Primary cilia are required for vertebrate cells to respond to specific intercellular signals. Here we define when and where primary cilia appear in the mouse embryo using a transgenic line that ...expresses ARL13B-mCherry in cilia and Centrin 2-GFP in centrosomes. Primary cilia first appear on cells of the epiblast at E6.0 and are subsequently present on all derivatives of the epiblast. In contrast, extraembryonic cells of the visceral endoderm and trophectoderm lineages have centrosomes but no cilia. Stem cell lines derived from embryonic lineages recapitulate the in vivo pattern: epiblast stem cells are ciliated, whereas trophoblast stem cells and extraembryonic endoderm (XEN) stem cells lack cilia. Basal bodies in XEN cells are mature and can form cilia when the AURKA-HDAC6 cilium disassembly pathway is inhibited. The lineage-dependent distribution of cilia is stable throughout much of gestation, defining which cells in the placenta and yolk sac are able to respond to Hedgehog ligands.
To explore the developmental reorganization of the three-dimensional genome of the brain in the context of neuropsychiatric disease, we monitored chromosomal conformations in differentiating neural ...progenitor cells. Neuronal and glial differentiation was associated with widespread developmental remodeling of the chromosomal contact map and included interactions anchored in common variant sequences that confer heritable risk for schizophrenia. We describe cell type-specific chromosomal connectomes composed of schizophrenia risk variants and their distal targets, which altogether show enrichment for genes that regulate neuronal connectivity and chromatin remodeling, and evidence for coordinated transcriptional regulation and proteomic interaction of the participating genes. Developmentally regulated chromosomal conformation changes at schizophrenia-relevant sequences disproportionally occurred in neurons, highlighting the existence of cell type-specific disease risk vulnerabilities in spatial genome organization.
The mechanisms by which common risk variants of small effect interact to contribute to complex genetic disorders are unclear. Here, we apply a genetic approach, using isogenic human induced ...pluripotent stem cells, to evaluate the effects of schizophrenia (SZ)-associated common variants predicted to function as SZ expression quantitative trait loci (eQTLs). By integrating CRISPR-mediated gene editing, activation and repression technologies to study one putative SZ eQTL (FURIN rs4702) and four top-ranked SZ eQTL genes (FURIN, SNAP91, TSNARE1 and CLCN3), our platform resolves pre- and postsynaptic neuronal deficits, recapitulates genotype-dependent gene expression differences and identifies convergence downstream of SZ eQTL gene perturbations. Our observations highlight the cell-type-specific effects of common variants and demonstrate a synergistic effect between SZ eQTL genes that converges on synaptic function. We propose that the links between rare and common variants implicated in psychiatric disease risk constitute a potentially generalizable phenomenon occurring more widely in complex genetic disorders.
Schizophrenia (SZ) is a psychiatric disorder with complex genetic risk dictated by interactions between hundreds of risk variants. Epigenetic factors, such as histone posttranslational modifications ...(PTMs), have been shown to play critical roles in many neurodevelopmental processes, and when perturbed may also contribute to the precipitation of disease. Here, we apply an unbiased proteomics approach to evaluate combinatorial histone PTMs in human induced pluripotent stem cell (hiPSC)-derived forebrain neurons from individuals with SZ. We observe hyperacetylation of H2A.Z and H4 in neurons derived from SZ cases, results that were confirmed in postmortem human brain. We demonstrate that the bromodomain and extraterminal (BET) protein, BRD4, is a bona fide 'reader' of H2A.Z acetylation, and further provide evidence that BET family protein inhibition ameliorates transcriptional abnormalities in patient-derived neurons. Thus, treatments aimed at alleviating BET protein interactions with hyperacetylated histones may aid in the prevention or treatment of SZ.
Mouse embryonic stem cells (mESCs) are key tools for genetic engineering, development of stem cell-based therapies and basic research on pluripotency and early lineage commitment. However, successful ...derivation of germline-competent embryonic stem cell lines has, until recently, been limited to a small number of inbred mouse strains. Recently, there have been considerable advances in the field of embryonic stem cell biology, particularly in the area of pluripotency maintenance in the epiblast from which the mESCs are derived. Here we describe a protocol for efficient derivation of germline-competent mESCs from any mouse strain, including strains previously deemed nonpermissive. We provide a protocol that is generally applicable to most inbred strains, as well as a variant for nonpermissive strains. By using this protocol, mESCs can be derived in 3 weeks and fully characterized after an additional 12 weeks, at efficiencies as high as 90% and in any strain background.
The mechanisms by which genetic risk variants interact with each other, as well as environmental factors, to contribute to complex genetic disorders remain unclear. We describe in detail our recently ...published approach to resolve distinct additive and synergistic transcriptomic effects after combinatorial manipulation of genetic variants and/or chemical perturbagens. Although first developed for CRISPR-based perturbation studies of isogenic human induced pluripotent stem cell-derived neurons, our methodology can be broadly applied to any RNA sequencing dataset, provided that raw read counts are available. Whereas other differential expression analyses reveal the effect of individual perturbations, here we specifically query interactions between two or more perturbagens, resolving the extent of non-additive (synergistic) interactions between perturbations. We discuss the careful experimental design required to resolve synergistic effects and considerations of statistical power and how to quantify observed synergy between experiments. Additionally, we speculate on potential future applications and explore the obvious limitations of this approach. Overall, by interrogating the effect of independent factors, alone and in combination, our analytic framework and experimental design facilitate the discovery of convergence and synergy downstream of gene and/or treatment perturbations hypothesized to contribute to complex diseases. We think that this protocol can be successfully applied by any scientist with bioinformatic skills and basic proficiency in the R programming language. Our computational pipeline ( https://github.com/nadschro/synergy-analysis ) is straightforward, does not require supercomputing support and can be conducted in a single day upon completion of RNA sequencing experiments.
To identify the molecular mechanisms and novel therapeutic targets of late-onset Alzheimer’s Disease (LOAD), we performed an integrative network analysis of multi-omics profiling of four cortical ...areas across 364 donors with varying cognitive and neuropathological phenotypes. Our analyses revealed thousands of molecular changes and uncovered neuronal gene subnetworks as the most dysregulated in LOAD. ATP6V1A was identified as a key regulator of a top-ranked neuronal subnetwork, and its role in disease-related processes was evaluated through CRISPR-based manipulation in human induced pluripotent stem cell-derived neurons and RNAi-based knockdown in Drosophila models. Neuronal impairment and neurodegeneration caused by ATP6V1A deficit were improved by a repositioned compound, NCH-51. This study provides not only a global landscape but also detailed signaling circuits of complex molecular interactions in key brain regions affected by LOAD, and the resulting network models will serve as a blueprint for developing next-generation therapeutic agents against LOAD.
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•Development of gene network models of four cortical areas affected by LOAD•Identification of region-specific molecular changes and gene subnetworks in LOAD•ATP6V1A is a top key regulator of a neuronal subnetwork most disrupted in LOAD•NCH-51 normalizes neuronal impairment and neurodegeneration caused by ATP6V1A deficit
Employing an integrative network biology approach, Wang et al. identify critical gene subnetworks associated with late-onset Alzheimer’s disease (LOAD) and predict ATP6V1A as a key regulator of a neuron-specific subnetwork most affected by LOAD. ATP6V1A deficit causes neuronal impairment and neurodegeneration, which are normalized by a predicted compound, NCH-51.
Neuropsychiatric disorders are highly heritable polygenic disorders arising from the complex interplay of highly penetrant rare variants and common variants of small effect. There is a large index of ...comorbidity and shared genetic risk between disorders, reflecting the pleiotropy of individual variants as well as predicted downstream pathway-level convergence. Importantly, the mechanism(s) through which psychiatric disease-associated variants interact to contribute to disease risk remains unknown. Human induced pluripotent stem cell (hiPSC)-based models are increasingly useful for the systematic study of the complex genetics associated with brain diseases, particularly when combined with CRISPR-mediated genomic engineering, which together facilitate isogenic comparisons of defined neuronal cell types. In this review, we discuss the latest CRISPR technologies and consider how they can be successfully applied to the functional characterization of the growing list genetic variants linked to psychiatric disease.
•An introduction to the complex genetic architectures of neuropsychiatric disease.•A review of state-of-the-art CRISPR mediated perturbations.•Applications of hiPSC- and CRISPR-based methodologies to for functional validation of neuropsychiatric disease risk loci.
Live imaging provides an essential methodology for understanding complex and dynamic cell behaviors and their underlying molecular mechanisms. Genetically-encoded reporter expressing mouse strains ...are an important tool for use in live imaging experiments. Such reporter strains can be engineered by placing cis-regulatory elements of interest to direct the expression of desired reporter genes. If these cis-regulatory elements are downstream targets, and thus activated as a consequence of signaling pathway activation, such reporters can provide read-outs of the signaling status of a cell. The Notch signaling pathway is an evolutionary conserved pathway operating in multiple developmental processes as well as being the basis for several congenital diseases. The transcription factor CBF1 is a central evolutionarily conserved component of the Notch signaling pathway. It binds the active form of the Notch receptor (NICD) and subsequently binds to cis-regulatory regions (CBF1 binding sites) in the promoters of Notch responsive genes. In this way, CBF1 binding sites represent a good target for the design of a Notch signaling reporter.
To generate a single-cell resolution Notch signaling reporter, we used a CBF responsive element to direct the expression of a nuclear-localized fluorescent protein. To do this, we linked 4 copies of a consensus CBF1 binding site to the basal simian virus 40 (SV40) promoter, placed this cassette in front of a fluorescent protein fusion comprising human histone H2B linked to the yellow fluorescent protein (YFP) Venus, one of the brightest available YFPs. We used the CBF:H2B-Venus construct to generate both transgenic embryonic mouse stem (ES) cell lines and a strain of transgenic mice that would report Notch signaling activity.
By using multiple CBF1 binding sites together with a subcellular-localized, genetically-encoded fluorescent protein, H2B-Venus, we have generated a transgenic strain of mice that faithfully recapitulates Notch signaling at single-cell resolution. This is the first mouse reporter strain in which individual cells transducing a Notch signal can be visualized. The improved resolution of this reporter makes it ideal for live imaging developmental processes regulated by the Notch signaling pathway as well as a short-term lineage tracer of Notch expressing cells due to the perdurance of the fluorescent reporter. Taken together, the CBF:H2B-Venus mouse strain is a unique tool to study and understand the morphogenetic events regulated by the Notch signaling pathway.
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