Publications by authors named "Seth Blackshaw"

138 Publications

Author Correction: A potential role for somatostatin signaling in regulating retinal neurogenesis.

Sci Rep 2021 Aug 16;11(1):16948. Epub 2021 Aug 16.

Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.

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http://dx.doi.org/10.1038/s41598-021-96291-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8368217PMC
August 2021

Ablation of lncRNA attenuates pathological hypertrophy and heart failure.

Theranostics 2021 6;11(16):7995-8007. Epub 2021 Jul 6.

Molecular Cardiology Program, Department of Biomedical Engineering, University of Alabama at Birmingham, School of Medicine and School of Engineering, Birmingham, AL 35294, USA.

The conserved long non-coding RNA (lncRNA) myocardial infarction associate transcript () was identified for its multiple single-nucleotide polymorphisms that are strongly associated with susceptibility to MI, but its role in cardiovascular biology remains elusive. Here we investigated whether regulates cardiac response to pathological hypertrophic stimuli. Both an angiotensin II (Ang II) infusion model and a transverse aortic constriction (TAC) model were used in adult WT and -null knockout (-KO) mice to induce pathological cardiac hypertrophy. Heart structure and function were evaluated by echocardiography and histological assessments. Gene expression in the heart was evaluated by RNA sequencing (RNA-seq), quantitative real-time RT-PCR (qRT-PCR), and Western blotting. Primary WT and -KO mouse cardiomyocytes were isolated and used in Ca transient and contractility measurements. Continuous Ang II infusion for 4 weeks induced concentric hypertrophy in WT mice, but to a lesser extent in -KO mice. Surgical TAC for 6 weeks resulted in decreased systolic function and heart failure in WT mice but not in -KO mice. In both models, -KO mice displayed reduced heart-weight to tibia-length ratio, cardiomyocyte cross-sectional area, cardiomyocyte apoptosis, and cardiac interstitial fibrosis and a better-preserved capillary density, as compared to WT mice. In addition, Ang II treatment led to significantly reduced mRNA and protein expression of the Ca cycling genes Sarcoplasmic/endoplasmic reticulum Ca ATPase 2a (SERCA2a) and ryanodine receptor 2 (RyR2) and a dramatic increase in global RNA splicing events in the left ventricle (LV) of WT mice, and these changes were largely blunted in -KO mice. Consistently, cardiomyocytes isolated from -KO mice demonstrated more efficient Ca cycling and greater contractility. Ablation of attenuates pathological hypertrophy and heart failure, in part, by enhancing cardiomyocyte contractility.
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http://dx.doi.org/10.7150/thno.50990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8315059PMC
August 2021

Transcriptomic Profiling of Control and Thyroid-Associated Orbitopathy (TAO) Orbital Fat and TAO Orbital Fibroblasts Undergoing Adipogenesis.

Invest Ophthalmol Vis Sci 2021 07;62(9):24

Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States.

Purpose: Orbital fat hyperplasia commonly occurs in thyroid-associated orbitopathy (TAO). To understand molecular mechanisms underlying orbital adipogenesis, we used transcriptomics to compare gene expression in controls and patients with TAO, as well as in orbital fibroblasts (OFs) undergoing adipogenic differentiation.

Methods: We performed bulk RNA sequencing (RNA-Seq) on intraconal orbital fat from controls and patients with TAO. We treated cultured OFs derived from patients with TAO with adipogenic media to induce adipogenesis. We used single nucleus RNA-Seq (snRNA-Seq) to profile treated and control OFs, identifying genes that are dynamically expressed during orbital adipogenesis in vitro, and compared these results to data from control and TAO orbital fat.

Results: Gene expression profiles in control and TAO orbital fat are distinct. Signaling pathways including PI3K-Akt signaling, cAMP signaling, AGE-RAGE signaling, regulation of lipolysis, and thyroid hormone signaling are enriched in orbital fat isolated from patients with TAO. SnRNA-Seq of orbital fibroblasts undergoing adipogenesis reveals differential expression of the adipocyte-specific genes FABP4/5, APOE, PPARG, and ADIPOQ during adipogenic differentiation. The insulin-like growth factor-1 receptor and Wnt signaling pathways appear to be enriched early in adipogenesis. Gene modules that are enriched in TAO orbital fat are upregulated in orbital adipocytes during differentiation in vitro, whereas genes that are enriched in control orbital fat are enriched in undifferentiated OFs.

Conclusions: We identified pathways enriched in TAO orbital fat, and dynamic changes in gene expression that occur during adipogenic differentiation of orbital fibroblasts. These findings may help guide functional studies of genes and pathways critical for orbital adipogenesis.
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http://dx.doi.org/10.1167/iovs.62.9.24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8297424PMC
July 2021

Cannabinoid signaling promotes the de-differentiation and proliferation of Müller glia-derived progenitor cells.

Glia 2021 Oct 7;69(10):2503-2521. Epub 2021 Jul 7.

Department of Neuroscience, College of Medicine, The Ohio State University, Columbus, Ohio, USA.

Endocannabinoids (eCB) are lipid-based neurotransmitters that are known to influence synaptic function in the visual system. eCBs are also known to suppress neuroinflammation in different pathological states. However, nothing is known about the roles of the eCB system during the transition of Müller glia (MG) into proliferating progenitor-like cells in the retina. Accordingly, we used the chick and mouse model to characterize expression patterns of eCB-related genes and applied pharmacological agents to investigate how the eCB system impacts glial reactivity and the capacity of MG to become Müller glia-derived progenitor cells (MGPCs). We queried single cell RNA-seq libraries to identify eCB-related genes and identify cells with dynamic patterns of expression in damaged retinas. MG and inner retinal neurons expressed the eCB receptor CNR1, as well as enzymes involved in eCB metabolism. In the chick, intraocular injections of cannabinoids, 2-Arachidonoylglycerol (2-AG) and Anandamide (AEA), stimulated the formation of MGPCs. Cannabinoid Receptor 1 (CNR1)-agonists and Monoglyceride Lipase-inhibitor promoted the formation of MGPCs, whereas CNR1-antagonist and inhibitors of eCB synthesis suppressed this process. In damaged mouse retinas where MG activate NFkB-signaling, activation of CNR1 decreased and inhibition of CNR1 increased NFkB, whereas levels of neuronal cell death were unaffected. Surprisingly, retinal microglia were largely unaffected by increases or decreases in eCB-signaling in both chick and mouse retinas. We conclude that the eCB system in the retina influences the reactivity of MG and the formation of proliferating MGPCs, but does not influence the reactivity of immune cells in the retina.
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http://dx.doi.org/10.1002/glia.24056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8373766PMC
October 2021

Control of neurogenic competence in mammalian hypothalamic tanycytes.

Sci Adv 2021 May 28;7(22). Epub 2021 May 28.

Solomon H. Snyder Department of Neuroscience, Johns Hopkins University, Baltimore, MD 21205, USA.

Hypothalamic tanycytes, radial glial cells that share many features with neuronal progenitors, can generate small numbers of neurons in the postnatal hypothalamus, but the identity of these neurons and the molecular mechanisms that control tanycyte-derived neurogenesis are unknown. In this study, we show that tanycyte-specific disruption of the NFI family of transcription factors () robustly stimulates tanycyte proliferation and tanycyte-derived neurogenesis. Single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) analysis reveals that NFI (nuclear factor I) factors repress Sonic hedgehog (Shh) and Wnt signaling in tanycytes and modulation of these pathways blocks proliferation and tanycyte-derived neurogenesis in -deficient mice. -deficient tanycytes give rise to multiple mediobasal hypothalamic neuronal subtypes that can mature, fire action potentials, receive synaptic inputs, and selectively respond to changes in internal states. These findings identify molecular mechanisms that control tanycyte-derived neurogenesis, which can potentially be targeted to selectively remodel the hypothalamic neural circuitry that controls homeostatic physiological processes.
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http://dx.doi.org/10.1126/sciadv.abg3777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163082PMC
May 2021

A potential role for somatostatin signaling in regulating retinal neurogenesis.

Sci Rep 2021 05 26;11(1):10962. Epub 2021 May 26.

Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.

Neuropeptides have been reported to regulate progenitor proliferation and neurogenesis in the central nervous system. However, these studies have typically been conducted using pharmacological agents in ex vivo preparations, and in vivo evidence for their developmental function is generally lacking. Recent scRNA-Seq studies have identified multiple neuropeptides and their receptors as being selectively expressed in neurogenic progenitors of the embryonic mouse and human retina. This includes Sstr2, whose ligand somatostatin is transiently expressed by immature retinal ganglion cells. By analyzing retinal explants treated with selective ligands that target these receptors, we found that Sstr2-dependent somatostatin signaling induces a modest, dose-dependent inhibition of photoreceptor generation, while correspondingly increasing the relative fraction of primary progenitor cells. These effects were confirmed by scRNA-Seq analysis of retinal explants but abolished in Sstr2-deficient retinas. Although no changes in the relative fraction of primary progenitors or photoreceptor precursors were observed in Sstr2-deficient retinas in vivo, scRNA-Seq analysis demonstrated accelerated differentiation of neurogenic progenitors. We conclude that, while Sstr2 signaling may act to negatively regulate retinal neurogenesis in combination with other retinal ganglion cell-derived secreted factors such as Shh, it is dispensable for normal retinal development.
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http://dx.doi.org/10.1038/s41598-021-90554-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8155210PMC
May 2021

An inducible Cre mouse for studying roles of the RPE in retinal physiology and disease.

JCI Insight 2021 May 10;6(9). Epub 2021 May 10.

Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, Irvine, California, USA.

The retinal pigment epithelium (RPE) provides vital metabolic support for retinal photoreceptor cells and is an important player in numerous retinal diseases. Gene manipulation in mice using the Cre-LoxP system is an invaluable tool for studying the genetic basis of these retinal diseases. However, existing RPE-targeted Cre mouse lines have critical limitations that restrict their reliability for studies of disease pathogenesis and treatment, including mosaic Cre expression, inducer-independent activity, off-target Cre expression, and intrinsic toxicity. Here, we report the generation and characterization of a knockin mouse line in which a P2A-CreERT2 coding sequence is fused with the native RPE-specific 65 kDa protein (Rpe65) gene for cotranslational expression of CreERT2. Cre+/- mice were able to recombine a stringent Cre reporter allele with more than 99% efficiency and absolute RPE specificity upon tamoxifen induction at both postnatal days (PD) 21 and 50. Tamoxifen-independent Cre activity was negligible at PD64. Moreover, tamoxifen-treated Cre+/- mice displayed no signs of structural or functional retinal pathology up to 4 months of age. Despite weak RPE65 expression from the knockin allele, visual cycle function was normal in Cre+/- mice. These data indicate that Rpe65CreERT2 mice are well suited for studies of gene function and pathophysiology in the RPE.
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http://dx.doi.org/10.1172/jci.insight.146604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262343PMC
May 2021

Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility using scRNA-Seq and scATAC-Seq.

J Vis Exp 2021 03 12(169). Epub 2021 Mar 12.

Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine; Department of Psychiatry and Behavioral Science, The Johns Hopkins Hospital; Department of Ophthalmology, The Johns Hopkins Hospital; Department of Neurology, The Johns Hopkins Hospital; Institute for Cell Engineering, Johns Hopkins University School of Medicine; Kavli Neuroscience Discovery Institute, Johns Hopkins University School of Medicine;

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during development and in disease states. Single-cell RNA sequencing allows us to comprehensively profile gene expression changes observed in retinal development and disease at a cellular level, while single-cell ATAC-Seq allows analysis of chromatin accessibility and transcription factor binding to be profiled at similar resolution. Here the use of these techniques in the developing retina is described, and MULTI-Seq is demonstrated, where individual samples are labeled with a modified oligonucleotide-lipid complex, enabling researchers to both increase the scope of individual experiments and substantially reduce costs.
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http://dx.doi.org/10.3791/62239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8356148PMC
March 2021

Atoh7-independent specification of retinal ganglion cell identity.

Sci Adv 2021 Mar 12;7(11). Epub 2021 Mar 12.

National Institute of Mental Health (NIMH), National Institutes of Health (NIH), Bethesda, MD, USA.

Retinal ganglion cells (RGCs) relay visual information from the eye to the brain. RGCs are the first cell type generated during retinal neurogenesis. Loss of function of the transcription factor , expressed in multipotent early neurogenic retinal progenitors leads to a selective and essentially complete loss of RGCs. Therefore, is considered essential for conferring competence on progenitors to generate RGCs. Despite the importance of Atoh7 in RGC specification, we find that inhibiting apoptosis in deficient mice by loss of function of only modestly reduces RGC numbers. Single-cell RNA sequencing of -deficient retinas shows that RGC differentiation is delayed but that the gene expression profile of RGC precursors is grossly normal. -deficient RGCs eventually mature, fire action potentials, and incorporate into retinal circuitry but exhibit severe axonal guidance defects. This study reveals an essential role for in RGC survival and demonstrates -dependent and independent mechanisms for RGC specification.
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http://dx.doi.org/10.1126/sciadv.abe4983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954457PMC
March 2021

Midkine is neuroprotective and influences glial reactivity and the formation of Müller glia-derived progenitor cells in chick and mouse retinas.

Glia 2021 Jun 10;69(6):1515-1539. Epub 2021 Feb 10.

Department of Neuroscience, College of Medicine, The Ohio State University, Columbus, Ohio, USA.

Recent studies suggest midkine (MDK) is involved in the development and regeneration of the zebrafish retina. We investigate the expression patterns of MDK and related factors, roles in neuronal survival, and influence upon the formation of Müller glia-derived progenitor cells (MGPCs) in chick and mouse model systems. By using single-cell RNA-sequencing, we find that MDK and pleiotrophin (PTN), a MDK-related cytokine, are upregulated by Müller glia (MG) during later stages of development in chick. While PTN is downregulated, MDK is dramatically upregulated in mature MG after retinal damage or FGF2 and insulin treatment. By comparison, MDK and PTN are downregulated by MG in damaged mouse retinas. In both chick and mouse retinas, exogenous MDK induces expression of cFos and pS6 in MG. In the chick, MDK significantly decreases numbers dying neurons, reactive microglia, and proliferating MGPCs, whereas PTN has no effect. Inhibition of MDK-signaling with Na VO blocks neuroprotective effects with an increase in the number of dying cells and negates the pro-proliferative effects on MGPCs in damaged retinas. Inhibitors of PP2A and Pak1, which are associated with MDK-signaling through integrin β1, suppressed the formation of MGPCs in damaged chick retinas. In mice, MDK promotes a small but significant increase in proliferating MGPCs in damaged retinas and potently decreases the number of dying cells. We conclude that MDK expression is dynamically regulated in Müller glia during embryonic maturation, following retinal injury, and during reprogramming into MGPCs. MDK mediates glial activity, neuronal survival, and the re-programming of Müller glia into proliferating MGPCs.
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http://dx.doi.org/10.1002/glia.23976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8194292PMC
June 2021

Temperature and species-dependent regulation of browning in retrobulbar fat.

Sci Rep 2021 02 4;11(1):3094. Epub 2021 Feb 4.

Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Retrobulbar fat deposits surround the posterior retina and optic nerve head, but their function and origin are obscure. We report that mouse retrobulbar fat is a neural crest-derived tissue histologically and transcriptionally resembles interscapular brown fat. In contrast, human retrobulbar fat closely resembles white adipose tissue. Retrobulbar fat is also brown in other rodents, which are typically housed at temperatures below thermoneutrality, but is white in larger animals. We show that retrobulbar fat in mice housed at thermoneutral temperature show reduced expression of the brown fat marker Ucp1, and histological properties intermediate between white and brown fat. We conclude that retrobulbar fat can potentially serve as a site of active thermogenesis, that this capability is both temperature and species-dependent, and that this may facilitate regulation of intraocular temperature.
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http://dx.doi.org/10.1038/s41598-021-82672-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862600PMC
February 2021

Dual midbrain and forebrain origins of thalamic inhibitory interneurons.

Elife 2021 Feb 1;10. Epub 2021 Feb 1.

Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, United Kingdom.

The ubiquitous presence of inhibitory interneurons in the thalamus of primates contrasts with the sparsity of interneurons reported in mice. Here, we identify a larger than expected complexity and distribution of interneurons across the mouse thalamus, where all thalamic interneurons can be traced back to two developmental programmes: one specified in the midbrain and the other in the forebrain. Interneurons migrate to functionally distinct thalamocortical nuclei depending on their origin: the abundant, midbrain-derived class populates the first and higher order sensory thalamus while the rarer, forebrain-generated class is restricted to some higher order associative regions. We also observe that markers for the midbrain-born class are abundantly expressed throughout the thalamus of the New World monkey marmoset. These data therefore reveal that, despite the broad variability in interneuron density across mammalian species, the blueprint of the ontogenetic organisation of thalamic interneurons of larger-brained mammals exists and can be studied in mice.
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http://dx.doi.org/10.7554/eLife.59272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906600PMC
February 2021

Gene regulatory networks controlling differentiation, survival, and diversification of hypothalamic Lhx6-expressing GABAergic neurons.

Commun Biol 2021 01 21;4(1):95. Epub 2021 Jan 21.

Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.

GABAergic neurons of the hypothalamus regulate many innate behaviors, but little is known about the mechanisms that control their development. We previously identified hypothalamic neurons that express the LIM homeodomain transcription factor Lhx6, a master regulator of cortical interneuron development, as sleep-promoting. In contrast to telencephalic interneurons, hypothalamic Lhx6 neurons do not undergo long-distance tangential migration and do not express cortical interneuronal markers such as Pvalb. Here, we show that Lhx6 is necessary for the survival of hypothalamic neurons. Dlx1/2, Nkx2-2, and Nkx2-1 are each required for specification of spatially distinct subsets of hypothalamic Lhx6 neurons, and that Nkx2-2+/Lhx6+ neurons of the zona incerta are responsive to sleep pressure. We further identify multiple neuropeptides that are enriched in spatially segregated subsets of hypothalamic Lhx6 neurons, and that are distinct from those seen in cortical neurons. These findings identify common and divergent molecular mechanisms by which Lhx6 controls the development of GABAergic neurons in the hypothalamus.
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http://dx.doi.org/10.1038/s42003-020-01616-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820013PMC
January 2021

Characterization of mWake expression in the murine brain.

J Comp Neurol 2021 06 10;529(8):1954-1987. Epub 2020 Nov 10.

Department of Neurology, Johns Hopkins University, Baltimore, Maryland, USA.

Structure-function analyses of the mammalian brain have historically relied on anatomically-based approaches. In these investigations, physical, chemical, or electrolytic lesions of anatomical structures are applied, and the resulting behavioral or physiological responses assayed. An alternative approach is to focus on the expression pattern of a molecule whose function has been characterized and then use genetic intersectional methods to optogenetically or chemogenetically manipulate distinct circuits. We previously identified WIDE AWAKE (WAKE) in Drosophila, a clock output molecule that mediates the temporal regulation of sleep onset and sleep maintenance. More recently, we have studied the mouse homolog, mWAKE/ANKFN1, and our data suggest that its basic role in the circadian regulation of arousal is conserved. Here, we perform a systematic analysis of the expression pattern of mWake mRNA, protein, and cells throughout the adult mouse brain. We find that mWAKE labels neurons in a restricted, but distributed manner, in multiple regions of the hypothalamus (including the suprachiasmatic nucleus, dorsomedial hypothalamus, and tuberomammillary nucleus region), the limbic system, sensory processing nuclei, and additional specific brainstem, subcortical, and cortical areas. Interestingly, mWAKE is also observed in non-neuronal ependymal cells. In addition, to describe the molecular identities and clustering of mWake cells, we provide detailed analyses of single cell RNA sequencing data from the hypothalamus, a region with particularly significant mWAKE expression. These findings lay the groundwork for future studies into the potential role of mWAKE cells in the rhythmic control of diverse behaviors and physiological processes.
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http://dx.doi.org/10.1002/cne.25066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8009828PMC
June 2021

Gene regulatory networks controlling vertebrate retinal regeneration.

Science 2020 11 1;370(6519). Epub 2020 Oct 1.

Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Injury induces retinal Müller glia of certain cold-blooded vertebrates, but not those of mammals, to regenerate neurons. To identify gene regulatory networks that reprogram Müller glia into progenitor cells, we profiled changes in gene expression and chromatin accessibility in Müller glia from zebrafish, chick, and mice in response to different stimuli. We identified evolutionarily conserved and species-specific gene networks controlling glial quiescence, reactivity, and neurogenesis. In zebrafish and chick, the transition from quiescence to reactivity is essential for retinal regeneration, whereas in mice, a dedicated network suppresses neurogenic competence and restores quiescence. Disruption of nuclear factor I transcription factors, which maintain and restore quiescence, induces Müller glia to proliferate and generate neurons in adult mice after injury. These findings may aid in designing therapies to restore retinal neurons lost to degenerative diseases.
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http://dx.doi.org/10.1126/science.abb8598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7899183PMC
November 2020

Zeb2 regulates the balance between retinal interneurons and Müller glia by inhibition of BMP-Smad signaling.

Dev Biol 2020 12 17;468(1-2):80-92. Epub 2020 Sep 17.

Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel; Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 69978, Israel. Electronic address:

The interplay between signaling molecules and transcription factors during retinal development is key to controlling the correct number of retinal cell types. Zeb2 (Sip1) is a zinc-finger multidomain transcription factor that plays multiple roles in central and peripheral nervous system development. Haploinsufficiency of ZEB2 causes Mowat-Wilson syndrome, a congenital disease characterized by intellectual disability, epilepsy and Hirschsprung disease. In the developing retina, Zeb2 is required for generation of horizontal cells and the correct number of interneurons; however, its potential function in controlling gliogenic versus neurogenic decisions remains unresolved. Here we present cellular and molecular evidence of the inhibition of Müller glia cell fate by Zeb2 in late stages of retinogenesis. Unbiased transcriptomic profiling of control and Zeb2-deficient early-postnatal retina revealed that Zeb2 functions in inhibiting Id1/2/4 and Hes1 gene expression. These neural progenitor factors normally inhibit neural differentiation and promote Müller glia cell fate. Chromatin immunoprecipitation (ChIP) supported direct regulation of Id1 by Zeb2 in the postnatal retina. Reporter assays and ChIP analyses in differentiating neural progenitors provided further evidence that Zeb2 inhibits Id1 through inhibition of Smad-mediated activation of Id1 transcription. Together, the results suggest that Zeb2 promotes the timely differentiation of retinal interneurons at least in part by repressing BMP-Smad/Notch target genes that inhibit neurogenesis. These findings show that Zeb2 integrates extrinsic cues to regulate the balance between neuronal and glial cell types in the developing murine retina.
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http://dx.doi.org/10.1016/j.ydbio.2020.09.006DOI Listing
December 2020

The cellular and molecular landscape of hypothalamic patterning and differentiation from embryonic to late postnatal development.

Nat Commun 2020 08 31;11(1):4360. Epub 2020 Aug 31.

Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.

The hypothalamus is a central regulator of many innate behaviors essential for survival, but the molecular mechanisms controlling hypothalamic patterning and cell fate specification are poorly understood. To identify genes that control hypothalamic development, we have used single-cell RNA sequencing (scRNA-Seq) to profile mouse hypothalamic gene expression across 12 developmental time points between embryonic day 10 and postnatal day 45. This identified genes that delineated clear developmental trajectories for all major hypothalamic cell types, and readily distinguished major regional subdivisions of the developing hypothalamus. By using our developmental dataset, we were able to rapidly annotate previously unidentified clusters from existing scRNA-Seq datasets collected during development and to identify the developmental origins of major neuronal populations of the ventromedial hypothalamus. We further show that our approach can rapidly and comprehensively characterize mutants that have altered hypothalamic patterning, identifying Nkx2.1 as a negative regulator of prethalamic identity. These data serve as a resource for further studies of hypothalamic development, physiology, and dysfunction.
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http://dx.doi.org/10.1038/s41467-020-18231-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459115PMC
August 2020

Epigenetic hallmarks of age-related macular degeneration are recapitulated in a photosensitive mouse model.

Hum Mol Genet 2020 08;29(15):2611-2624

Gavin Herbert Eye Institute and the Department of Ophthalmology, University of California-Irvine, Irvine, CA 92697, USA.

Age-related macular degeneration (AMD) is a chronic, multifactorial disorder and a leading cause of blindness in the elderly. Characterized by progressive photoreceptor degeneration in the central retina, disease progression involves epigenetic changes in chromatin accessibility resulting from environmental exposures and chronic stress. Here, we report that a photosensitive mouse model of acute stress-induced photoreceptor degeneration recapitulates the epigenetic hallmarks of human AMD. Global epigenomic profiling was accomplished by employing an Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq), which revealed an association between decreased chromatin accessibility and stress-induced photoreceptor cell death in our mouse model. The epigenomic changes induced by light damage include reduced euchromatin and increased heterochromatin abundance, resulting in transcriptional and translational dysregulation that ultimately drives photoreceptor apoptosis and an inflammatory reactive gliosis in the retina. Of particular interest, pharmacological inhibition of histone deacetylase 11 (HDAC11) and suppressor of variegation 3-9 homolog 2 (SUV39H2), key histone-modifying enzymes involved in promoting reduced chromatin accessibility, ameliorated light damage in our mouse model, supporting a causal link between decreased chromatin accessibility and photoreceptor degeneration, thereby elucidating a potential new therapeutic strategy to combat AMD.
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http://dx.doi.org/10.1093/hmg/ddaa158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7471509PMC
August 2020

A single cell transcriptional atlas of early synovial joint development.

Development 2020 07 20;147(14). Epub 2020 Jul 20.

Institute for Cell Engineering, Johns Hopkins School of Medicine, Baltimore MD 21205, USA

Synovial joint development begins with the formation of the interzone, a region of condensed mesenchymal cells at the site of the prospective joint. Recently, lineage-tracing strategies have revealed that Gdf5-lineage cells native to and from outside the interzone contribute to most, if not all, of the major joint components. However, there is limited knowledge of the specific transcriptional and signaling programs that regulate interzone formation and fate diversification of synovial joint constituents. To address this, we have performed single cell RNA-Seq analysis of 7329 synovial joint progenitor cells from the developing murine knee joint from E12.5 to E15.5. By using a combination of computational analytics, hybridization and characterization of prospectively isolated populations, we have identified the transcriptional profiles of the major developmental paths for joint progenitors. Our freely available single cell transcriptional atlas will serve as a resource for the community to uncover transcriptional programs and cell interactions that regulate synovial joint development.
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http://dx.doi.org/10.1242/dev.185777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7390639PMC
July 2020

Single-Cell Analysis of Human Retina Identifies Evolutionarily Conserved and Species-Specific Mechanisms Controlling Development.

Dev Cell 2020 05 7;53(4):473-491.e9. Epub 2020 May 7.

John F. Hardesty, MD, Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.devcel.2020.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8015270PMC
May 2020

NF-κB signaling regulates the formation of proliferating Müller glia-derived progenitor cells in the avian retina.

Development 2020 05 22;147(10). Epub 2020 May 22.

Department of Neuroscience, College of Medicine, The Ohio State University, Columbus, OH 43210, USA

Retinal regeneration is robust in some cold-blooded vertebrates, but this process is ineffective in warm-blooded vertebrates. Understanding the mechanisms that suppress the reprogramming of Müller glia into neurogenic progenitors is key to harnessing the regenerative potential of the retina Inflammation and reactive microglia are known to influence the formation of Müller glia-derived progenitor cells (MGPCs), but the mechanisms underlying this interaction are unknown. We used a chick model to investigate nuclear factor kappa B (NF-κB) signaling, a critical regulator of inflammation, during the reprogramming of Müller glia into proliferating progenitors. We find that components of the NF-κB pathway are dynamically regulated by Müller glia after neuronal damage or treatment with growth factors. Inhibition of NF-κB enhances, whereas activation suppresses, the formation of proliferating MGPCs. Following microglia ablation, the effects of NF-κB-agonists on MGPC-formation are reversed, suggesting that signals provided by reactive microglia influence how NF-κB impacts Müller glia reprogramming. We propose that NF-κB is an important signaling 'hub' that suppresses the reprogramming of Müller glia into proliferating MGPCs and this 'hub' coordinates signals provided by reactive microglia.
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http://dx.doi.org/10.1242/dev.183418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325431PMC
May 2020

Tanycyte ablation in the arcuate nucleus and median eminence increases obesity susceptibility by increasing body fat content in male mice.

Glia 2020 10 16;68(10):1987-2000. Epub 2020 Mar 16.

Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Tanycytes are radial glial cells located in the mediobasal hypothalamus. Recent studies have proposed that tanycytes play an important role in hypothalamic control of energy homeostasis, although this has not been directly tested. Here, we report the phenotype of mice in which tanycytes of the arcuate nucleus and median eminence were conditionally ablated in adult mice. Although the cerebrospinal fluid-hypothalamic barrier was rendered more permeable following tanycyte ablation, neither the blood-hypothalamic barrier nor leptin-induced pSTAT3 activation in hypothalamic parenchyma were affected. We observed a significant increase in visceral fat distribution accompanying insulin insensitivity in male mice, without significant effect on either body weight or food intake. A high-fat diet tended to accelerate overall body weight gain in tanycyte-ablated mice, but the development of visceral adiposity and insulin insensitivity was comparable to wildtype. Thermoneutral housing exacerbated fat accumulation and produced a shift away from fat oxidation in tanycyte-ablated mice. These results clarify the extent to which tanycytes regulate energy balance, and demonstrate a role for tanycytes in regulating fat metabolism.
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http://dx.doi.org/10.1002/glia.23817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7423758PMC
October 2020

ASCOT identifies key regulators of neuronal subtype-specific splicing.

Nat Commun 2020 01 9;11(1):137. Epub 2020 Jan 9.

Kavli Neuroscience Discovery Institute, Johns Hopkins University, Baltimore, MD, USA.

Public archives of next-generation sequencing data are growing exponentially, but the difficulty of marshaling this data has led to its underutilization by scientists. Here, we present ASCOT, a resource that uses annotation-free methods to rapidly analyze and visualize splice variants across tens of thousands of bulk and single-cell data sets in the public archive. To demonstrate the utility of ASCOT, we identify novel cell type-specific alternative exons across the nervous system and leverage ENCODE and GTEx data sets to study the unique splicing of photoreceptors. We find that PTBP1 knockdown and MSI1 and PCBP2 overexpression are sufficient to activate many photoreceptor-specific exons in HepG2 liver cancer cells. This work demonstrates how large-scale analysis of public RNA-Seq data sets can yield key insights into cell type-specific control of RNA splicing and underscores the importance of considering both annotated and unannotated splicing events.
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http://dx.doi.org/10.1038/s41467-019-14020-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952364PMC
January 2020

Photic generation of 11--retinal in bovine retinal pigment epithelium.

J Biol Chem 2019 12 6;294(50):19137-19154. Epub 2019 Nov 6.

Gavin Herbert Eye Institute, Department of Ophthalmology, University of California, Irvine, California 92697

Photoisomerization of the 11--retinal chromophore of rod and cone visual pigments to an all--configuration is the initiating event for vision in vertebrates. The regeneration of 11--retinal, necessary for sustained visual function, is an endergonic process normally conducted by specialized enzyme systems. However, 11--retinal also can be formed through reverse photoisomerization from all--retinal. A nonvisual opsin known as retinal pigment epithelium (RPE)-retinal G-protein-coupled receptor (RGR) was previously shown to mediate visual chromophore regeneration in photic conditions, but conflicting results have cast doubt on its role as a photoisomerase. Here, we describe high-level production of 11--retinal from RPE membranes stimulated by illumination at a narrow band of wavelengths. This activity was associated with RGR and enhanced by cellular retinaldehyde-binding protein (CRALBP), which binds the 11--retinal produced by RGR and prevents its re-isomerization to all--retinal. The activity was recapitulated with cells heterologously expressing RGR and with purified recombinant RGR. Using an RGR variant, K255A, we confirmed that a Schiff base linkage at Lys-255 is critical for substrate binding and isomerization. Single-cell RNA-Seq analysis of the retina and RPE tissue confirmed that RGR is expressed in human and bovine RPE and Müller glia, whereas mouse RGR is expressed in RPE but not in Müller glia. These results provide key insights into the mechanisms of physiological retinoid photoisomerization and suggest a novel mechanism by which RGR, in concert with CRALBP, regenerates the visual chromophore in the RPE under sustained light conditions.
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http://dx.doi.org/10.1074/jbc.RA119.011169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6916499PMC
December 2019

Clarin-1 expression in adult mouse and human retina highlights a role of Müller glia in Usher syndrome.

J Pathol 2020 02 4;250(2):195-204. Epub 2019 Dec 4.

Department of Ophthalmology, University of Florida, Gainesville, FL, USA.

Usher syndrome type 3 (USH3) is an autosomal recessively inherited disorder caused by mutations in the gene clarin-1 (CLRN1), leading to combined progressive hearing loss and retinal degeneration. The cellular distribution of CLRN1 in the retina remains uncertain, either because its expression levels are low or because its epitopes are masked. Indeed, in the adult mouse retina, Clrn1 mRNA is developmentally downregulated, detectable only by RT-PCR. In this study we used the highly sensitive RNAscope in situ hybridization assay and single-cell RNA-sequencing techniques to investigate the distribution of Clrn1 and CLRN1 in mouse and human retina, respectively. We found that Clrn1 transcripts in mouse tissue are localized to the inner retina during postnatal development and in adult stages. The pattern of Clrn1 mRNA cellular expression is similar in both mouse and human adult retina, with CLRN1 transcripts being localized in Müller glia, and not photoreceptors. We generated a novel knock-in mouse with a hemagglutinin (HA) epitope-tagged CLRN1 and showed that CLRN1 is expressed continuously at the protein level in the retina. Following enzymatic deglycosylation and immunoblotting analysis, we detected a single CLRN1-specific protein band in homogenates of mouse and human retina, consistent in size with the main CLRN1 isoform. Taken together, our results implicate Müller glia in USH3 pathology, placing this cell type to the center of future mechanistic and therapeutic studies to prevent vision loss in this disease. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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http://dx.doi.org/10.1002/path.5360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003947PMC
February 2020

Tissue- and Species-Specific Patterns of RNA metabolism in Post-Mortem Mammalian Retina and Retinal Pigment Epithelium.

Sci Rep 2019 10 15;9(1):14821. Epub 2019 Oct 15.

Gavin Herbert Eye Institute and the Department of Ophthalmology, University of California-Irvine, Irvine, CA, 92657, USA.

Accurate analysis of gene expression in human tissues using RNA sequencing is dependent on the quality of source material. One major source of variation in mRNA quality is post-mortem time. While it is known that individual transcripts show differential post-mortem stability, few studies have directly and comprehensively analyzed mRNA stability following death, and in particular the extent to which tissue- and species-specific factors influence post-mortem mRNA stability are poorly understood. This knowledge is particularly important for ocular tissues studies, where tissues obtained post-mortem are frequently used for research or therapeutic applications. To directly investigate this question, we profiled mRNA levels in both neuroretina and retinal pigment epithelium (RPE) from mouse and baboon over a series of post-mortem intervals. We found substantial changes in gene expression as early as 15 minutes in the mouse and as early as three hours in the baboon eye tissues. Importantly, our findings demonstrate both tissue- and species- specific patterns of RNA metabolism, by identifying a set of genes that are either rapidly degraded or very stable in both species and/or tissues. Taken together, the data from this study lay the foundation for understanding RNA regulation post-mortem and provide novel insights into RNA metabolism in the tissues of the mammalian eye.
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http://dx.doi.org/10.1038/s41598-019-51379-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794289PMC
October 2019

PanoView: An iterative clustering method for single-cell RNA sequencing data.

PLoS Comput Biol 2019 08 30;15(8):e1007040. Epub 2019 Aug 30.

Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD, United States of America.

Single-cell RNA-sequencing (scRNA-seq) provides new opportunities to gain a mechanistic understanding of many biological processes. Current approaches for single cell clustering are often sensitive to the input parameters and have difficulty dealing with cell types with different densities. Here, we present Panoramic View (PanoView), an iterative method integrated with a novel density-based clustering, Ordering Local Maximum by Convex hull (OLMC), that uses a heuristic approach to estimate the required parameters based on the input data structures. In each iteration, PanoView will identify the most confident cell clusters and repeat the clustering with the remaining cells in a new PCA space. Without adjusting any parameter in PanoView, we demonstrated that PanoView was able to detect major and rare cell types simultaneously and outperformed other existing methods in both simulated datasets and published single-cell RNA-sequencing datasets. Finally, we conducted scRNA-Seq analysis of embryonic mouse hypothalamus, and PanoView was able to reveal known cell types and several rare cell subpopulations.
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http://dx.doi.org/10.1371/journal.pcbi.1007040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6742414PMC
August 2019
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