF family members that were induced during optic nerve regeneration. Hundreds of genes, including the Sp/KLF family members KLF6a and KLF7a, were identified. KLF6a and KLF7a are both able to bind to the alpha1 tubulin promoter and transactivate its expression. Morpholino antisense oligos were used to knockdown KLF6a and KLF7a expression in adult zebrafish RGCs. Knockdown of these proteins decreases alpha1 tubulin promoter transgene expression in RGCs during optic nerve regeneration. In addition, retinal explants from KLF6a and KLF7a morpholino treated eyes demonstrated reduced axon regeneration as comoared to controls. These studies establish KLF6a and KLF7a as important transcriptional regulators of optic axon regeneration and provide an approach for investigating the requirement for additional regeneration associated genes in successful re-growth of damaged optic axons.Ph.D.Biological SciencesNeurosciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126842/2/3276317.pd