Gene Regulation and Mitochondrial Activity During White and Brown Adipogenesis Are Modulated by KDM5 Histone Demethylase.

Body fat accumulation differs between males and females and is influenced by both gonadal sex (ovaries vs testes) and chromosomal sex (XX vs XY). We previously showed that an X chromosome gene, Kdm5c, is expressed at higher levels in females compared to males and correlates with adiposity in mice and humans. Kdm5c encodes a KDM5 histone demethylase that regulates gene expression by modulating histone methylation at gene promoters and enhancers. Here, we use chemical inhibition and genetic knockdown to identify a role for KDM5 activity during early stages of white and brown preadipocyte differentiation, with specific effects on white adipocyte clonal expansion, and white and brown adipocyte gene expression and mitochondrial activity. In white adipogenesis, KDM5 activity modulates H3K4 histone methylation at the Dlk1 gene promoter to repress gene expression and promote progression from preadipocytes to mature adipocytes. In brown adipogenesis, KDM5 activity modulates H3K4 methylation and gene expression of Ucp1, which is required for thermogenesis. Unbiased transcriptome analysis revealed that KDM5 activity regulates genes associated with cell cycle regulation and mitochondrial function, and this was confirmed by functional analyses of cell proliferation and cellular bioenergetics. Using genetic knockdown, we demonstrate that KDM5C is the likely KDM5 family member that is responsible for regulation of white and brown preadipocyte programming. Given that KDM5C levels are higher in females compared to males, our findings suggest that sex differences in white and brown preadipocyte gene regulation may contribute to sex differences in adipose tissue function.

The impact of chromosomal sex on cardiometabolic health and disease.

Many aspects of metabolism are sex-biased, from gene expression in metabolic tissues to the prevalence and presentation of cardiometabolic diseases. The influence of hormones produced by male and female gonads has been widely documented, but recent studies have begun to elucidate the impact of genetic sex (XX or XY chromosomes) on cellular and organismal metabolism. XX and XY cells have differential gene dosage conferred by specific genes that escape X chromosome inactivation or the presence of Y chromosome genes that are absent from XX cells. Studies in mouse models that dissociate chromosomal and gonadal sex have uncovered mechanisms for sex-biased epigenetic, transcriptional, and post-transcriptional regulation of gene expression in conditions such as obesity, atherosclerosis, pulmonary hypertension, autoimmune disease, and Alzheimer's disease.

A "Four Core Genotypes" rat model to distinguish mechanisms underlying sex-biased phenotypes and diseases.

Background: We have generated a rat model similar to the Four Core Genotypes mouse model, allowing comparison of XX and XY rats with the same type of gonad. The model detects novel sex chromosome effects (XX vs. XY) that contribute to sex differences in any rat phenotype. Methods: XY rats were produced with an autosomal transgene of Sry , the testis-determining factor gene, which were fathers of XX and XY progeny with testes. In other rats, CRISPR-Cas9 technology was used to remove Y chromosome factors that initiate testis differentiation, producing fertile XY gonadal females that have XX and XY progeny with ovaries. These groups can be compared to detect sex differences caused by sex chromosome complement (XX vs. XY) and/or by gonadal hormones (rats with testes vs. ovaries). Results: We have measured numerous phenotypes to characterize this model, including gonadal histology, breeding performance, anogenital distance, levels of reproductive hormones, body and organ weights, and central nervous system sexual dimorphisms. Serum testosterone levels were comparable in adult XX and XY gonadal males. Numerous phenotypes previously found to be sexually differentiated by the action of gonadal hormones were found to be similar in XX and XY rats with the same type of gonad, suggesting that XX and XY rats with the same type of gonad have comparable levels of gonadal hormones at various stages of development. Conclusion: The results establish a powerful new model to discriminate sex chromosome and gonadal hormone effects that cause sexual differences in rat physiology and disease.

Chromosomal and gonadal sex drive sex differences in lipids and hepatic gene expression in response to hypercholesterolemia and statin treatment.

BACKGROUND: Biological sex impacts susceptibility and presentation of cardiovascular disease, which remains the leading cause of death for both sexes. To reduce cardiovascular disease risk, statin drugs are commonly prescribed to reduce circulating cholesterol levels through inhibition of cholesterol synthesis. The effectiveness of statin therapy differs between individuals with a sex bias in the frequency of adverse effects. Limited information is available regarding the mechanisms driving sex-specific responses to hypercholesterolemia or statin treatment. METHODS: Four Core Genotypes mice (XX and XY mice with ovaries and XX and XY mice with testes) on a hypercholesteremic Apoe-/- background were fed a chow diet without or with simvastatin for 8 weeks. Plasma lipid levels were quantified and hepatic differential gene expression was evaluated with RNA-sequencing to identify the independent effects of gonadal and chromosomal sex. RESULTS: In a hypercholesterolemic state, gonadal sex influenced the expression levels of more than 3000 genes, and chromosomal sex impacted expression of nearly 1400 genes, which were distributed across all autosomes as well as the sex chromosomes. Gonadal sex uniquely influenced the expression of ER stress response genes, whereas chromosomal and gonadal sex influenced fatty acid metabolism gene expression in hypercholesterolemic mice. Sex-specific effects on gene regulation in response to statin treatment included a compensatory upregulation of cholesterol biosynthetic gene expression in mice with XY chromosome complement, regardless of presence of ovaries or testes. CONCLUSION: Gonadal and chromosomal sex have independent effects on the hepatic transcriptome to influence different cellular pathways in a hypercholesterolemic environment. Furthermore, chromosomal sex in particular impacted the cellular response to statin treatment. An improved understanding of how gonadal and chromosomal sex influence cellular response to disease conditions and in response to drug treatment is critical to optimize disease management for all individuals.

Illuminating the Mechanisms Underlying Sex Differences in Cardiovascular Disease.

Sex is a key risk factor for many types of cardiovascular disease. It is imperative to understand the mechanisms underlying sex differences to devise optimal preventive and therapeutic approaches for all individuals. Both biological sex (determined by sex chromosomes and gonadal hormones) and gender (social and cultural behaviors associated with femininity or masculinity) influence differences between men and women in disease susceptibility and pathology. Here, we focus on the application of experimental mouse models that elucidate the influence of 2 components of biological sex-sex chromosome complement (XX or XY) and gonad type (ovaries or testes). These models have revealed that in addition to well-known effects of gonadal hormones, sex chromosome complement influences cardiovascular risk factors, such as plasma cholesterol levels and adiposity, as well as the development of atherosclerosis and pulmonary hypertension. One mechanism by which sex chromosome dosage influences cardiometabolic traits is through sex-biased expression of X chromosome genes that escape X inactivation. These include chromatin-modifying enzymes that regulate gene expression throughout the genome. The identification of factors that determine sex-biased gene expression and cardiometabolic traits will expand our mechanistic understanding of cardiovascular disease processes and provide insight into sex differences that remain throughout the lifespan as gonadal hormone levels alter with age.

Kidney injury-mediated disruption of intestinal lymphatics involves dicarbonyl-modified lipoproteins.

Kidney disease affects intestinal structure and function. Although intestinal lymphatics are central in absorption and remodeling of dietary and synthesized lipids/lipoproteins, little is known about how kidney injury impacts the intestinal lymphatic network, or lipoproteins transported therein. To study this, we used puromycin aminoglycoside-treated rats and NEP25 transgenic mice to show that proteinuric injury expanded the intestinal lymphatic network, activated lymphatic endothelial cells and increased mesenteric lymph flow. The lymph was found to contain increased levels of cytokines, immune cells, and isolevuglandin (a highly reactive dicarbonyl) and to have a greater output of apolipoprotein AI. Plasma levels of cytokines and isolevuglandin were not changed. However, isolevuglandin was also increased in the ileum of proteinuric animals, and intestinal epithelial cells exposed to myeloperoxidase produced more isolevuglandin. Apolipoprotein AI modified by isolevuglandin directly increased lymphatic vessel contractions, activated lymphatic endothelial cells, and enhanced the secretion of the lymphangiogenic promoter vascular endothelial growth factor-C by macrophages. Inhibition of isolevuglandin synthesis by a carbonyl scavenger reduced intestinal isolevuglandin adduct level and lymphangiogenesis. Thus, our data reveal a novel mediator, isolevuglandin modified apolipoprotein AI, and uncover intestinal lymphatic network structure and activity as a new pathway in the crosstalk between kidney and intestine that may contribute to the adverse impact of kidney disease on other organs.

X chromosome dosage of histone demethylase KDM5C determines sex differences in adiposity.

Males and females differ in body composition and fat distribution. Using a mouse model that segregates gonadal sex (ovaries and testes) from chromosomal sex (XX and XY), we showed that XX chromosome complement in combination with a high-fat diet led to enhanced weight gain in the presence of male or female gonads. We identified the genomic dosage of Kdm5c, an X chromosome gene that escapes X chromosome inactivation, as a determinant of the X chromosome effect on adiposity. Modulating Kdm5c gene dosage in XX female mice to levels that are normally present in males resulted in reduced body weight, fat content, and food intake to a degree similar to that seen with altering the entire X chromosome dosage. In cultured preadipocytes, the levels of KDM5C histone demethylase influenced chromatin accessibility (ATAC-Seq), gene expression (RNA-Seq), and adipocyte differentiation. Both in vitro and in vivo, Kdm5c dosage influenced gene expression involved in extracellular matrix remodeling, which is critical for adipocyte differentiation and adipose tissue expansion. In humans, adipose tissue KDM5C mRNA levels and KDM5C genetic variants were associated with body mass. These studies demonstrate that the sex-dependent dosage of Kdm5c contributes to male/female differences in adipocyte biology and highlight X-escape genes as a critical component of female physiology.

High-density lipoproteins induce miR-223-3p biogenesis and export from myeloid cells: Role of scavenger receptor BI-mediated lipid transfer.

BACKGROUND AND AIMS: We recently showed that miR-223-3p on high-density lipoproteins (HDL) is exported to endothelial cells, where it inhibits inflammation. However, the origin of miR-223-3p on HDL is unknown. We hypothesize that HDL-associated miR-223-3p originates in myeloid cells and is exported to HDL in a scavenger receptor BI (SR-BI)-dependent manner. METHODS: Polymorphonuclear neutrophils (PMNs) and human monocyte derived macrophages (HMDMs) were incubated with native HDL (nHDL) or discoidal reconstituted HDL (rHDL). Total RNA was isolated before and after incubation. Mature and primary miR-223-3p (pri-mir-223-3p) levels were quantified by real-time PCR. RESULTS: Incubation with nHDL and rHDL increased miR-223-3p export from PMNs and HMDMs. In PMNs, nHDL but not rHDL, increased mature and pri-mir-223-3p. Incubation with HDL also increased Dicer mRNA, a critical regulator of miRNA biogenesis. Incubation of HMDMs with nHDL did not increase cellular levels of mature miR-223-3p, but significantly increased pri-mir-223 levels. Incubation with rHDL had no effect on either mature or pri-mir-223-3p levels. Activated PMNs increased miR-223-3p export to HDL and the production of reactive oxygen species and activated protein kinase C. Blocking HDL binding to SR-BI increased miR-223-3p export to HDL in both PMNs and HMDMs, but did not affect mature and primary miR-223-3p levels. Chemical inhibition of cholesterol flux by Block Lipid Transport (BLT)-1 inhibited HDL-induced pri-mir-223 expression in PMNs. CONCLUSIONS: HDL-associated miR-223-3p originates in PMNs and macrophages. HDL stimulates miR-223-3p biogenesis in PMNs in a process that is regulated by SR-BI-mediated lipid flux.

Dual inhibition of endothelial miR-92a-3p and miR-489-3p reduces renal injury-associated atherosclerosis.

BACKGROUND AND AIMS: Cardiovascular disease (CVD) is the leading cause of death in chronic kidney disease (CKD) patients, however, the underlying mechanisms that link CKD and CVD are not fully understood and limited treatment options exist in this high-risk population. microRNAs (miRNA) are critical regulators of gene expression for many biological processes in atherosclerosis, including endothelial dysfunction and inflammation. We hypothesized that renal injury-induced endothelial miRNAs promote atherosclerosis. Here, we demonstrate that dual inhibition of endothelial miRNAs inhibits atherosclerosis in the setting of renal injury. METHODS: Aortic endothelial miRNAs were analyzed in apolipoprotein E-deficient (Apoe-/-) mice with renal damage (5/6 nephrectomy, 5/6Nx) by real-time PCR. Endothelial miR-92a-3p and miR-489-3p were inhibited by locked-nucleic acid (LNA) miRNA inhibitors complexed to HDL. RESULTS: Renal injury significantly increased endothelial miR-92a-3p levels in Apoe-/-;5/6Nx mice. Dual inhibition of miR-92a-3p and miR-489-3p in Apoe-/-;5/6Nx with a single injection of HDL + LNA inhibitors significantly reduced atherosclerotic lesion area by 28.6% compared to HDL + LNA scramble (LNA-Scr) controls. To examine the impact of dual LNA treatment on aortic endothelial gene expression, total RNA sequencing was completed, and multiple putative target genes and pathways were identified to be significantly altered, including the STAT3 immune response pathway. Among the differentially expressed genes, Tgfb2 and Fam220a were identified as putative targets of miR-489-3p and miR-92a-3p, respectively. Both Tgfb2 and Fam220a were significantly increased in aortic endothelium after miRNA inhibition in vivo compared to HDL + LNA-Scr controls. Furthermore, Tgfb2 and Fam220a were validated with gene reporter assays as direct targets of miR-489-3p and miR-92a-3p, respectively. In human coronary artery endothelial cells, over-expression and inhibition of miR-92a-3p decreased and increased FAM220A expression, respectively. Moreover, miR-92a-3p overexpression increased STAT3 phosphorylation, likely through direct regulation of FAM220A, a negative regulator of STAT3 phosphorylation. CONCLUSIONS: These results support endothelial miRNAs as therapeutic targets and dual miRNA inhibition as viable strategy to reduce CKD-associated atherosclerosis.

Migration pathways of sacral neural crest during development of lower urogenital tract innervation.

The migration and fate of cranial and vagal neural crest-derived progenitor cells (NCPCs) have been extensively studied; however, much less is known about sacral NCPCs particularly in regard to their distribution in the urogenital system. To construct a spatiotemporal map of NCPC migration pathways into the developing lower urinary tract, we utilized the Sox10-H2BVenus transgene to visualize NCPCs expressing Sox10. Our aim was to define the relationship of Sox10-expressing NCPCs relative to bladder innervation, smooth muscle differentiation, and vascularization through fetal development into adulthood. Sacral NCPC migration is a highly regimented, specifically timed process, with several potential regulatory mileposts. Neuronal differentiation occurs concomitantly with sacral NCPC migration, and neuronal cell bodies are present even before the pelvic ganglia coalesce. Sacral NCPCs reside within the pelvic ganglia anlagen through 13.5 days post coitum (dpc), after which they begin streaming into the bladder body in progressive waves. Smooth muscle differentiation and vascularization of the bladder initiate prior to innervation and appear to be independent processes. In adult bladder, the majority of Sox10+ cells express the glial marker S100ß, consistent with Sox10 being a glial marker in other tissues. However, rare Sox10+ NCPCs are seen in close proximity to blood vessels and not all are S100ß+, suggesting either glial heterogeneity or a potential nonglial role for Sox10+ cells along vasculature. Taken together, the developmental atlas of Sox10+ NCPC migration and distribution profile of these cells in adult bladder provided here will serve as a roadmap for future investigation in mouse models of lower urinary tract dysfunction.

Porous Silicon and Polymer Nanocomposites for Delivery of Peptide Nucleic Acids as Anti-MicroRNA Therapies.

Self-assembled polymer/porous silicon nanocomposites overcome intracellular and systemic barriers for in vivo application of peptide nucleic acid (PNA) anti-microRNA therapeutics. Porous silicon (PSi) is leveraged as a biodegradable scaffold with high drug-cargo-loading capacity. Functionalization with a diblock polymer improves PSi nanoparticle colloidal stability, in vivo pharmacokinetics, and intracellular bioavailability through endosomal escape, enabling PNA to inhibit miR-122 in vivo.

An illustrated anatomical ontology of the developing mouse lower urogenital tract.

Malformation of the urogenital tract represents a considerable paediatric burden, with many defects affecting the lower urinary tract (LUT), genital tubercle and associated structures. Understanding the molecular basis of such defects frequently draws on murine models. However, human anatomical terms do not always superimpose on the mouse, and the lack of accurate and standardised nomenclature is hampering the utility of such animal models. We previously developed an anatomical ontology for the murine urogenital system. Here, we present a comprehensive update of this ontology pertaining to mouse LUT, genital tubercle and associated reproductive structures (E10.5 to adult). Ontology changes were based on recently published insights into the cellular and gross anatomy of these structures, and on new analyses of epithelial cell types present in the pelvic urethra and regions of the bladder. Ontology changes include new structures, tissue layers and cell types within the LUT, external genitalia and lower reproductive structures. Representative illustrations, detailed text descriptions and molecular markers that selectively label muscle, nerves/ganglia and epithelia of the lower urogenital system are also presented. The revised ontology will be an important tool for researchers studying urogenital development/malformation in mouse models and will improve our capacity to appropriately interpret these with respect to the human situation.

Macrophage deficiency of Akt2 reduces atherosclerosis in Ldlr null mice.

Macrophages play crucial roles in the formation of atherosclerotic lesions. Akt, a serine/threonine protein kinase B, is vital for cell proliferation, migration, and survival. Macrophages express three Akt isoforms, Akt1, Akt2, and Akt3, but the roles of Akt1 and Akt2 in atherosclerosis in vivo remain unclear. To dissect the impact of macrophage Akt1 and Akt2 on early atherosclerosis, we generated mice with hematopoietic deficiency of Akt1 or Akt2. After 8 weeks on Western diet, Ldlr(-/-) mice reconstituted with Akt1(-/-) fetal liver cells (Akt1(-/-)→Ldlr(-/-)) had similar atherosclerotic lesion areas compared with control mice transplanted with WT cells (WT→Ldlr(-/-)). In contrast, Akt2(-/-)→Ldlr(-/-) mice had dramatically reduced atherosclerotic lesions compared with WT→Ldlr(-/-) mice of both genders. Similarly, in the setting of advanced atherosclerotic lesions, Akt2(-/-)→Ldlr(-/-) mice had smaller aortic lesions compared with WT→Ldlr(-/-) and Akt1(-/-)→Ldlr(-/-) mice. Importantly, Akt2(-/-)→Ldlr(-/-) mice had reduced numbers of proinflammatory blood monocytes expressing Ly-6C(hi) and chemokine C-C motif receptor 2. Peritoneal macrophages isolated from Akt2(-/-) mice were skewed toward an M2 phenotype and showed decreased expression of proinflammatory genes and reduced cell migration. Our data demonstrate that loss of Akt2 suppresses the ability of macrophages to undergo M1 polarization reducing both early and advanced atherosclerosis.

HDL-transferred microRNA-223 regulates ICAM-1 expression in endothelial cells.

High-density lipoproteins (HDL) have many biological functions, including reducing endothelial activation and adhesion molecule expression. We recently reported that HDL transport and deliver functional microRNAs (miRNA). Here we show that HDL suppresses expression of intercellular adhesion molecule 1 (ICAM-1) through the transfer of miR-223 to endothelial cells. After incubation of endothelial cells with HDL, mature miR-223 levels are significantly increased in endothelial cells and decreased on HDL. However, miR-223 is not transcribed in endothelial cells and is not increased in cells treated with HDL from miR-223(-/-) mice. HDL inhibit ICAM-1 protein levels, but not in cells pretreated with miR-223 inhibitors. ICAM-1 is a direct target of HDL-transferred miR-223 and this is the first example of an extracellular miRNA regulating gene expression in cells where it is not transcribed. Collectively, we demonstrate that HDL's anti-inflammatory properties are conferred, in part, through HDL-miR-223 delivery and translational repression of ICAM-1 in endothelial cells.

A Uchl1-Histone2BmCherry:GFP-gpi BAC transgene for imaging neuronal progenitors.

Uchl1 encodes the protein gene product 9.5 antigen (PGP9.5) that is a widely used to identify migrating neural progenitors in the PNS, mature neurons of the central and peripheral nervous systems, as well as neuroendocrine cells. To facilitate analysis of developing peripheral neurons, we linked regulatory regions of Uchl1 carried within a 160kb bacterial artificial chromosome (BAC) to the dual fluorescent reporter H2BmCherry:GFP-gpi. The Uchl1-H2BmCherry:GFP-gpi transgene exhibits robust expression and allows clear discrimination of individual cells and cellular processes in cranial ganglia, sympathetic chain, the enteric nervous system (ENS), and autonomic ganglia of the urogenital system. The transgene also labels subsets of cells in endocrine tissues where earlier in situ hybridization (ISH) studies have previously identified expression of this deubiquinating enzyme. The Uchl1-H2BmCherry:GFP-gpi transgene will be a powerful tool for static and live imaging, as well as isolation of viable neural progenitors to investigate processes of autonomic neurogenesis.

A genome-wide screen to identify transcription factors expressed in pelvic Ganglia of the lower urinary tract.

Relative positions of neurons within mature murine pelvic ganglia based on expression of neurotransmitters have been described. However the spatial organization of developing innervation in the murine urogenital tract (UGT) and the gene networks that regulate specification and maturation of neurons within the pelvic ganglia of the lower urinary tract (LUT) are unknown. We used whole-mount immunohistochemistry and histochemical stains to localize neural elements in 15.5 days post coitus (dpc) fetal mice. To identify potential regulatory factors expressed in pelvic ganglia, we surveyed expression patterns for known or probable transcription factors (TF) annotated in the mouse genome by screening a whole-mount in situ hybridization library of fetal UGTs. Of the 155 genes detected in pelvic ganglia, 88 encode TFs based on the presence of predicted DNA-binding domains. Neural crest (NC)-derived progenitors within the LUT were labeled by Sox10, a well-known regulator of NC development. Genes identified were categorized based on patterns of restricted expression in pelvic ganglia, pelvic ganglia and urethral epithelium, or pelvic ganglia and urethral mesenchyme. Gene expression patterns and the distribution of Sox10+, Phox2b+, Hu+, and PGP9.5+ cells within developing ganglia suggest previously unrecognized regional segregation of Sox10+ progenitors and differentiating neurons in early development of pelvic ganglia. Reverse transcription-PCR of pelvic ganglia RNA from fetal and post-natal stages demonstrated that multiple TFs maintain post-natal expression, although Pax3 is extinguished before weaning. Our analysis identifies multiple potential regulatory genes including TFs that may participate in segregation of discrete lineages within pelvic ganglia. The genes identified here are attractive candidate disease genes that may now be further investigated for their roles in malformation syndromes or in LUT dysfunction.

An optimized procedure for fluorescence-activated cell sorting (FACS) isolation of autonomic neural progenitors from visceral organs of fetal mice.

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the intestine and lower urinary tract. Both during development and in mature tissues these cells are often widely dispersed throughout tissues so that isolation of discrete populations using methods like laser capture micro-dissection is difficult. They can however be directly visualized by expression of fluorescent reporters driven from regulatory regions of neuron-specific genes like Tyrosine hydroxylase (TH). We describe a method optimized for high yields of viable TH+ neuronal progenitors from fetal mouse visceral tissues, including intestine and lower urogenital tract (LUT), based on dissociation and fluorescence-activated cell sorting (FACS). The Th gene encodes the rate-limiting enzyme for production of catecholamines. Enteric neuronal progenitors begin to express TH during their migration in the fetal intestine and TH is also present in a subset of adult pelvic ganglia neurons . The first appearance of this lineage and the distribution of these neurons in other aspects of the LUT, and their isolation has not been described. Neuronal progenitors expressing TH can be readily visualized by expression of EGFP in mice carrying the transgene construct Tg(Th-EGFP)DJ76Gsat/Mmnc. We imaged expression of this transgene in fetal mice to document the distribution of TH+ cells in the developing LUT at 15.5 days post coitus (dpc), designating the morning of plug detection as 0.5 dpc, and observed that a subset of neuronal progenitors in the coalescing pelvic ganglia express EGFP. To isolate LUT TH+ neuronal progenitors, we optimized methods that were initially used to purify neural crest stem cells from fetal mouse intestine. Prior efforts to isolate NC-derived populations relied upon digestion with a cocktail of collagenase and trypsin to obtain cell suspensions for flow cytometry. In our hands these methods produced cell suspensions from the LUT with relatively low viability. Given the already low incidence of neuronal progenitors in fetal LUT tissues, we set out to optimize dissociation methods such that cell survival in the final dissociates would be increased. We determined that gentle dissociation in Accumax (Innovative Cell Technologies, Inc), manual filtering, and flow sorting at low pressures allowed us to achieve consistently greater survival (>70% of total cells) with subsequent yields of neuronal progenitors sufficient for downstream analysis. The method we describe can be broadly applied to isolate a variety of neuronal populations from either fetal or adult murine tissues.