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To form functional circuits, neurons must settle in their appropriate cellular locations and then project and elaborate neurites to contact their target synaptic neuropils. Laminar organization within the vertebrate retinal inner plexiform layer (IPL) facilitates pre- and postsynaptic neurite targeting, yet the precise mechanisms underlying establishment of functional IPL subdomains are not well understood. Here we explore mechanisms defining the compartmentalization of OFF and ON neurites generally, and OFF and ON direction-selective neurites specifically, within the developing IPL. We show that semaphorin 6A (Sema6A), a repulsive axon guidance cue, is required for delineation of OFF versus ON circuits within the IPL: in the Sema6a null IPL, the boundary between OFF and ON domains is blurred. Furthermore, Sema6A expressed by retinal ganglion cells (RGCs) directs laminar segregation of OFF and ON starburst amacrine cell (SAC) dendritic scaffolds, which themselves serve as a substrate upon which other retinal neurites elaborate. These results demonstrate that RGCs, the first neuron-type born within the retina, play an active role in functional specialization of the IPL., (© 2024. Published by The Company of Biologists Ltd.)

Oligodendrocyte progenitor cells (OPCs) are highly dynamic, widely distributed glial cells of the central nervous system (CNS) that are responsible for generating myelinating oligodendrocytes during development. By also generating new oligodendrocytes in the adult CNS, OPCs allow formation of new myelin sheaths in response to environmental and behavioral changes and play a crucial role in regenerating myelin following demyelination (remyelination). However, the rates of OPC proliferation and differentiation decline dramatically with aging, which may impair homeostasis, remyelination, and adaptive myelination during learning. To determine how aging influences OPCs, we generated a novel transgenic mouse line that expresses membrane-anchored EGFP under the endogenous promoter/enhancer of Matrilin-4 ( Matn4-mEGFP ) and performed high-throughput single-cell RNA sequencing, providing enhanced resolution of transcriptional changes during key transitions from quiescence to proliferation and differentiation across the lifespan. Comparative analysis of OPCs isolated from mice aged 30 to 720 days, revealed that aging induces distinct inflammatory transcriptomic changes in OPCs in different states, including enhanced activation of HIF-1α and Wnt pathways. Inhibition of these pathways in acutely isolated OPCs from aged animals restored their ability to differentiate, suggesting that this enhanced signaling may contribute to the decreased regenerative potential of OPCs with aging. This Matn4-mEGFP mouse line and single-cell mRNA datasets of cortical OPCs across ages help to define the molecular changes guiding their behavior in various physiological and pathological contexts.

Background: Sweat chloride concentration is used both for CF diagnosis and for tracking CFTR modulator efficacy over time, but the relationship between sweat chloride and lung health is heterogeneous and informed by CFTR genotype. Here, we endeavored to characterize ion transport in eccrine sweat glands (ESGs)., Methods: First, ESGs were microdissected from a non-CF skin donor to analyze individual glands. We established primary cultures of ESG cells via conditional reprogramming for functional testing of ion transport by short circuit current measurement and examined cell composition by single-cell RNA-sequencing (scRNA-seq) comparing with whole dissociated ESGs. Secondly, we cultured nasal epithelial (NE) cells and ESGs from two people with CF (pwCF) to assess modulator efficacy. Finally, NEs and ESGs were grown from one person with the CFTR genotype F312del/F508del to explore genotype-phenotype heterogeneity., Results: ESG primary cells from individuals without CF demonstrated robust ENaC and CFTR function. scRNA-seq demonstrated both secretory and ductal ESG markers in cultured ESG cells. In both NEs and ESGs from pwCF homozygous for F508del, minimal baseline CFTR function was observed, and treatment with CFTR modulators significantly enhanced function. Notably, NEs from an individual bearing F312del/F508del exhibited significant baseline CFTR function, whereas ESGs from the same person displayed minimal CFTR function, consistent with observed phenotype., Conclusions: This study has established a novel primary culture technique for ESGs that allows for functional ion transport measurement to assess modulator efficacy and evaluate genotype-phenoytpe heterogeneity. To our knowledge, this is the first reported application of conditional reprogramming and scRNA-seq of microdissected ESGs., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

It is important to model biological variation when analyzing spatial transcriptomics data from multiple samples. One approach to multi-sample analysis is to spatially align samples, but this is a challenging problem. Here, we provide an alignment-free framework for generalizing a one-sample spatial factorization model to multi-sample data. Using this framework, we develop a method, called multi-sample non-negative spatial factorization (mNSF) that extends the one-sample non-negative spatial factorization (NSF) framework to a multi-sample dataset. Our model allows for a sample-specific model for the spatial correlation structure and extracts a low-dimensional representation of the data. We illustrate the performance of mNSF by simulation studies and real data. mNSF identifies true factors in simulated data, identifies shared anatomical regions across samples in real data and reveals region-specific biological functions. mNSFs performance is similar to alignment based methods when alignment is possible, but extends analysis to situations where spatial alignment is impossible. We expect multi-sample factorization methods to be a powerful class of methods for analyzing spatially resolved transcriptomics data., Competing Interests: Conflicts of Interest None.

The enteric nervous system (ENS), a collection of neural cells contained in the wall of the gut, is of fundamental importance to gastrointestinal and systemic health. According to the prevailing paradigm, the ENS arises from progenitor cells migrating from the neural crest and remains largely unchanged thereafter. Here, we show that the lineage composition of maturing ENS changes with time, with a decline in the canonical lineage of neural-crest derived neurons and their replacement by a newly identified lineage of mesoderm-derived neurons. Single cell transcriptomics and immunochemical approaches establish a distinct expression profile of mesoderm-derived neurons. The dynamic balance between the proportions of neurons from these two different lineages in the post-natal gut is dependent on the availability of their respective trophic signals, GDNF-RET and HGF-MET. With increasing age, the mesoderm-derived neurons become the dominant form of neurons in the ENS, a change associated with significant functional effects on intestinal motility which can be reversed by GDNF supplementation. Transcriptomic analyses of human gut tissues show reduced GDNF-RET signaling in patients with intestinal dysmotility which is associated with reduction in neural crest-derived neuronal markers and concomitant increase in transcriptional patterns specific to mesoderm-derived neurons. Normal intestinal function in the adult gastrointestinal tract therefore appears to require an optimal balance between these two distinct lineages within the ENS., Competing Interests: SK, MS, JS, AS, SN, LB, CZ, AB, ZW, GL, JL, MK, SB, MA, ML, EV, LG, PP No competing interests declared

Non-negative matrix factorization (NMF) is an unsupervised learning method well suited to high-throughput biology. However, inferring biological processes from an NMF result still requires additional post hoc statistics and annotation for interpretation of learned features. Here, we introduce a suite of computational tools that implement NMF and provide methods for accurate and clear biological interpretation and analysis. A generalized discussion of NMF covering its benefits, limitations and open questions is followed by four procedures for the Bayesian NMF algorithm Coordinated Gene Activity across Pattern Subsets (CoGAPS). Each procedure will demonstrate NMF analysis to quantify cell state transitions in a public domain single-cell RNA-sequencing dataset. The first demonstrates PyCoGAPS, our new Python implementation that enhances runtime for large datasets, and the second allows its deployment in Docker. The third procedure steps through the same single-cell NMF analysis using our R CoGAPS interface. The fourth introduces a beginner-friendly CoGAPS platform using GenePattern Notebook, aimed at users with a working conceptual knowledge of data analysis but without a basic proficiency in the R or Python programming language. We also constructed a user-facing website to serve as a central repository for information and instructional materials about CoGAPS and its application programming interfaces. The expected timing to setup the packages and conduct a test run is around 15 min, and an additional 30 min to conduct analyses on a precomputed result. The expected runtime on the user's desired dataset can vary from hours to days depending on factors such as dataset size or input parameters., (© 2023. Springer Nature Limited.)

Background: RNA velocity analysis of single cells offers the potential to predict temporal dynamics from gene expression. In many systems, RNA velocity has been observed to produce a vector field that qualitatively reflects known features of the system. However, the limitations of RNA velocity estimates are still not well understood., Results: We analyze the impact of different steps in the RNA velocity workflow on direction and speed. We consider both high-dimensional velocity estimates and low-dimensional velocity vector fields mapped onto an embedding. We conclude the transition probability method for mapping velocity estimates onto an embedding is effectively interpolating in the embedding space. Our findings reveal a significant dependence of the RNA velocity workflow on smoothing via the k-nearest-neighbors (k-NN) graph of the observed data. This reliance results in considerable estimation errors for both direction and speed in both high- and low-dimensional settings when the k-NN graph fails to accurately represent the true data structure; this is an unknown feature of real data. RNA velocity performs poorly at estimating speed in both low- and high-dimensional spaces, except in very low noise settings. We introduce a novel quality measure that can identify when RNA velocity should not be used., Conclusions: Our findings emphasize the importance of choices in the RNA velocity workflow and highlight critical limitations of data analysis. We advise against over-interpreting expression dynamics using RNA velocity, particularly in terms of speed. Finally, we emphasize that the use of RNA velocity in assessing the correctness of a low-dimensional embedding is circular., (© 2023. The Author(s).)

The lacrimal gland is of central interest in ophthalmology both as the source of the aqueous component of tear fluid and as the site of autoimmune pathology in the context of Sjogren's syndrome (SjS). To provide a foundational description of mouse lacrimal gland cell types and their patterns of gene expression, we have analyzed single-cell transcriptomes from wild-type (Balb/c) mice and from two genetically based SjS models, MRL/lpr and NOD (nonobese diabetic) .H2b , and defined the localization of multiple cell-type-specific protein and mRNA markers. This analysis has uncovered a previously undescribed cell type, Car6+ cells, which are located at the junction of the acini and the connecting ducts. More than a dozen secreted polypeptides that are likely to be components of tear fluid are expressed by acinar cells and show pronounced sex differences in expression. Additional examples of gene expression heterogeneity within a single cell type were identified, including a gradient of Claudin4 along the length of the ductal system and cell-to-cell heterogeneity in transcription factor expression within acinar and myoepithelial cells. The patterns of expression of channels, transporters, and pumps in acinar, Car6+, and ductal cells make strong predictions regarding the mechanisms of water and electrolyte secretion. In MRL/lpr and NOD.H2b lacrimal glands, distinctive changes in parenchymal gene expression and in immune cell subsets reveal widespread interferon responses, a T cell-dominated infiltrate in the MRL/lpr model, and a mixed B cell and T cell infiltrate in the NOD.H2b model.

The receptor tyrosine kinase RET plays a critical role in the fate specification of enteric neural crest-derived cells (ENCDCs) during enteric nervous system (ENS) development. RET loss of function (LoF) is associated with Hirschsprung disease (HSCR), which is marked by aganglionosis of the gastrointestinal (GI) tract. Although the major phenotypic consequences and the underlying transcriptional changes from Ret LoF in the developing ENS have been described, cell type- and state-specific effects are unknown. We performed single-cell RNA sequencing on an enriched population of ENCDCs from the developing GI tract of Ret null heterozygous and homozygous mice at embryonic day (E)12.5 and E14.5. We demonstrate four significant findings: 1) Ret -expressing ENCDCs are a heterogeneous population comprising ENS progenitors as well as glial- and neuronal-committed cells; 2) neurons committed to a predominantly inhibitory motor neuron developmental trajectory are not produced under Ret LoF, leaving behind a mostly excitatory motor neuron developmental program; 3) expression patterns of HSCR-associated and Ret gene regulatory network genes are impacted by Ret LoF; and 4) Ret deficiency leads to precocious differentiation and reduction in the number of proliferating ENS precursors. Our results support a model in which Ret contributes to multiple distinct cellular phenotypes during development of the ENS, including the specification of inhibitory neuron subtypes, cell cycle dynamics of ENS progenitors, and the developmental timing of neuronal and glial commitment.

Psychedelics are a broad class of drugs defined by their ability to induce an altered state of consciousness 1,2 . These drugs have been used for millennia in both spiritual and medicinal contexts, and a number of recent clinical successes have spurred a renewed interest in developing psychedelic therapies 3-9 . Nevertheless, a unifying mechanism that can account for these shared phenomenological and therapeutic properties remains unknown. Here we demonstrate in mice that the ability to reopen the social reward learning critical period is a shared property across psychedelic drugs. Notably, the time course of critical period reopening is proportional to the duration of acute subjective effects reported in humans. Furthermore, the ability to reinstate social reward learning in adulthood is paralleled by metaplastic restoration of oxytocin-mediated long-term depression in the nucleus accumbens. Finally, identification of differentially expressed genes in the 'open state' versus the 'closed state' provides evidence that reorganization of the extracellular matrix is a common downstream mechanism underlying psychedelic drug-mediated critical period reopening. Together these results have important implications for the implementation of psychedelics in clinical practice, as well as the design of novel compounds for the treatment of neuropsychiatric disease., (© 2023. The Author(s).)

The diversity of visual input processed by the mammalian visual system requires the generation of many distinct retinal ganglion cell (RGC) types, each tuned to a particular feature. The molecular code needed to generate this cell-type diversity is poorly understood. Here, we focus on the molecules needed to specify one type of retinal cell: the upward-preferring ON direction-selective ganglion cell (up-oDSGC) of the mouse visual system. Single-cell transcriptomic profiling of up- and down-oDSGCs shows that the transcription factor Tbx5 is selectively expressed in up-oDSGCs. The loss of Tbx5 in up-oDSGCs results in a selective defect in the formation of up-oDSGCs and a corresponding inability to detect vertical motion. A downstream effector of Tbx5, Sfrp1, is also critical for vertical motion detection but not up-oDSGC formation. These results advance our understanding of the molecular mechanisms that specify a rare retinal cell type and show how disrupting this specification leads to a corresponding defect in neural circuitry and behavior., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Recent studies suggested that microglia, the primary brain immune cells, can affect circuit connectivity and neuronal function 1,2 . Microglia infiltrate the neuroepithelium early in embryonic development and are maintained in the brain throughout adulthood 3,4 . Several maternal environmental factors-such as an aberrant microbiome, immune activation and poor nutrition-can influence prenatal brain development 5,6 . Nevertheless, it is unknown how changes in the prenatal environment instruct the developmental trajectory of infiltrating microglia, which in turn affect brain development and function. Here we show that, after maternal immune activation (MIA) in mice, microglia from the offspring have a long-lived decrease in immune reactivity (blunting) across the developmental trajectory. The blunted immune response was accompanied by changes in chromatin accessibility and reduced transcription factor occupancy of the open chromatin. Single-cell RNA-sequencing analysis revealed that MIA does not induce a distinct subpopulation but, rather, decreases the contribution to inflammatory microglia states. Prenatal replacement of microglia from MIA offspring with physiological infiltration of naive microglia ameliorated the immune blunting and restored a decrease in presynaptic vesicle release probability onto dopamine receptor type-two medium spiny neurons, indicating that aberrantly formed microglia due to an adverse prenatal environment affect the long-term microglia reactivity and proper striatal circuit development., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Heterozygous, loss of function mutations in positive regulators of the Transforming Growth Factor-β (TGF-β) pathway cause hereditary forms of thoracic aortic aneurysm. It is unclear whether and how the initial signaling deficiency triggers secondary signaling upregulation in the remaining functional branches of the pathway, and if this contributes to maladaptive vascular remodeling. To examine this process in a mouse model in which time-controlled, partial interference with postnatal TGF-β signaling in vascular smooth muscle cells (VSMCs) could be assessed, we used a VSMC-specific tamoxifen-inducible system, and a conditional allele, to inactivate Smad3 at 6 weeks of age, after completion of perinatal aortic development. This intervention induced dilation and histological abnormalities in the aortic root, with minor involvement of the ascending aorta. To analyze early and late events associated with disease progression, we performed a comparative single cell transcriptomic analysis at 10- and 18-weeks post-deletion, when aortic dilation is undetectable and moderate, respectively. At the early time-point, Smad3 -inactivation resulted in a broad reduction in the expression of extracellular matrix components and critical components of focal adhesions, including integrins and anchoring proteins, which was reflected histologically by loss of connections between VSMCs and elastic lamellae. At the later time point, however, expression of several transcripts belonging to the same functional categories was normalized or even upregulated; this occurred in association with upregulation of transcripts coding for TGF-β ligands, and persistent downregulation of negative regulators of the pathway. To interrogate how VSMC heterogeneity may influence this transition, we examined transcriptional changes in each of the four VSMC subclusters identified, regardless of genotype, as partly reflecting the proximal-to-distal anatomic location based on in situ RNA hybridization. The response to Smad3 -deficiency varied depending on subset, and VSMC subsets over-represented in the aortic root, the site most vulnerable to dilation, most prominently upregulated TGF-β ligands and pro-pathogenic factors such as thrombospondin-1, angiotensin converting enzyme, and pro-inflammatory mediators. These data suggest that Smad3 is required for maintenance of focal adhesions, and that loss of contacts with the extracellular matrix has consequences specific to each VSMC subset, possibly contributing to the regional susceptibility to dilation in the aorta., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bramel, Creamer, Saqib, Camejo Nunez, Bagirzadeh, Roker, Goff and MacFarlane.)

Mosquitoes locate and approach humans based on the activity of odorant receptors (ORs) expressed on olfactory receptor neurons (ORNs). Olfactogenetic experiments in Anopheles gambiae mosquitoes revealed that the ectopic expression of an AgOR (AgOR2) in ORNs dampened the activity of the expressing neuron. This contrasts with studies in Drosophila melanogaster in which the ectopic expression of non-native ORs in ORNs confers ectopic neuronal responses without interfering with native olfactory physiology. RNA-seq analyses comparing wild-type antennae to those ectopically expressing AgOR2 in ORNs indicated that nearly all AgOR transcripts were significantly downregulated (except for AgOR2). Additional experiments suggest that AgOR2 protein rather than mRNA mediates this downregulation. Using in situ hybridization, we find that AgOR gene choice is active into adulthood and that AgOR2 expression inhibits AgORs from turning on at this late stage. Our study shows that the ORNs of Anopheles mosquitoes (in contrast to Drosophila) are sensitive to a currently unexplored mechanism of AgOR regulation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Hair cell (HC) loss within the inner ear cochlea is a leading cause for deafness in humans. Before the onset of hearing, immature supporting cells (SCs) in neonatal mice have some limited capacity for HC regeneration. Here, we show that in organoid culture, transient activation of the progenitor-specific RNA binding protein LIN28B and Activin antagonist follistatin (FST) enhances regenerative competence of maturing/mature cochlear SCs by reprogramming them into progenitor-like cells. Transcriptome profiling and mechanistic studies reveal that LIN28B drives SC reprogramming, while FST is required to counterbalance hyperactivation of transforming growth factor-β-type signaling by LIN28B. Last, we show that LIN28B and FST coactivation enhances spontaneous cochlear HC regeneration in neonatal mice and that LIN28B may be part of an endogenous repair mechanism that primes SCs for HC regeneration. These findings indicate that SC dedifferentiation is critical for HC regeneration and identify LIN28B and FST as main regulators.

Background: The cell cycle is a highly conserved, continuous process which controls faithful replication and division of cells. Single-cell technologies have enabled increasingly precise measurements of the cell cycle both as a biological process of interest and as a possible confounding factor. Despite its importance and conservation, there is no universally applicable approach to infer position in the cell cycle with high-resolution from single-cell RNA-seq data., Results: Here, we present tricycle, an R/Bioconductor package, to address this challenge by leveraging key features of the biology of the cell cycle, the mathematical properties of principal component analysis of periodic functions, and the use of transfer learning. We estimate a cell-cycle embedding using a fixed reference dataset and project new data into this reference embedding, an approach that overcomes key limitations of learning a dataset-dependent embedding. Tricycle then predicts a cell-specific position in the cell cycle based on the data projection. The accuracy of tricycle compares favorably to gold-standard experimental assays, which generally require specialized measurements in specifically constructed in vitro systems. Using internal controls which are available for any dataset, we show that tricycle predictions generalize to datasets with multiple cell types, across tissues, species, and even sequencing assays., Conclusions: Tricycle generalizes across datasets and is highly scalable and applicable to atlas-level single-cell RNA-seq data., (© 2021. The Author(s).)

Radial glial progenitor cells (RGCs) in the dorsal telencephalon directly or indirectly produce excitatory projection neurons and macroglia of the neocortex. Recent evidence shows that the pool of RGCs is more heterogeneous than originally thought and that progenitor subpopulations can generate particular neuronal cell types. Using single-cell RNA sequencing, we have studied gene expression patterns of RGCs with different neurogenic behavior at early stages of cortical development. At this early age, some RGCs rapidly produce postmitotic neurons, whereas others self-renew and undergo neurogenic divisions at a later age. We have identified candidate genes that are differentially expressed among these early RGC subpopulations, including the transcription factor Sox9. Using in utero electroporation in embryonic mice of either sex, we demonstrate that elevated Sox9 expression in progenitors affects RGC cell cycle duration and leads to the generation of upper layer cortical neurons. Our data thus reveal molecular differences between progenitor cells with different neurogenic behavior at early stages of corticogenesis and indicates that Sox9 is critical for the maintenance of RGCs to regulate the generation of upper layer neurons. SIGNIFICANCE STATEMENT The existence of heterogeneity in the pool of RGCs and its relationship with the generation of cellular diversity in the cerebral cortex has been an interesting topic of debate for many years. Here we describe the existence of RGCs with reduced neurogenic behavior at early embryonic ages presenting a particular molecular signature. This molecular signature consists of differential expression of some genes including the transcription factor Sox9, which has been found to be a specific regulator of this subpopulation of progenitor cells. Functional experiments perturbing expression levels of Sox9 reveal its instructive role in the regulation of the neurogenic behavior of RGCs and its relationship with the generation of upper layer projection neurons at later ages., (Copyright © 2021 Fabra-Beser et al.)

Haematopoiesis relies on tightly controlled gene expression patterns as development proceeds through a series of progenitors. While the regulation of hematopoietic development has been well studied, the role of noncoding elements in this critical process is a developing field. In particular, the discovery of new regulators of lymphopoiesis could have important implications for our understanding of the adaptive immune system and disease. Here we elucidate how a noncoding element is capable of regulating a broadly expressed transcription factor, Ikaros, in a lymphoid lineage-specific manner, such that it imbues Ikaros with the ability to specify the lymphoid lineage over alternate fates. Deletion of the Daedalus locus, which is proximal to Ikaros, led to a severe reduction in early lymphoid progenitors, exerting control over the earliest fate decisions during lymphoid lineage commitment. Daedalus locus deletion led to alterations in Ikaros isoform expression and a significant reduction in Ikaros protein. The Daedalus locus may function through direct DNA interaction as Hi-C analysis demonstrated an interaction between the two loci. Finally, we identify an Ikaros-regulated erythroid-lymphoid checkpoint that is governed by Daedalus in a lymphoid-lineage-specific manner. Daedalus appears to act as a gatekeeper of Ikaros's broad lineage-specifying functions, selectively stabilizing Ikaros activity in the lymphoid lineage and permitting diversion to the erythroid fate in its absence. These findings represent a key illustration of how a transcription factor with broad lineage expression must work in concert with noncoding elements to orchestrate hematopoietic lineage commitment., Competing Interests: Competing interest statement: R.A.F. is a consultant for GSK and Zai Lab Ltd.

Somatic LINE-1 (L1) retrotransposition has been detected in early embryos, adult brains, and the gastrointestinal (GI) tract, and many cancers, including epithelial GI tumors. We previously found numerous somatic L1 insertions in paired normal and GI cancerous tissues. Here, using a modified method of single-cell analysis for somatic L1 insertions, we studied adenocarcinomas of colon, pancreas, and stomach, and found a variable number of somatic L1 insertions in tumors of the same type from patient to patient. We detected no somatic L1 insertions in single cells of 5 of 10 tumors studied. In three tumors, aneuploid cells were detected by FACS. In one pancreatic tumor, there were many more L1 insertions in aneuploid than in euploid tumor cells. In one gastric cancer, both aneuploid and euploid cells contained large numbers of likely clonal insertions. However, in a second gastric cancer with aneuploid cells, no somatic L1 insertions were found. We suggest that when the cellular environment is favorable to retrotransposition, aneuploidy predisposes tumor cells to L1 insertions, and retrotransposition may occur at the transition from euploidy to aneuploidy. Seventeen percent of insertions were also present in normal cells, similar to findings in genomic DNA from normal tissues of GI tumor patients. We provide evidence that: 1) The number of L1 insertions in tumors of the same type is highly variable, 2) most somatic L1 insertions in GI cancer tissues are absent from normal tissues, and 3) under certain conditions, somatic L1 retrotransposition exhibits a propensity for occurring in aneuploid cells., Competing Interests: The authors declare no competing interest.

Parallel processing circuits are thought to dramatically expand the network capabilities of the nervous system. Magnocellular and parvocellular oxytocin neurons have been proposed to subserve two parallel streams of social information processing, which allow a single molecule to encode a diverse array of ethologically distinct behaviors. Here we provide the first comprehensive characterization of magnocellular and parvocellular oxytocin neurons in male mice, validated across anatomical, projection target, electrophysiological, and transcriptional criteria. We next use novel multiple feature selection tools in Fmr1-KO mice to provide direct evidence that normal functioning of the parvocellular but not magnocellular oxytocin pathway is required for autism-relevant social reward behavior. Finally, we demonstrate that autism risk genes are enriched in parvocellular compared with magnocellular oxytocin neurons. Taken together, these results provide the first evidence that oxytocin-pathway-specific pathogenic mechanisms account for social impairments across a broad range of autism etiologies., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In the hippocampus, a widely accepted model posits that the dentate gyrus improves learning and memory by enhancing discrimination between inputs. To test this model, we studied conditional knockout mice in which the vast majority of dentate granule cells (DGCs) fail to develop - including nearly all DGCs in the dorsal hippocampus - secondary to eliminating Wntless ( Wls ) in a subset of cortical progenitors with Gfap-Cre . Other cells in the Wls fl/- ;Gfap-Cre hippocampus were minimally affected, as determined by single nucleus RNA sequencing. CA3 pyramidal cells, the targets of DGC-derived mossy fibers, exhibited normal morphologies with a small reduction in the numbers of synaptic spines. Wls fl/- ;Gfap-Cre mice have a modest performance decrement in several complex spatial tasks, including active place avoidance. They were also modestly impaired in one simpler spatial task, finding a visible platform in the Morris water maze. These experiments support a role for DGCs in enhancing spatial learning and memory., Competing Interests: AR, CT, CJ, TK, SH, JW, ZH, MA, SM, GS, LG, MW, MP, AF, JN No competing interests declared, (© 2020, Rattner et al.)

Introduction: The microtubule-associated protein tau (MAPT) gene is considered a strong genetic risk factor for Parkinson's disease (PD) in Caucasians. MAPT is located within an inversion region of high linkage disequilibrium designated as H1 and H2 haplotype, and contains eight other genes which have been implicated in neurodegeneration. The aim of the current study was to identify common coding variants in strong linkage disequilibrium (LD) within the associated loci on chr17q21 harboring MAPT., Methods: Sanger sequencing of coding exons in 90 Caucasian late-onset PD (LOPD) patients was performed. Specific gene sequencing for LRRC37A, LRRC37A2, ARL17A and ARL17B was not possible given the high homology, presence of pseudogenes and copy number variants that are in the region, and therefore four genes (NSF, KANSL1, SPPL2C, and CRHR1) were included in the analysis. Coding variants from these four genes that did not perfectly tag (r 2  = 1) the MAPT H1/H2 haplotype were genotyped in an independent replication series of Caucasian PD cases (N = 851) and controls (N = 730)., Results: In the 90 LOPD cases we identified 30 coding variants. Eleven non-synonymous variants tagged the MAPT H1/H2 haplotype, including two SPPL2C variants (rs12185233 and rs12373123) that had high pathogenic combined annotation dependent depletion (CADD) scores of >20. In the replication series, the non-synonymous KANSL1 rs17585974 variant was in very strong LD with MAPT H1/H2 and had a high CADD score of 24.7., Conclusion: We have identified several non-synonymous variants across neighboring genes of MAPT that may warrant further genetic and functional investigation within the biological etiology of PD., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Motivation: Dimension reduction techniques are widely used to interpret high-dimensional biological data. Features learned from these methods are used to discover both technical artifacts and novel biological phenomena. Such feature discovery is critically importent in analysis of large single-cell datasets, where lack of a ground truth limits validation and interpretation. Transfer learning (TL) can be used to relate the features learned from one source dataset to a new target dataset to perform biologically driven validation by evaluating their use in or association with additional sample annotations in that independent target dataset., Results: We developed an R/Bioconductor package, projectR, to perform TL for analyses of genomics data via TL of clustering, correlation and factorization methods. We then demonstrate the utility TL for integrated data analysis with an example for spatial single-cell analysis., Availability and Implementation: projectR is available on Bioconductor and at https://github.com/genesofeve/projectR., Contact: gsteinobrien@jhmi.edu or ejfertig@jhmi.edu., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The development of single-cell RNA sequencing (scRNA-seq) has allowed high-resolution analysis of cell-type diversity and transcriptional networks controlling cell-fate specification. To identify the transcriptional networks governing human retinal development, we performed scRNA-seq analysis on 16 time points from developing retina as well as four early stages of retinal organoid differentiation. We identified evolutionarily conserved patterns of gene expression during retinal progenitor maturation and specification of all seven major retinal cell types. Furthermore, we identified gene-expression differences between developing macula and periphery and between distinct populations of horizontal cells. We also identified species-specific patterns of gene expression during human and mouse retinal development. Finally, we identified an unexpected role for ATOH7 expression in regulation of photoreceptor specification during late retinogenesis. These results provide a roadmap to future studies of human retinal development and may help guide the design of cell-based therapies for treating retinal dystrophies., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We previously discovered in mouse adipocytes an lncRNA (the homolog of human LINC00116) regulating adipogenesis that contains a highly conserved coding region. Here, we show human protein expression of a peptide within LINC00116, and demonstrate that this peptide modulates triglyceride clearance in human adipocytes by regulating lipolysis and mitochondrial β-oxidation. This gene has previously been identified as mitoregulin (MTLN). We conclude that MTLN has a regulatory role in adipocyte metabolism as demonstrated by systemic lipid phenotypes in knockout mice. We also assert its adipocyte-autonomous phenotypes in both isolated murine adipocytes as well as human stem cell-derived adipocytes. MTLN directly interacts with the β subunit of the mitochondrial trifunctional protein, an enzyme critical in the β-oxidation of long-chain fatty acids. Our human and murine models contend that MTLN could be an avenue for further therapeutic research, albeit not without caveats, for example, by promoting white adipocyte triglyceride clearance in obese subjects., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Diabetes is a common complication of cystic fibrosis (CF) that affects approximately 20% of adolescents and 40%-50% of adults with CF. The age at onset of CF-related diabetes (CFRD) (marked by clinical diagnosis and treatment initiation) is an important measure of the disease process. DNA variants associated with age at onset of CFRD reside in and near SLC26A9. Deep sequencing of the SLC26A9 gene in 762 individuals with CF revealed that 2 common DNA haplotypes formed by the risk variants account for the association with diabetes. Single-cell RNA sequencing (scRNA-Seq) indicated that SLC26A9 is predominantly expressed in pancreatic ductal cells and frequently coexpressed with CF transmembrane conductance regulator (CFTR) along with transcription factors that have binding sites 5' of SLC26A9. These findings were replicated upon reanalysis of scRNA-Seq data from 4 independent studies. DNA fragments derived from the 5' region of SLC26A9-bearing variants from the low-risk haplotype generated 12%-20% higher levels of expression in PANC-1 and CFPAC-1 cells compared with the high- risk haplotype. Taken together, our findings indicate that an increase in SLC26A9 expression in ductal cells of the pancreas delays the age at onset of diabetes, suggesting a CFTR-agnostic treatment for a major complication of CF.

Recent genome-wide association studies (GWAS) identified numerous schizophrenia (SZ) and Alzheimer's disease (AD) associated loci, most outside protein-coding regions and hypothesized to affect gene transcription. We used a massively parallel reporter assay to screen, 1,049 SZ and 30 AD variants in 64 and nine loci, respectively for allele differences in driving reporter gene expression. A library of synthetic oligonucleotides assaying each allele five times was transfected into K562 chronic myelogenous leukemia lymphoblasts and SK-SY5Y human neuroblastoma cells. One hundred forty eight variants showed allelic differences in K562 and 53 in SK-SY5Y cells, on average 2.6 variants per locus. Nine showed significant differences in both lines, a modest overlap reflecting different regulatory landscapes of these lines that also differ significantly in chromatin marks. Eight of nine were in the same direction. We observe no preference for risk alleles to increase or decrease expression. We find a positive correlation between the number of SNPs in linkage disequilibrium and the proportion of functional SNPs supporting combinatorial effects that may lead to haplotype selection. Our results prioritize future functional follow up of disease associated SNPs to determine the driver GWAS variant(s), at each locus and enhance our understanding of gene regulation dynamics., (© 2019 Wiley Periodicals, Inc.)

Autoimmune uveoretinitis is a significant cause of visual loss, and mouse models offer unique opportunities to study its disease mechanisms. Aire -/- mice fail to express self-antigens in the thymus, exhibit reduced central tolerance, and develop a spontaneous, chronic, and progressive uveoretinitis. Using single-cell RNA sequencing (scRNA-seq), we characterized wild-type and Aire -/- retinas to define, in a comprehensive and unbiased manner, the cell populations and gene expression patterns associated with disease. Based on scRNA-seq, immunostaining, and in situ hybridization, we infer that 1) the dominant effector response in Aire -/- retinas is Th1-driven, 2) a subset of monocytes convert to either a macrophage/microglia state or a dendritic cell state, 3) the development of tertiary lymphoid structures constitutes part of the Aire -/- retinal phenotype, 4) all major resident retinal cell types respond to interferon gamma (IFNG) by changing their patterns of gene expression, and 5) Muller glia up-regulate specific genes in response to IFN gamma and may act as antigen-presenting cells.

Chromatin modifiers act to coordinate gene expression changes critical to neuronal differentiation from neural stem/progenitor cells (NSPCs). Lysine-specific methyltransferase 2D (KMT2D) encodes a histone methyltransferase that promotes transcriptional activation and is frequently mutated in cancers and in the majority (>70%) of patients diagnosed with the congenital, multisystem intellectual disability disorder Kabuki syndrome 1 (KS1). Critical roles for KMT2D are established in various non-neural tissues, but the effects of KMT2D loss in brain cell development have not been described. We conducted parallel studies of proliferation, differentiation, transcription, and chromatin profiling in KMT2D-deficient human and mouse models to define KMT2D-regulated functions in neurodevelopmental contexts, including adult-born hippocampal NSPCs in vivo and in vitro. We report cell-autonomous defects in proliferation, cell cycle, and survival, accompanied by early NSPC maturation in several KMT2D-deficient model systems. Transcriptional suppression in KMT2D-deficient cells indicated strong perturbation of hypoxia-responsive metabolism pathways. Functional experiments confirmed abnormalities of cellular hypoxia responses in KMT2D-deficient neural cells and accelerated NSPC maturation in vivo. Together, our findings support a model in which loss of KMT2D function suppresses expression of oxygen-responsive gene programs important to neural progenitor maintenance, resulting in precocious neuronal differentiation in a mouse model of KS1.

Tumor heterogeneity provides a complex challenge to cancer treatment and is a critical component of therapeutic response, disease recurrence, and patient survival. Single-cell RNA-sequencing (scRNA-seq) technologies have revealed the prevalence of intratumor and intertumor heterogeneity. Computational techniques are essential to quantify the differences in variation of these profiles between distinct cell types, tumor subtypes, and patients to fully characterize intratumor and intertumor molecular heterogeneity. In this study, we adapted our algorithm for pathway dysregulation, Expression Variation Analysis (EVA), to perform multivariate statistical analyses of differential variation of expression in gene sets for scRNA-seq. EVA has high sensitivity and specificity to detect pathways with true differential heterogeneity in simulated data. EVA was applied to several public domain scRNA-seq tumor datasets to quantify the landscape of tumor heterogeneity in several key applications in cancer genomics such as immunogenicity, metastasis, and cancer subtypes. Immune pathway heterogeneity of hematopoietic cell populations in breast tumors corresponded to the amount of diversity present in the T-cell repertoire of each individual. Cells from head and neck squamous cell carcinoma (HNSCC) primary tumors had significantly more heterogeneity across pathways than cells from metastases, consistent with a model of clonal outgrowth. Moreover, there were dramatic differences in pathway dysregulation across HNSCC basal primary tumors. Within the basal primary tumors, there was increased immune dysregulation in individuals with a high proportion of fibroblasts present in the tumor microenvironment. These results demonstrate the broad utility of EVA to quantify intertumor and intratumor heterogeneity from scRNA-seq data without reliance on low-dimensional visualization. SIGNIFICANCE: This study presents a robust statistical algorithm for evaluating gene expression heterogeneity within pathways or gene sets in single-cell RNA-seq data., (©2019 American Association for Cancer Research.)

Precise temporal control of gene expression in neuronal progenitors is necessary for correct regulation of neurogenesis and cell fate specification. However, the cellular heterogeneity of the developing CNS has posed a major obstacle to identifying the gene regulatory networks that control these processes. To address this, we used single-cell RNA sequencing to profile ten developmental stages encompassing the full course of retinal neurogenesis. This allowed us to comprehensively characterize changes in gene expression that occur during initiation of neurogenesis, changes in developmental competence, and specification and differentiation of each major retinal cell type. We identify the NFI transcription factors (Nfia, Nfib, and Nfix) as selectively expressed in late retinal progenitor cells and show that they control bipolar interneuron and Müller glia cell fate specification and promote proliferative quiescence., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Analysis of gene expression in single cells allows for decomposition of cellular states as low-dimensional latent spaces. However, the interpretation and validation of these spaces remains a challenge. Here, we present scCoGAPS, which defines latent spaces from a source single-cell RNA-sequencing (scRNA-seq) dataset, and projectR, which evaluates these latent spaces in independent target datasets via transfer learning. Application of developing mouse retina to scRNA-Seq reveals intrinsic relationships across biological contexts and assays while avoiding batch effects and other technical features. We compare the dimensions learned in this source dataset to adult mouse retina, a time-course of human retinal development, select scRNA-seq datasets from developing brain, chromatin accessibility data, and a murine-cell type atlas to identify shared biological features. These tools lay the groundwork for exploratory analysis of scRNA-seq data via latent space representations, enabling a shift in how we compare and identify cells beyond reliance on marker genes or ensemble molecular identity., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The mammalian CNS is capable of tolerating chronic hypoxia, but cell type-specific responses to this stress have not been systematically characterized. In the Norrin KO ( Ndp KO ) mouse, a model of familial exudative vitreoretinopathy (FEVR), developmental hypovascularization of the retina produces chronic hypoxia of inner nuclear-layer (INL) neurons and Muller glia. We used single-cell RNA sequencing, untargeted metabolomics, and metabolite labeling from 13 C-glucose to compare WT and Ndp KO retinas. In Ndp KO retinas, we observe gene expression responses consistent with hypoxia in Muller glia and retinal neurons, and we find a metabolic shift that combines reduced flux through the TCA cycle with increased synthesis of serine, glycine, and glutathione. We also used single-cell RNA sequencing to compare the responses of individual cell types in Ndp KO retinas with those in the hypoxic cerebral cortex of mice that were housed for 1 week in a reduced oxygen environment (7.5% oxygen). In the hypoxic cerebral cortex, glial transcriptome responses most closely resemble the response of Muller glia in the Ndp KO retina. In both retina and brain, vascular endothelial cells activate a previously dormant tip cell gene expression program, which likely underlies the adaptive neoangiogenic response to chronic hypoxia. These analyses of retina and brain transcriptomes at single-cell resolution reveal both shared and cell type-specific changes in gene expression in response to chronic hypoxia, implying both shared and distinct cell type-specific physiologic responses., Competing Interests: The authors declare no conflict of interest.

Background: Massively parallel reporter assays (MPRAs) have emerged as a popular means for understanding noncoding variation in a variety of conditions. While a large number of experiments have been described in the literature, analysis typically uses ad-hoc methods. There has been little attention to comparing performance of methods across datasets., Results: We present the mpralm method which we show is calibrated and powerful, by analyzing its performance on multiple MPRA datasets. We show that it outperforms existing statistical methods for analysis of this data type, in the first comprehensive evaluation of statistical methods on several datasets. We investigate theoretical and real-data properties of barcode summarization methods and show an unappreciated impact of summarization method for some datasets. Finally, we use our model to conduct a power analysis for this assay and show substantial improvements in power by performing up to 6 replicates per condition, whereas sequencing depth has smaller impact; we recommend to always use at least 4 replicates. An R package is available from the Bioconductor project., Conclusions: Together, these results inform recommendations for differential analysis, general group comparisons, and power analysis and will help improve design and analysis of MPRA experiments.

Omics data contain signals from the molecular, physical, and kinetic inter- and intracellular interactions that control biological systems. Matrix factorization (MF) techniques can reveal low-dimensional structure from high-dimensional data that reflect these interactions. These techniques can uncover new biological knowledge from diverse high-throughput omics data in applications ranging from pathway discovery to timecourse analysis. We review exemplary applications of MF for systems-level analyses. We discuss appropriate applications of these methods, their limitations, and focus on the analysis of results to facilitate optimal biological interpretation. The inference of biologically relevant features with MF enables discovery from high-throughput data beyond the limits of current biological knowledge - answering questions from high-dimensional data that we have not yet thought to ask., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Vascular endothelial cell (EC) function depends on appropriate organ-specific molecular and cellular specializations. To explore genomic mechanisms that control this specialization, we have analyzed and compared the transcriptome, accessible chromatin, and DNA methylome landscapes from mouse brain, liver, lung, and kidney ECs. Analysis of transcription factor (TF) gene expression and TF motifs at candidate cis -regulatory elements reveals both shared and organ-specific EC regulatory networks. In the embryo, only those ECs that are adjacent to or within the central nervous system (CNS) exhibit canonical Wnt signaling, which correlates precisely with blood-brain barrier (BBB) differentiation and Zic3 expression. In the early postnatal brain, single-cell RNA-seq of purified ECs reveals (1) close relationships between veins and mitotic cells and between arteries and tip cells, (2) a division of capillary ECs into vein-like and artery-like classes, and (3) new endothelial subtype markers, including new validated tip cell markers., Competing Interests: MS, JH, CL, RC, JN, AR, LG, JE No competing interests declared, JN Reviewing editor, eLife, (© 2018, Sabbagh et al.)

Genetic variation modulating risk of sporadic Parkinson disease (PD) has been primarily explored through genome-wide association studies (GWASs). However, like many other common genetic diseases, the impacted genes remain largely unknown. Here, we used single-cell RNA-seq to characterize dopaminergic (DA) neuron populations in the mouse brain at embryonic and early postnatal time points. These data facilitated unbiased identification of DA neuron subpopulations through their unique transcriptional profiles, including a postnatal neuroblast population and substantia nigra (SN) DA neurons. We use these population-specific data to develop a scoring system to prioritize candidate genes in all 49 GWAS intervals implicated in PD risk, including genes with known PD associations and many with extensive supporting literature. As proof of principle, we confirm that the nigrostriatal pathway is compromised in Cplx1-null mice. Ultimately, this systematic approach establishes biologically pertinent candidates and testable hypotheses for sporadic PD, informing a new era of PD genetic research., (Copyright © 2018 American Society of Human Genetics. All rights reserved.)

Single-cell RNA sequencing has generated catalogs of transcriptionally defined neuronal subtypes of the brain. However, the cellular processes that contribute to neuronal subtype specification and transcriptional heterogeneity remain unclear. By comparing the gene expression profiles of single layer 6 corticothalamic neurons in somatosensory cortex, we show that transcriptional subtypes primarily reflect axonal projection pattern, laminar position within the cortex, and neuronal activity state. Pseudotemporal ordering of 1,023 cellular responses to sensory manipulation demonstrates that changes in expression of activity-induced genes both reinforced cell-type identity and contributed to increased transcriptional heterogeneity within each cell type. This is due to cell-type biased choices of transcriptional states following manipulation of neuronal activity. These results reveal that axonal projection pattern, laminar position, and activity state define significant axes of variation that contribute both to the transcriptional identity of individual neurons and to the transcriptional heterogeneity within each neuronal subtype., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Commitment to the innate lymphoid cell (ILC) lineage is determined by Id2, a transcriptional regulator that antagonizes T and B cell-specific gene expression programs. Yet how Id2 expression is regulated in each ILC subset remains poorly understood. We identified a cis-regulatory element demarcated by a long non-coding RNA (lncRNA) that controls the function and lineage identity of group 1 ILCs, while being dispensable for early ILC development and homeostasis of ILC2s and ILC3s. The locus encoding this lncRNA, which we termed Rroid, directly interacted with the promoter of its neighboring gene, Id2, in group 1 ILCs. Moreover, the Rroid locus, but not the lncRNA itself, controlled the identity and function of ILC1s by promoting chromatin accessibility and deposition of STAT5 at the promoter of Id2 in response to interleukin (IL)-15. Thus, non-coding elements responsive to extracellular cues unique to each ILC subset represent a key regulatory layer for controlling the identity and function of ILCs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cell type-specific changes in neuronal excitability have been proposed to contribute to the selective degeneration of corticospinal neurons in amyotrophic lateral sclerosis (ALS) and to neocortical hyperexcitability, a prominent feature of both inherited and sporadic variants of the disease, but the mechanisms underlying selective loss of specific cell types in ALS are not known. We analyzed the physiological properties of distinct classes of cortical neurons in the motor cortex of hSOD1 G93A mice of both sexes and found that they all exhibit increases in intrinsic excitability that depend on disease stage. Targeted recordings and in vivo calcium imaging further revealed that neurons adapt their functional properties to normalize cortical excitability as the disease progresses. Although different neuron classes all exhibited increases in intrinsic excitability, transcriptional profiling indicated that the molecular mechanisms underlying these changes are cell type specific. The increases in excitability in both excitatory and inhibitory cortical neurons show that selective dysfunction of neuronal cell types cannot account for the specific vulnerability of corticospinal motor neurons in ALS. Furthermore, the stage-dependent alterations in neuronal function highlight the ability of cortical circuits to adapt as disease progresses. These findings show that both disease stage and cell type must be considered when developing therapeutic strategies for treating ALS. SIGNIFICANCE STATEMENT It is not known why certain classes of neurons preferentially die in different neurodegenerative diseases. It has been proposed that the enhanced excitability of affected neurons is a major contributor to their selective loss. We show using a mouse model of amyotrophic lateral sclerosis (ALS), a disease in which corticospinal neurons exhibit selective vulnerability, that changes in excitability are not restricted to this neuronal class and that excitability does not increase monotonically with disease progression. Moreover, although all neuronal cell types tested exhibited abnormal functional properties, analysis of their gene expression demonstrated cell type-specific responses to the ALS-causing mutation. These findings suggest that therapies for ALS may need to be tailored for different cell types and stages of disease., (Copyright © 2017 the authors 0270-6474/17/379038-17$15.00/0.)

Kabuki syndrome is a Mendelian intellectual disability syndrome caused by mutations in either of two genes (KMT2D and KDM6A) involved in chromatin accessibility. We previously showed that an agent that promotes chromatin opening, the histone deacetylase inhibitor (HDACi) AR-42, ameliorates the deficiency of adult neurogenesis in the granule cell layer of the dentate gyrus and rescues hippocampal memory defects in a mouse model of Kabuki syndrome (Kmt2d +/βGeo ). Unlike a drug, a dietary intervention could be quickly transitioned to the clinic. Therefore, we have explored whether treatment with a ketogenic diet could lead to a similar rescue through increased amounts of beta-hydroxybutyrate, an endogenous HDACi. Here, we report that a ketogenic diet in Kmt2d +/βGeo mice modulates H3ac and H3K4me3 in the granule cell layer, with concomitant rescue of both the neurogenesis defect and hippocampal memory abnormalities seen in Kmt2d +/βGeo mice; similar effects on neurogenesis were observed on exogenous administration of beta-hydroxybutyrate. These data suggest that dietary modulation of epigenetic modifications through elevation of beta-hydroxybutyrate may provide a feasible strategy to treat the intellectual disability seen in Kabuki syndrome and related disorders., Competing Interests: J.S.B. and H.T.B. have a pending patent for the use of a ketogenic diet and injection of BHB for treatment of Mendelian disorders of the epigenetic machinery.

The development of the central nervous system (CNS) is a complex orchestration of stem cells, transcription factors, growth/differentiation factors, and epigenetic control. Noncoding RNAs have been identified, classified, and studied for their functional roles in many systems including the CNS. In particular, the class of long noncoding RNAs (lncRNAs) has generated both enthusiasm and skepticism due to the unexpected discovery, the diversity of mechanisms, and the lower level of expression than found in protein-coding RNAs. Here we describe evidence supporting the role of lncRNAs in driving CNS-specific differentiation. It is clear that lncRNAs exhibit a functional diversity that makes their study and compartmentalization more challenging than other classes of noncoding RNAs. We predict, however, that lncRNAs will be essential for the characterization of discrete neuronal cell types in the age of single-cell transcriptomics and that these regulatory RNAs contribute to the multitude of functional mechanisms during CNS differentiation that will rival the diversities of protein-based mechanisms., (Copyright © 2016 ISDN. Published by Elsevier Ltd. All rights reserved.)

Neutrophils, eosinophils and 'classical' monocytes collectively account for about 70% of human blood leukocytes and are among the shortest-lived cells in the body. Precise regulation of the lifespan of these myeloid cells is critical to maintain protective immune responses and minimize the deleterious consequences of prolonged inflammation. However, how the lifespan of these cells is strictly controlled remains largely unknown. Here we identify a long non-coding RNA that we termed Morrbid, which tightly controls the survival of neutrophils, eosinophils and classical monocytes in response to pro-survival cytokines in mice. To control the lifespan of these cells, Morrbid regulates the transcription of the neighbouring pro-apoptotic gene, Bcl2l11 (also known as Bim), by promoting the enrichment of the PRC2 complex at the Bcl2l11 promoter to maintain this gene in a poised state. Notably, Morrbid regulates this process in cis, enabling allele-specific control of Bcl2l11 transcription. Thus, in these highly inflammatory cells, changes in Morrbid levels provide a locus-specific regulatory mechanism that allows rapid control of apoptosis in response to extracellular pro-survival signals. As MORRBID is present in humans and dysregulated in individuals with hypereosinophilic syndrome, this long non-coding RNA may represent a potential therapeutic target for inflammatory disorders characterized by aberrant short-lived myeloid cell lifespan.

The number of long noncoding RNAs (lncRNAs) has grown rapidly; however, our understanding of their function remains limited. Although cultured cells have facilitated investigations of lncRNA function at the molecular level, the use of animal models provides a rich context in which to investigate the phenotypic impact of these molecules. Promising initial studies using animal models demonstrated that lncRNAs influence a diverse number of phenotypes, ranging from subtle dysmorphia to viability. Here, we highlight the diversity of animal models and their unique advantages, discuss the use of animal models to profile lncRNA expression, evaluate experimental strategies to manipulate lncRNA function in vivo, and review the phenotypes attributable to lncRNAs. Despite a limited number of studies leveraging animal models, lncRNAs are already recognized as a notable class of molecules with important implications for health and disease.

Background: Analysis of the functional consequences and treatment response of rare CFTR variants is challenging due to the limited availability of primary airways cells., Methods: A Flp recombination target (FRT) site for stable expression of CFTR was incorporated into an immortalized CF bronchial epithelial cell line (CFBE41o-). CFTR cDNA was integrated into the FRT site. Expression was evaluated by western blotting and confocal microscopy and function measured by short circuit current. RNA sequencing was used to compare the transcriptional profile of the resulting CF8Flp cell line to primary cells and tissues., Results: Functional CFTR was expressed from integrated cDNA at the FRT site of the CF8Flp cell line at levels comparable to that seen in native airway cells. CF8Flp cells expressing WT-CFTR have a stable transcriptome comparable to that of primary cultured airway epithelial cells, including genes that play key roles in CFTR pathways., Conclusion: CF8Flp cells provide a viable substitute for primary CF airway cells for the analysis of CFTR variants in a native context., (Copyright © 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

The regulatory potential of RNA has never ceased to amaze: from RNA catalysis, to RNA-mediated splicing, to RNA-based silencing of an entire chromosome during dosage compensation. More recently, thousands of long noncoding RNA (lncRNA) transcripts have been identified, the majority with unknown function. Thus, it is tempting to think that these lncRNAs represent a cadre of new factors that function through ribonucleic mechanisms. Some evidence points to several lncRNAs with tantalizing physiological contributions and thought-provoking molecular modalities. However, dissecting the RNA biology of lncRNAs has been difficult, and distinguishing the independent contributions of functional RNAs from underlying DNA elements, or the local act of transcription, is challenging. Here, we aim to survey the existing literature and highlight future approaches that will be needed to link the RNA-based biology and mechanisms of lncRNAs in vitro and in vivo., (© 2015 Goff and Rinn; Published by Cold Spring Harbor Laboratory Press.)

Long noncoding RNAs (lncRNAs) have been implicated in numerous cellular processes including brain development. However, the in vivo expression dynamics and molecular pathways regulated by these loci are not well understood. Here, we leveraged a cohort of 13 lncRNAnull mutant mouse models to investigate the spatiotemporal expression of lncRNAs in the developing and adult brain and the transcriptome alterations resulting from the loss of these lncRNA loci. We show that several lncRNAs are differentially expressed both in time and space, with some presenting highly restricted expression in only selected brain regions. We further demonstrate altered regulation of genes for a large variety of cellular pathways and processes upon deletion of the lncRNA loci. Finally, we found that 4 of the 13 lncRNAs significantly affect the expression of several neighboring proteincoding genes in a cis-like manner. By providing insight into the endogenous expression patterns and the transcriptional perturbations caused by deletion of the lncRNA locus in the developing and postnatal mammalian brain, these data provide a resource to facilitate future examination of the specific functional relevance of these genes in neural development, brain function, and disease.