ABSTRACT
Direct neuronal reprogramming is a promising approach to regenerate neurons from local glial cells. However, mechanisms of epigenome remodeling and co-factors facilitating this process are unclear. In this study, we combined single-cell multiomics with genome-wide profiling of three-dimensional nuclear architecture and DNA methylation in mouse astrocyte-to-neuron reprogramming mediated by Neurogenin2 (Ngn2) and its phosphorylation-resistant form (PmutNgn2), respectively. We show that Ngn2 drives multilayered chromatin remodeling at dynamic enhancer-gene interaction sites. PmutNgn2 leads to higher reprogramming efficiency and enhances epigenetic remodeling associated with neuronal maturation. However, the differences in binding sites or downstream gene activation cannot fully explain this effect. Instead, we identified Yy1, a transcriptional co-factor recruited by direct interaction with Ngn2 to its target sites. Upon deletion of Yy1, activation of neuronal enhancers, genes and ultimately reprogramming are impaired without affecting Ngn2 binding. Thus, our work highlights the key role of interactors of proneural factors in direct neuronal reprogramming.
Subject(s)
Astrocytes , Basic Helix-Loop-Helix Transcription Factors , Cellular Reprogramming , Nerve Tissue Proteins , Neurons , YY1 Transcription Factor , Animals , YY1 Transcription Factor/metabolism , YY1 Transcription Factor/genetics , Astrocytes/metabolism , Mice , Cellular Reprogramming/physiology , Neurons/metabolism , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Epigenome , Chromatin Assembly and Disassembly , Epigenesis, Genetic , Cells, CulturedABSTRACT
Activation of neural stem cells (NSCs) correlates with improved functional outcomes in mouse models of injury. In the murine brain, NSCs have been extensively characterized and comprise (1) primitive NSCs (pNSCs) and (2) definitive NSCs (dNSCs). pNSCs are the earliest cells in the NSC lineage giving rise to dNSCs in the embryonic and adult mouse brain. pNSCs are quiescent under baseline conditions and can be activated upon injury. Herein, we asked whether human pNSCs and dNSCs can be isolated during the maturation of human cerebral organoids (COs) and activated by drugs known to regulate mouse NSC behavior. We demonstrate that self-renewing, multipotent pNSC and dNSC populations are present in human COs and express genes previously characterized in mouse NSCs. The drug NWL283, an inhibitor of apoptosis, reduced cell death in COs but did not improve NSC survival. Metformin, a drug used to treat type II diabetes that is known to promote NSC activation in mice, was found to expand human NSC pools. Together, these findings are the first to identify and characterize human pNSCs, advancing our understanding of the human NSC lineage and highlighting drugs that enhance their activity.
Subject(s)
Neural Stem Cells , Organoids , Humans , Neural Stem Cells/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/drug effects , Organoids/metabolism , Organoids/cytology , Organoids/drug effects , Animals , Mice , Cell Differentiation , Metformin/pharmacology , Cells, Cultured , Brain/metabolism , Brain/cytologyABSTRACT
Purpose: The blood-retinal barrier (BRB) restricts the delivery of intravenous therapeutics to the retina, necessitating innovative approaches for treating retinal disorders. This study sought to explore the potential of focused ultrasound (FUS) to non-invasively deliver intravenously administered gold nanoparticles (AuNPs) across the BRB. FUS-BRB modulation can offer a novel method for targeted retinal therapy. Methods: AuNPs of different sizes and shapes were characterized, and FUS parameters were optimized to permeate the BRB without causing retinal damage in a rodent model. The delivery of 70-kDa dextran and AuNPs to the retinal ganglion cell (RGC) layer was visualized using confocal and two-photon microscopy, respectively. Histological and statistical analyses were conducted to assess the effectiveness and safety of the procedure. Results: FUS-BRB modulation resulted in the delivery of dextran and AuNPs to the RGC and inner nuclear layer. Smaller AuNPs reached the retinal layers to a greater extent than larger ones. The delivery of dextran and AuNPs across the BRB with FUS was achieved without significant retinal damage. Conclusions: This investigation provides the first evidence, to our knowledge, of FUS-mediated AuNP delivery across the BRB, establishing a foundation for a targeted and non-invasive approach to retinal treatment. The results contribute to developing promising non-invasive therapeutic strategies in ophthalmology to treat retinal diseases. Translational Relevance: Modifying the BRB with ultrasound offers a targeted and non-invasive delivery strategy of intravenous therapeutics to the retina.
Subject(s)
Blood-Retinal Barrier , Gold , Metal Nanoparticles , Retinal Ganglion Cells , Animals , Gold/chemistry , Gold/administration & dosage , Retinal Ganglion Cells/cytology , Metal Nanoparticles/administration & dosage , Metal Nanoparticles/chemistry , Dextrans/administration & dosage , Dextrans/chemistry , Drug Delivery Systems/methods , Rats , Microscopy, Confocal/methods , MaleABSTRACT
The retina is exquisitely patterned, with neuronal somata positioned at regular intervals to completely sample the visual field. Here, we show that phosphatase and tensin homolog (Pten) controls starburst amacrine cell spacing by modulating vesicular trafficking of cell adhesion molecules and Wnt proteins. Single-cell transcriptomics and double-mutant analyses revealed that Pten and Down syndrome cell adhesion molecule Dscam) are co-expressed and function additively to pattern starburst amacrine cell mosaics. Mechanistically, Pten loss accelerates the endocytic trafficking of DSCAM, FAT3, and MEGF10 off the cell membrane and into endocytic vesicles in amacrine cells. Accordingly, the vesicular proteome, a molecular signature of the cell of origin, is enriched in exocytosis, vesicle-mediated transport, and receptor internalization proteins in Pten conditional knockout (PtencKO) retinas. Wnt signaling molecules are also enriched in PtencKO retinal vesicles, and the genetic or pharmacological disruption of Wnt signaling phenocopies amacrine cell patterning defects. Pten thus controls vesicular trafficking of cell adhesion and signaling molecules to establish retinal amacrine cell mosaics.
Subject(s)
Amacrine Cells , Cell Adhesion , Endocytosis , PTEN Phosphohydrolase , Retina , Wnt Signaling Pathway , Animals , PTEN Phosphohydrolase/metabolism , PTEN Phosphohydrolase/genetics , Retina/metabolism , Mice , Amacrine Cells/metabolism , Mice, Knockout , Protein Transport , Wnt Proteins/metabolism , Cell Adhesion Molecules/metabolism , Cell Adhesion Molecules/geneticsABSTRACT
The cerebral cortex develops through a carefully conscripted series of cellular and molecular events that culminate in the production of highly specialized neuronal and glial cells. During development, cortical neurons and glia acquire a precise cellular arrangement and architecture to support higher-order cognitive functioning. Decades of study using rodent models, naturally gyrencephalic animal models, human pathology specimens, and, recently, human cerebral organoids, reveal that rodents recapitulate some but not all the cellular and molecular features of human cortices. Whereas rodent cortices are smooth-surfaced or lissencephalic, larger mammals, including humans and nonhuman primates, have highly folded/gyrencephalic cortices that accommodate an expansion in neuronal mass and increase in surface area. Several genes have evolved to drive cortical gyrification, arising from gene duplications or de novo origins, or by alterations to the structure/function of ancestral genes or their gene regulatory regions. Primary cortical folds arise in stereotypical locations, prefigured by a molecular "blueprint" that is set up by several signaling pathways (e.g., Notch, Fgf, Wnt, PI3K, Shh) and influenced by the extracellular matrix. Mutations that affect neural progenitor cell proliferation and/or neurogenesis, predominantly of upper-layer neurons, perturb cortical gyrification. Below we review the molecular drivers of cortical folding and their roles in disease.
ABSTRACT
[This corrects the article DOI: 10.3389/fnins.2022.919462.].
ABSTRACT
The proneural transcription factor Achaete-scute complex-like 1 (Ascl1) is a major regulator of neural fate decisions, implicated both in neurogenesis and oligodendrogliogenesis. Focusing on its neurogenic activity, Ascl1 has been widely used to reprogram non-neuronal cells into induced neurons. In vitro, Ascl1 induces efficient reprogramming of proliferative astroglia from the early postnatal cerebral cortex into interneuron-like cells. Here, we examined whether Ascl1 can similarly induce neuronal reprogramming of glia undergoing proliferation in the postnatal mouse cerebral cortex in vivo. Toward this goal, we targeted cortical glia during the peak of proliferative expansion (i.e., postnatal day 5) by injecting a retrovirus encoding for Ascl1 into the mouse cerebral cortex. In contrast to the efficient reprogramming observed in vitro, in vivo Ascl1-transduced glial cells were converted into doublecortin-immunoreactive neurons only with very low efficiency. However, we noted a drastic shift in the relative number of retrovirus-transduced Sox10-positive oligodendrocyte progenitor cells (OPCs) as compared to glial fibrillary acidic protein (GFAP)-positive astrocytes. Genetic fate mapping demonstrated that this increase in OPCs was not due to Ascl1-mediated astrocyte-to-OPC fate conversion. Rather, EdU incorporation experiments revealed that Ascl1 caused a selective increase in proliferative activity of OPCs, but not astrocytes. Our data indicate that rather than inducing neuronal reprogramming of glia in the early postnatal cortex, Ascl1 is a selective enhancer of OPC proliferation.
ABSTRACT
Direct neuronal reprogramming, the process whereby a terminally differentiated cell is converted into an induced neuron without traversing a pluripotent state, has tremendous therapeutic potential for a host of neurodegenerative diseases. While there is strong evidence for astrocyte-to-neuron conversion in vitro, in vivo studies in the adult brain are less supportive or controversial. Here, we set out to enhance the efficacy of neuronal conversion of adult astrocytes in vivo by optimizing the neurogenic capacity of a driver transcription factor encoded by the proneural gene Ascl1. Specifically, we mutated six serine phospho-acceptor sites in Ascl1 to alanines (Ascl1 SA 6) to prevent phosphorylation by proline-directed serine/threonine kinases. Native Ascl1 or Ascl1 SA 6 were expressed in adult, murine cortical astrocytes under the control of a glial fibrillary acidic protein (GFAP) promoter using adeno-associated viruses (AAVs). When targeted to the cerebral cortex in vivo, mCherry+ cells transduced with AAV8-GFAP-Ascl1 SA 6-mCherry or AAV8-GFAP-Ascl1-mCherry expressed neuronal markers within 14 days post-transduction, with Ascl1 SA 6 promoting the formation of more mature dendritic arbors compared to Ascl1. However, mCherry expression disappeared by 2-months post-transduction of the AAV8-GFAP-mCherry control-vector. To circumvent reporter issues, AAV-GFAP-iCre (control) and AAV-GFAP-Ascl1 (or Ascl1 SA 6)-iCre constructs were generated and injected into the cerebral cortex of Rosa reporter mice. In all comparisons of AAV capsids (AAV5 and AAV8), GFAP promoters (long and short), and reporter mice (Rosa-zsGreen and Rosa-tdtomato), Ascl1 SA 6 transduced cells more frequently expressed early- (Dcx) and late- (NeuN) neuronal markers. Furthermore, Ascl1 SA 6 repressed the expression of astrocytic markers Sox9 and GFAP more efficiently than Ascl1. Finally, we co-transduced an AAV expressing ChR2-(H134R)-YFP, an optogenetic actuator. After channelrhodopsin photostimulation, we found that Ascl1 SA 6 co-transduced astrocytes exhibited a significantly faster decay of evoked potentials to baseline, a neuronal feature, when compared to iCre control cells. Taken together, our findings support an enhanced neuronal conversion efficiency of Ascl1 SA 6 vs. Ascl1, and position Ascl1 SA 6 as a critical transcription factor for future studies aimed at converting adult brain astrocytes to mature neurons to treat disease.
ABSTRACT
Vision commences in the retina with rod and cone photoreceptors that detect and convert light to electrical signals. The irreversible loss of photoreceptors due to neurodegenerative disease leads to visual impairment and blindness. Interventions now in development include transplanting photoreceptors, committed photoreceptor precursors, or retinal pigment epithelial (RPE) cells, with the latter protecting photoreceptors from dying. However, introducing exogenous human cells in a clinical setting faces both regulatory and supply chain hurdles. Recent work has shown that abnormalities in central cell metabolism pathways are an underlying feature of most neurodegenerative disorders, including those in the retina. Reversal of key metabolic alterations to drive retinal repair thus represents a novel strategy to treat vision loss based on cell regeneration. Here, we review the connection between photoreceptor degeneration and alterations in cell metabolism, along with new insights into how metabolic reprogramming drives both retinal development and repair following damage. The potential impact of metabolic reprogramming on retinal regeneration is also discussed, specifically in the context of how metabolic switches drive both retinal development and the activation of retinal glial cells known as Müller glia. Müller glia display latent regenerative properties in teleost fish, however, their capacity to regenerate new photoreceptors has been lost in mammals. Thus, re-activating the regenerative properties of Müller glia in mammals represents an exciting new area that integrates research into developmental cues, central metabolism, disease mechanisms, and glial cell biology. In addition, we discuss this work in relation to the latest insights gleaned from other tissues (brain, muscle) and regenerative species (zebrafish).
ABSTRACT
The retinal pigmented epithelium (RPE) plays a critical role in photoreceptor survival and function. RPE deficits are implicated in a wide range of diseases that result in vision loss, including age-related macular degeneration (AMD) and Stargardt disease, affecting millions worldwide. Subretinal delivery of RPE cells is considered a promising avenue for treatment, and encouraging results from animal trials have supported recent progression into the clinic. However, the limited survival and engraftment of transplanted RPE cells delivered as a suspension continues to be a major challenge. While RPE delivery as epithelial sheets exhibits improved outcomes, this comes at the price of increased complexity at both the production and transplant stages. In order to combine the benefits of both approaches, we have developed size-controlled, scaffold-free RPE microtissues (RPE-µTs) that are suitable for scalable production and delivery via injection. RPE-µTs retain key RPE molecular markers, and interestingly, in comparison to conventional monolayer cultures, they show significant increases in the transcription and secretion of pigment-epithelium-derived factor (PEDF), which is a key trophic factor known to enhance the survival and function of photoreceptors. Furthermore, these microtissues readily spread in vitro on a substrate analogous to Bruch's membrane, suggesting that RPE-µTs may collapse into a sheet upon transplantation. We anticipate that this approach may provide an alternative cell delivery system to improve the survival and integration of RPE transplants, while also retaining the benefits of low complexity in production and delivery.
Subject(s)
Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/transplantation , Tissue Engineering/methods , Cell Adhesion , Cell Line , Cells, Cultured , Choroid/cytology , Eye Proteins/metabolism , Human Embryonic Stem Cells , Humans , Macular Degeneration/therapy , Nerve Growth Factors/metabolism , Retina/cytology , Retina/metabolism , Retinal Pigment Epithelium/cytology , Serpins/metabolismABSTRACT
Emerging evidence suggests that intracellular molecules and organelles transfer between cells during embryonic development, tissue homeostasis and disease. We and others recently showed that transplanted and host photoreceptors engage in bidirectional transfer of intracellular material in the recipient retina, a process termed material transfer (MT). We used cell transplantation, advanced tissue imaging approaches, genetic and pharmacologic interventions and primary cell culture to characterize and elucidate the mechanism of MT. We show that MT correlates with donor cell persistence and the accumulation of donor-derived proteins, mitochondria and transcripts in acceptor cells in vivo. MT requires cell contact in vitro and is associated with the formation of stable microtubule-containing protrusions, termed photoreceptor nanotubes (Ph NTs), that connect donor and host cells in vivo and in vitro. Ph NTs mediate GFP transfer between connected cells in vitro. Furthermore, interfering with Ph NT outgrowth by targeting Rho GTPase-dependent actin remodelling inhibits MT in vivo. Collectively, our observations provide evidence for horizontal exchange of intracellular material via nanotube-like connections between neurons in vivo.
Subject(s)
Photoreceptor Cells, Vertebrate/metabolism , Photoreceptor Cells, Vertebrate/ultrastructure , Retina/cytology , Actins/metabolism , Animals , Biological Transport , Cell Survival , Extracellular Vesicles , Female , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Male , Mice, Inbred C57BL , Mice, Transgenic , Mitochondria/metabolism , Retina/physiology , Retinoblastoma/metabolism , Retinoblastoma/pathology , Transducin/metabolism , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolismABSTRACT
Asymmetric neuronal expansion is thought to drive evolutionary transitions between lissencephalic and gyrencephalic cerebral cortices. We report that Neurog2 and Ascl1 proneural genes together sustain neurogenic continuity and lissencephaly in rodent cortices. Using transgenic reporter mice and human cerebral organoids, we found that Neurog2 and Ascl1 expression defines a continuum of four lineage-biased neural progenitor cell (NPC) pools. Double+ NPCs, at the hierarchical apex, are least lineage restricted due to Neurog2-Ascl1 cross-repression and display unique features of multipotency (more open chromatin, complex gene regulatory network, G2 pausing). Strikingly, selectively eliminating double+ NPCs by crossing Neurog2-Ascl1 split-Cre mice with diphtheria toxin-dependent "deleter" strains locally disrupts Notch signaling, perturbs neurogenic symmetry, and triggers cortical folding. In support of our discovery that double+ NPCs are Notch-ligand-expressing "niche" cells that control neurogenic periodicity and cortical folding, NEUROG2, ASCL1, and HES1 transcript distribution is modular (adjacent high/low zones) in gyrencephalic macaque cortices, prefiguring future folds.
Subject(s)
Cell Differentiation/physiology , Neocortex/embryology , Neocortex/physiology , Neurogenesis/physiology , Neurons/physiology , Animals , Cells, Cultured , Female , Humans , Macaca fascicularis , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , NIH 3T3 Cells , Neocortex/cytology , Pregnancy , Time-Lapse Imaging/methodsABSTRACT
Direct neuronal reprogramming is an innovative new technology that involves the conversion of somatic cells to induced neurons (iNs) without passing through a pluripotent state. The capacity to make new neurons in the brain, which previously was not achievable, has created great excitement in the field as it has opened the door for the potential treatment of incurable neurodegenerative diseases and brain injuries such as stroke. These neurological disorders are associated with frank neuronal loss, and as new neurons are not made in most of the adult brain, treatment options are limited. Developmental biologists have paved the way for the field of direct neuronal reprogramming by identifying both intrinsic cues, primarily transcription factors (TFs) and miRNAs, and extrinsic cues, including growth factors and other signaling molecules, that induce neurogenesis and specify neuronal subtype identities in the embryonic brain. The striking observation that postmitotic, terminally differentiated somatic cells can be converted to iNs by mis-expression of TFs or miRNAs involved in neural lineage development, and/or by exposure to growth factors or small molecule cocktails that recapitulate the signaling environment of the developing brain, has opened the door to the rapid expansion of new neuronal reprogramming methodologies. Furthermore, the more recent applications of neuronal lineage conversion strategies that target resident glial cells in situ has expanded the clinical potential of direct neuronal reprogramming techniques. Herein, we present an overview of the history, accomplishments, and therapeutic potential of direct neuronal reprogramming as revealed over the last two decades.
ABSTRACT
Historically, the mammalian brain was thought to lack stem cells as no new neurons were found to be made in adulthood. That dogma changed â¼25 years ago with the identification of neural stem cells (NSCs) in the adult rodent forebrain. However, unlike rapidly self-renewing mature tissues (e.g., blood, intestinal crypts, skin), the majority of adult NSCs are quiescent, and those that become 'activated' are restricted to a few neurogenic zones that repopulate specific brain regions. Conversely, embryonic NSCs are actively proliferating and neurogenic. Investigations into the molecular control of the quiescence-to-proliferation-to-differentiation continuum in the embryonic and adult brain have identified proneural genes encoding basic-helix-loop-helix (bHLH) transcription factors (TFs) as critical regulators. These bHLH TFs initiate genetic programs that remove NSCs from quiescence and drive daughter neural progenitor cells (NPCs) to differentiate into specific neural cell subtypes, thereby contributing to the enormous cellular diversity of the adult brain. However, new insights have revealed that proneural gene activities are context-dependent and tightly regulated. Here we review how proneural bHLH TFs are regulated, with a focus on the murine cerebral cortex, drawing parallels where appropriate to other organisms and neural tissues. We discuss upstream regulatory events, post-translational modifications (phosphorylation, ubiquitinylation), protein-protein interactions, epigenetic and metabolic mechanisms that govern bHLH TF expression, stability, localization, and consequent transactivation of downstream target genes. These tight regulatory controls help to explain paradoxical findings of changes to bHLH activity in different cellular contexts.
ABSTRACT
Despite a growing appreciation for microglial influences on the developing brain, the responsiveness of microglia to insults during gestation remains less well characterized, especially in the embryo when microglia themselves are still maturing. Here, we asked if fetal microglia could coordinate an innate immune response to an exogenous insult. Using time-lapse imaging, we showed that hypothalamic microglia actively surveyed their environment by near-constant "touching" of radial glia projections. However, following an insult (i.e., IUE or AAV transduction), this seemingly passive touching became more intimate and long lasting, ultimately resulting in the retraction of radial glial projections and degeneration into small pieces. Mechanistically, the TAM receptors MERTK and AXL were upregulated in microglia following the insult, and Annexin V treatment inhibited radial glia breakage and engulfment by microglia. These data demonstrate a remarkable responsiveness of embryonic microglia to insults during gestation, a critical window for neurodevelopment.
Subject(s)
Embryo, Mammalian/metabolism , Ependymoglial Cells/physiology , Hypothalamus/embryology , Hypothalamus/physiology , Microglia/physiology , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , c-Mer Tyrosine Kinase/metabolism , Animals , Brain/embryology , Embryonic Development , Gene Expression Regulation, Developmental , Immunity, Innate , Mice , Mice, Transgenic , Optical Imaging/methods , Axl Receptor Tyrosine KinaseABSTRACT
The cysteine protease inhibitor Cystatin C (CST3) is highly expressed in the brains of multiple sclerosis (MS) patients and C57BL/6J mice with experimental autoimmune encephalomyelitis (EAE; a model of MS), but its roles in the diseases are unknown. Here, we show that CST3 plays a detrimental function in myelin oligodendrocyte glycoprotein 35-55 (MOG35-55)-induced EAE but only in female animals. Female Cst3 null mice display significantly lower clinical signs of disease compared to wild-type (WT) littermates. This difference is associated with reduced interleukin-6 production and lower expression of key proteins (CD80, CD86, major histocompatibility complex [MHC] II, LC3A/B) involved in antigen processing, presentation, and co-stimulation in antigen-presenting cells (APCs). In contrast, male WT and Cst3-/- mice and cells show no differences in EAE signs or APC function. Further, the sex-dependent effect of CST3 in EAE is sensitive to gonadal hormones. Altogether, we have shown that CST3 has a sex-dependent role in MOG35-55-induced EAE.
Subject(s)
Cystatin C/metabolism , Encephalomyelitis, Autoimmune, Experimental/immunology , Multiple Sclerosis/immunology , Animals , Female , Mice , Sex FactorsABSTRACT
Gliomas are a diverse group of brain tumors comprised of malignant cells ('tumor' cells) and non-malignant 'normal' cells, including neural (neurons, glia), inflammatory (microglia, macrophage) and vascular cells. Tumor heterogeneity arises in part because, within the glioma mass, both 'tumor' and 'normal' cells secrete factors that form a unique microenvironment to influence tumor progression. Extracellular vesicles (EVs) are critical mediators of intercellular communication between immediate cellular neighbors and distantly located cells in healthy tissues/organs and in tumors, including gliomas. EVs mediate cell-cell signaling as carriers of nucleic acid, lipid and protein cargo, and their content is unique to cell types and physiological states. EVs secreted by non-malignant neural cells have important physiological roles in the healthy brain, which can be altered or co-opted to promote tumor progression and metastasis, acting in combination with glioma-secreted EVs. The cell-type specificity of EV content means that 'vesiculome' data can potentially be used to trace the cell of origin. EVs may also serve as biomarkers to be exploited for disease diagnosis and to assess therapeutic progress. In this review, we discuss how EVs mediate intercellular communication in glioma, and their potential role as biomarkers and readouts of a therapeutic response.
ABSTRACT
Despite clear physiological roles, the ventromedial hypothalamus (VMH) developmental programs are poorly understood. Here, we asked whether the proneural gene achaete-scute homolog 1 (Ascl1) contributes to VMH development. Ascl1 transcripts were detected in embryonic day (E) 10.5 to postnatal day 0 VMH neural progenitors. The elimination of Ascl1 reduced the number of VMH neurons at E12.5 and E15.5, particularly within the VMH-central (VMHC) and -dorsomedial (VMHDM) subdomains, and resulted in a VMH cell fate change from glutamatergic to GABAergic. We observed a loss of Neurog3 expression in Ascl1-/- hypothalamic progenitors and an upregulation of Neurog3 when Ascl1 was overexpressed. We also demonstrated a glutamatergic to GABAergic fate switch in Neurog3-null mutant mice, suggesting that Ascl1 might act via Neurog3 to drive VMH cell fate decisions. We also showed a concomitant increase in expression of the central GABAergic fate determinant Dlx1/2 in the Ascl1-null hypothalamus. However, Ascl1 was not sufficient to induce an ectopic VMH fate when overexpressed outside the normal window of competency. Combined, Ascl1 is required but not sufficient to specify the neurotransmitter identity of VMH neurons, acting in a transcriptional cascade with Neurog3.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , GABAergic Neurons/metabolism , Synaptic Transmission/genetics , Ventromedial Hypothalamic Nucleus/embryology , Ventromedial Hypothalamic Nucleus/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Differentiation/genetics , Cells, Cultured , Female , Gene Knockout Techniques , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurogenesis/geneticsABSTRACT
The tuberal hypothalamus is comprised of the dorsomedial, ventromedial, and arcuate nuclei, as well as parts of the lateral hypothalamic area, and it governs a wide range of physiologies. During neurogenesis, tuberal hypothalamic neurons are thought to be born in a dorsal-to-ventral and outside-in pattern, although the accuracy of this description has been questioned over the years. Moreover, the intrinsic factors that control the timing of neurogenesis in this region are poorly characterized. Proneural genes, including Achate-scute-like 1 (Ascl1) and Neurogenin 3 (Neurog3) are widely expressed in hypothalamic progenitors and contribute to lineage commitment and subtype-specific neuronal identifies, but the potential role of Neurogenin 2 (Neurog2) remains unexplored. Birthdating in male and female mice showed that tuberal hypothalamic neurogenesis begins as early as E9.5 in the lateral hypothalamic and arcuate and rapidly expands to dorsomedial and ventromedial neurons by E10.5, peaking throughout the region by E11.5. We confirmed an outside-in trend, except for neurons born at E9.5, and uncovered a rostrocaudal progression but did not confirm a dorsal-ventral patterning to tuberal hypothalamic neuronal birth. In the absence of Neurog2, neurogenesis stalls, with a significant reduction in early-born BrdU+ cells but no change at later time points. Further, the loss of Ascl1 yielded a similar delay in neuronal birth, suggesting that Ascl1 cannot rescue the loss of Neurog2 and that these proneural genes act independently in the tuberal hypothalamus. Together, our findings show that Neurog2 functions as a classical proneural gene to regulate the temporal progression of tuberal hypothalamic neurogenesis.SIGNIFICANCE STATEMENT Here, we investigated the general timing and pattern of neurogenesis within the tuberal hypothalamus. Our results confirmed an outside-in trend of neurogenesis and uncovered a rostrocaudal progression. We also showed that Neurog2 acts as a classical proneural gene and is responsible for regulating the birth of early-born neurons within the ventromedial hypothalamus, acting independently of Ascl1 In addition, we revealed a role for Neurog2 in cell fate specification and differentiation of ventromedial -specific neurons. Last, Neurog2 does not have cross-inhibitory effects on Neurog1, Neurog3, and Ascl1 These findings are the first to reveal a role for Neurog2 in hypothalamic development.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/biosynthesis , Hypothalamus, Middle/cytology , Hypothalamus, Middle/metabolism , Nerve Tissue Proteins/biosynthesis , Neurogenesis/physiology , Neurons/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Female , Hypothalamus, Middle/embryology , Mice , Mice, Transgenic , Nerve Tissue Proteins/genetics , PregnancyABSTRACT
Müller glia are specialized retinal cells with stem cell properties in fish and frogs but not in mammals. Current efforts to develop gene therapies to activate mammalian Müller glia for retinal repair will require safe and effective delivery strategies for recombinant adeno-associated viruses (AAVs), vectors of choice for clinical translation. Intravitreal and subretinal injections are currently used for AAV gene delivery in the eye, but less invasive methods efficiently targeting Müller glia have yet to be developed. Methods: As gene delivery strategies have been more extensively studied in the brain, to validate our vectors, we initially compared the glial tropism of AAV-PHP.eB, an AAV9 that crosses the blood-brain and blood-retinal barriers, for its ability to drive fluorescent protein expression in glial cells in both the brain and retina. We then tested the glial transduction of AAV2/8-GFAP-mCherry, a virus that does not cross blood-brain and blood-retinal barriers, for its effectiveness in transducing Müller glia in murine retinal explants ex vivo. For in vivo assays we used larger rat eyes, performing invasive intravitreal injections, and non-invasive intravenous delivery using focused ultrasound (FUS) (pressure amplitude: 0.360 - 0.84 MPa) and microbubbles (Definity, 0.2 ml/kg). Results: We showed that AAV-PHP.eB carrying a ubiquitous promoter (CAG) and green fluorescent protein (GFP) reporter, readily crossed the blood-brain and blood-retinal barriers after intravenous delivery in mice. However, murine Müller glia did not express GFP, suggesting that they were not transduced by AAV-PHP.eB. We thus tested an AAV2/8 variant, which was selected based on its safety record in multiple clinical trials, adding a glial fibrillary acidic protein (GFAP) promoter and mCherry (red fluorescent protein) reporter. We confirmed the glial specificity of AAV2/8-GFAP-mCherry, showing effective expression of mCherry in astrocytes after intracranial injection in the mouse brain, and of Müller glia in murine retinal explants. For in vivo experiments we switched to rats because of their larger size, injecting AAV2/8-GFAP-mCherry intravitreally, an invasive procedure, demonstrating passage across the inner limiting membrane, leading to Müller glia transduction. We then tested an alternative non-invasive delivery approach targeting a different barrier - the inner blood-retinal-barrier, applying focused ultrasound (FUS) to the retina after intravenous injection of AAV2/8 and microbubbles in rats, using magnetic resonance imaging (MRI) for FUS targeting. FUS permeabilized the rat blood-retinal-barrier and allowed the passage of macromolecules to the retina (Evans blue, IgG, IgM), with minimal extravasation of platelets and red blood cells. Intravenous injection of microbubbles and AAV2/8-GFAP-mCherry followed by FUS resulted in mCherry expression in rat Müller glia. However, systemic delivery of AAV2/8 also had off-target effects, transducing several murine peripheral organs, particularly the liver. Conclusions: Retinal permeabilisation via FUS in the presence of microbubbles is effective for delivering AAV2/8 across the inner blood-retinal-barrier, targeting Müller glia, which is less invasive than intravitreal injections that bypass the inner limiting membrane. However, implementing FUS in the clinic will require a comprehensive consideration of any off-target tropism of the AAV in peripheral organs, combined ideally, with the development of Müller glia-specific promoters.