Cooperative interactions enable singular olfactory receptor expression in mouse olfactory neurons

The monogenic and monoallelic expression of only one out of >1000 mouse olfactory receptor (ORs) genes requires the formation of large heterochromatic chromatin domains that sequester the OR gene clusters. Within these domains, intergenic transcriptional enhancers evade heterochromatic silencing and converge into interchromosomal hubs that assemble over the transcriptionally active OR. The significance of this nuclear organization in OR choice remains elusive. Here, we show that transcription factors Lhx2 and Ebf specify OR enhancers by binding in a functionally cooperative fashion to stereotypically spaced motifs that defy heterochromatin. Specific displacement of Lhx2 and Ebf from OR enhancers resulted in pervasive, long-range, and trans downregulation of OR transcription, whereas pre-assembly of a multi-enhancer hub increased the frequency of OR choice in cis. Our data provide genetic support for the requirement and sufficiency of interchromosomal interactions in singular OR choice and generate general regulatory principles for stochastic, mutually exclusive gene expression programs

The choroid plexus and cerebrospinal fluid: Emerging roles in development, disease, and therapy

Although universally recognized as the source of cerebrospinal fluid (CSF), the choroid plexus (ChP) has been one of the most understudied tissues in neuroscience. The reasons for this are multiple and varied, including historical perceptions about passive and permissive roles for the ChP, experimental issues, and lack of clinical salience. However, recent work on the ChP and instructive signals in the CSF have sparked new hypotheses about how the ChP and CSF provide unexpected means for regulating nervous system structure and function in health and disease, as well as new ChP-based therapeutic approaches using pluripotent stem cell technology. This minisymposium combines new and established investigators to capture some of the newfound excitement surrounding the ChP-CSF system

A hybrid microfluidic-vacuum device for direct interfacing with conventional cell culture methods

<p>Abstract</p> <p>Background</p> <p>Microfluidics is an enabling technology with a number of advantages over traditional tissue culture methods when precise control of cellular microenvironment is required. However, there are a number of practical and technical limitations that impede wider implementation in routine biomedical research. Specialized equipment and protocols required for fabrication and setting up microfluidic experiments present hurdles for routine use by most biology laboratories.</p> <p>Results</p> <p>We have developed and validated a novel microfluidic device that can directly interface with conventional tissue culture methods to generate and maintain controlled soluble environments in a Petri dish. It incorporates separate sets of fluidic channels and vacuum networks on a single device that allows reversible application of microfluidic gradients onto wet cell culture surfaces. Stable, precise concentration gradients of soluble factors were generated using simple microfluidic channels that were attached to a perfusion system. We successfully demonstrated real-time optical live/dead cell imaging of neural stem cells exposed to a hydrogen peroxide gradient and chemotaxis of metastatic breast cancer cells in a growth factor gradient.</p> <p>Conclusion</p> <p>This paper describes the design and application of a versatile microfluidic device that can directly interface with conventional cell culture methods. This platform provides a simple yet versatile tool for incorporating the advantages of a microfluidic approach to biological assays without changing established tissue culture protocols.</p

Parsing patterns: emerging roles of tissue self-organization in health and disease

Patterned morphologies, such as segments, spirals, stripes, and spots, frequently emerge during embryogenesis through self-organized coordination between cells. Yet, complex patterns also emerge in adults, suggesting that the capacity for spontaneous self-organization is a ubiquitous property of biological tissues. We review current knowledge on the principles and mechanisms of self-organized patterning in embryonic tissues and explore how these principles and mechanisms apply to adult tissues that exhibit features of patterning. We discuss how and why spontaneous pattern generation is integral to homeostasis and healing of tissues, illustrating it with examples from regenerative biology. We examine how aberrant self-organization underlies diverse pathological states, including inflammatory skin disorders and tumors. Lastly, we posit that based on such blueprints, targeted engineering of pattern-driving molecular circuits can be leveraged for synthetic biology and the generation of organoids with intricate patterns

Human neural stem cell growth and differentiation in a gradient-generating microfluidic device

This paper describes a gradient-generating microfluidic platform for optimizing proliferation and differentiation of neural stem cells (NSCs) in culture. Microfluidic technology has great potential to improve stem cell (SC) cultures, whose promise in cell–based therapies is limited by the inability to precisely control their behavior in culture. Compared to traditional culture tools, microfluidic platforms should provide much greater control over cell microenvironment and rapid optimization of media composition using relatively small numbers of cells. Our platform exposes cells to a concentration gradient of growth factors under continuous flow, thus minimizing autocrine and paracrine signaling. Human NSCs (hNSCs) from the developing cerebral cortex were cultured for more than 1 week in the microfluidic device while constantly exposed to a continuous gradient of a growth factor (GF) mixture containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF). Proliferation and differentiation of NSCs into astrocytes were monitored by time-lapse microscopy and immunocytochemistry. The NSCs remained healthy throughout the entire culture period, and importantly, proliferated and differentiated in a graded and proportional fashion that varied directly with GF concentration. These concentration-dependent cellular responses were quantitatively similar to those measured in control chambers built into the device and in parallel cultures using traditional 6-well plates. This gradient-generating microfluidic platform should be useful for a wide range of basic and applied studies on cultured cells, including SCs.This paper was supported by seed grants from UCI CORCLR, UCI College of Medicine/Biomedical Engineering, and a Roman Reed Research Award from the State of California

BMP4 Sufficiency to Induce Choroid Plexus Epithelial Fate from Embryonic Stem Cell-Derived Neuroepithelial Progenitors

Choroid plexus epithelial cells (CPECs) have essential developmental and homeostatic roles related to the cerebrospinal fluid (CSF) and blood-CSF barrier they produce. Accordingly, CPEC dysfunction has been implicated in many neurological disorders, such as Alzheimer’s disease, and transplant studies have provided proof-of-concept for CPEC-based therapies. However, such therapies have been hindered by the inability to expand or generate CPECs in culture. During development, CPECs differentiate from preneurogenic neuroepithelial cells and require Bone Morphogenetic Protein (BMP) signaling, but whether BMPs suffice for CPEC induction is unknown. Here we provide evidence for BMP4 sufficiency to induce CPEC fate from neural progenitors derived from mouse embryonic stem cells (ESCs). CPEC specification by BMP4 was restricted to an early time period after neural induction in culture, with peak CPEC competency correlating to neuroepithelial cells rather than radial glia. In addition to molecular, cellular, and ultrastructural criteria, derived CPECs (dCPECs) had functions that were indistinguishable from primary CPECs, including self-assembly into secretory vesicles and integration into endogenous choroid plexus epithelium following intraventricular injection. We then used BMP4 to generate dCPECs from human ESC-derived neuroepithelial cells. These findings demonstrate BMP4 sufficiency to instruct CPEC fate, expand the repertoire of stem cell-derived neural derivatives in culture, and herald dCPEC-based therapeutic applications aimed at the unique interface between blood, CSF, and brain governed by CPECs

Histone H2A ubiquitination resulting from Brap loss of function connects multiple aging hallmarks and accelerates neurodegeneration

Aging is an intricate process characterized by multiple hallmarks including stem cell exhaustion, genome instability, epigenome alteration, impaired proteostasis, and cellular senescence. Whereas each of these traits is detrimental at the cellular level, it remains unclear how they are interconnected to cause systemic organ deterioration. Here we show that abrogating Brap, a BRCA1-associated protein essential for neurogenesis, results in persistent DNA double-strand breaks and elevation of histone H2A mono- and poly-ubiquitination (H2Aub). These defects extend to cellular senescence and proteasome-mediated histone H2A proteolysis with alterations in cells' proteomic and epigenetic states. Brap deletion in the mouse brain causes neuroinflammation, impaired proteostasis, accelerated neurodegeneration, and substantially shortened the lifespan. We further show the elevation of H2Aub also occurs in human brain tissues with Alzheimer's disease. These data together suggest that chromatin aberrations mediated by H2Aub may act as a nexus of multiple aging hallmarks and promote tissue-wide degeneration

Genome sequencing analysis identifies new loci associated with Lewy body dementia and provides insights into its genetic architecture

The genetic basis of Lewy body dementia (LBD) is not well understood. Here, we performed whole-genome sequencing in large cohorts of LBD cases and neurologically healthy controls to study the genetic architecture of this understudied form of dementia and to generate a resource for the scientific community. Genome-wide association analysis identified five independent risk loci, whereas genome-wide gene-aggregation tests implicated mutations in the gene GBA. Genetic risk scores demonstrate that LBD shares risk profiles and pathways with Alzheimer’s and Parkinson’s disease, providing a deeper molecular understanding of the complex genetic architecture of this age-related neurodegenerative condition

Genome sequencing analysis identifies new loci associated with Lewy body dementia and provides insights into its genetic architecture

The genetic basis of Lewy body dementia (LBD) is not well understood. Here, we performed whole-genome sequencing in large cohorts of LBD cases and neurologically healthy controls to study the genetic architecture of this understudied form of dementia, and to generate a resource for the scientific community. Genome-wide association analysis identified five independent risk loci, whereas genome-wide gene-aggregation tests implicated mutations in the gene GBA. Genetic risk scores demonstrate that LBD shares risk profiles and pathways with Alzheimer's disease and Parkinson's disease, providing a deeper molecular understanding of the complex genetic architecture of this age-related neurodegenerative condition

Genome-wide structural variant analysis identifies risk loci for non-Alzheimer’s dementias

We characterized the role of structural variants, a largely unexplored type of genetic variation, in two non-Alzheimer’s dementias, namely Lewy body dementia (LBD) and frontotemporal dementia (FTD)/amyotrophic lateral sclerosis (ALS). To do this, we applied an advanced structural variant calling pipeline (GATK-SV) to short-read whole-genome sequence data from 5,213 European-ancestry cases and 4,132 controls. We discovered, replicated, and validated a deletion in TPCN1 as a novel risk locus for LBD and detected the known structural variants at the C9orf72 and MAPT loci as associated with FTD/ALS. We also identified rare pathogenic structural variants in both LBD and FTD/ALS. Finally, we assembled a catalog of structural variants that can be mined for new insights into the pathogenesis of these understudied forms of dementia