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SummaryViral genetic tools to target specific brain cell types in humans and non-genetic model organisms will transform basic neuroscience and targeted gene therapy. Here we used comparative epigenetics to identify thousands of human neuronal subclass-specific putative enhancers to regulate viral tools, and 34% of these were conserved in mouse. We established an AAV platform to evaluate cellular specificity of functional enhancers by multiplexed fluorescent in situ hybridization (FISH) and single cell RNA sequencing. Initial testing in mouse neocortex yields a functional enhancer discovery success rate of over 30%. We identify enhancers with specificity for excitatory and inhibitory classes and subclasses including PVALB, LAMP5, and VIP/LAMP5 cells, some of which maintain specificityin vivoorex vivoin monkey and human neocortex. Finally, functional enhancers can be proximal or distal to cellular marker genes, conserved or divergent across species, and could yield brain-wide specificity greater than the most selective marker genes.

Nervous systems are composed of various cell types, but the extent of cell type diversity is poorly understood. We constructed a cellular taxonomy of one cortical region, primary visual cortex, in adult mice on the basis of single-cell RNA sequencing. We identified 49 transcriptomic cell types, including 23 GABAergic, 19 glutamatergic and 7 non-neuronal types. We also analyzed cell type-specific mRNA processing and characterized genetic access to these transcriptomic types by many transgenic Cre lines. Finally, we found that some of our transcriptomic cell types displayed specific and differential electrophysiological and axon projection properties, thereby confirming that the single-cell transcriptomic signatures can be associated with specific cellular properties.

The human neocortex is created from diverse intermixed progenitors in the prenatal germinal zones. These progenitors have been difficult to characterize since progenitors—particularly radial glia (RG)—are rare, and are defined by a combination of intracellular markers, position and morphology. To circumvent these problems we developed a method called FRISCR for transcriptome profiling of individual fixed, stained and sorted cells. After validation of FRISCR using human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These RG could be classified into ventricular zone-enriched RG (vRG) that express ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that express HOPX. Our study identifies the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks driving the lineage of human neocortical progenitors. Furthermore, FRISCR allows targeted single-cell transcriptomic profiling of tissues that lack live-cell markers.

Organoid techniques provide unique platforms to model brain development and neurological disorders. While several methods for recapitulating corticogenesis have been described, a system modeling human medial ganglionic eminence (MGE) development, a critical ventral brain domain producing cortical interneurons and related lineages, has been lacking until recently. Here, we describe the generation of MGE and cortex-specific organoids from human pluripotent stem cells that recapitulate the development of MGE and cortex domains respectively. Population and single-cell RNA-seq profiling combined with bulk ATAC-seq analyses revealed transcriptional and chromatin accessibility dynamics and lineage relationships during MGE and cortical organoid development. Furthermore, MGE and cortical organoids generated physiologically functional neurons and neuronal networks. Finally, fusing region-specific organoids followed by live-imaging enabled analysis of human interneuron migration and integration. Together, our study provides a platform for generating domain-specific brain organoids, for modeling human interneuron migration, and offers deeper insight into molecular dynamics during human brain development.

During human brain development, multiple signaling pathways generate diverse cell types with varied regional identities. Here, we integrate single-cell RNA sequencing and clonal analyses to reveal lineage trees and molecular signals underlying early forebrain and mid/hindbrain cell differentiation from human embryonic stem cells (hESCs). Clustering single cell transcriptomic data identified 41 distinct populations of progenitors, neuronal, and non-neural cells across our differentiation time course. Comparisons with primary mouse and human gene expression data demonstrated rostral and caudal progenitor and neuronal identities from early brain development. Bayesian analyses inferred a unified cell type lineage tree that bifurcates between cortical and mid/hindbrain cell types. Two methods of clonal analyses confirmed these findings and further revealed the importance of Wnt/beta-catenin signaling in controlling this lineage decision. Together, these findings provide a rich transcriptome-based lineage map for studying human brain development and modeling developmental disorders.

To better understand the mechanisms that regulate stem cell identity and function, we sought to identify genes that are preferentially expressed by stem cells and critical for their function in multiple tissues. Prdm16 is a transcription factor that regulates leukaemogenesis, palatogenesis and brown-fat development, but which was not known to be required for stem cell function. We demonstrate that Prdm16 is preferentially expressed by stem cells throughout the nervous and haematopoietic systems and is required for their maintenance. In the haematopoietic and nervous systems, Prdm16 deficiency led to changes in the levels of reactive oxygen species (ROS), depletion of stem cells, increased cell death and altered cell-cycle distribution. In neural stem/progenitor cells, Prdm16 binds to the Hgf promoter, and Hgf expression declined in the absence of Prdm16. Addition of recombinant HGF to Prdm16-deficient neural stem cells in cell culture reduced the depletion of these cells and partially rescued the increase in ROS levels. Administration of the anti-oxidant, N-acetyl-cysteine, to Prdm16-deficient mice partially rescued defects in neural stem/progenitor cell function and neural development. Prdm16 therefore promotes stem cell maintenance in multiple tissues, partly by modulating oxidative stress.

High levels of aldehyde dehydrogenase (ALDH) activity have been proposed to be a common feature of stem cells. Adult hematopoietic, neural, and cancer stem cells have all been reported to have high ALDH activity, detected using Aldefluor, a fluorogenic substrate for ALDH. This activity has been attributed to Aldh1a1, an enzyme that is expressed at high levels in stem cells and that has been suggested to regulate stem cell function. Nonetheless, Aldh1a1 function in stem cells has never been tested genetically. We observed that Aldh1a1 was preferentially expressed in mouse hematopoietic stem cells (HSCs) and expression increased with age. Hematopoietic cells from Aldh1a1-deficient mice exhibited increased sensitivity to cyclophosphamide in a non–cell-autonomous manner, consistent with its role in cyclophosphamide metabolism in the liver. However, Aldh1a1 deficiency did not affect hematopoiesis, HSC function, or the capacity to reconstitute irradiated recipients in young or old adult mice. Aldh1a1 deficiency also did not affect Aldefluor staining of hematopoietic cells. Finally, Aldh1a1 deficiency did not affect the function of stem cells from the adult central or peripheral nervous systems. Aldh1a1 is not a critical regulator of adult stem cell function or Aldefluor staining in mice.

Active migration of polymorphonuclear leukocytes (PMN) through the intestinal crypt epithelium is a hallmark of inflammatory bowel disease and correlates with patient symptoms. Previous in vitro studies have shown that PMN transepithelial migration results in increased epithelial permeability. In this study, we modeled PMN transepithelial migration across T84 monolayers and demonstrated that enhanced paracellular permeability to small solutes occurred in the absence of transepithelial migration but required both PMN contact with the epithelial cell basolateral membrane and a transepithelial chemotactic gradient. Early events that occurred before PMN entering the paracellular space included increased permeability to small solutes (

We show that the vacuolar protein sorting gene VPS44 is identical to NHX1, a gene that encodes a sodium/proton exchanger. The Saccharomyces cerevisiae protein Nhx1p shows high homology to mammalian sodium/proton exchangers of the NHE family. Nhx1p is thought to transport sodium ions into the prevacuole compartment in exchange for protons. Pulse-chase experiments show that approximately 35% of the newly synthesized soluble vacuolar protein carboxypeptidase Y is missorted in nhx1 delta cells, and is secreted from the cell. nhx1 delta cells accumulate late Golgi, prevacuole, and lysosome markers in an aberrant structure next to the vacuole, and late Golgi proteins are proteolytically cleaved more rapidly than in wild-type cells. Our results show that efficient transport out of the prevacuolar compartment requires Nhx1p, and that nhx1 delta cells exhibit phenotypes characteristic of the "class E" group of vps mutants. In addition, we show that Nhx1p is required for protein trafficking even in the absence of the vacuolar ATPase. Our analysis of Nhx1p provides the first evidence that a sodium/proton exchange protein is important for correct protein sorting, and that intraorganellar ion balance may be important for endosomal function in yeast.

Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae requires the function of the endosomal syntaxin, Pep12p. Many vacuolar proteins, such as the soluble vacuolar hydrolase, carboxypeptidase Y (CPY), traverse the prevacuolar compartment (PVC) en route to the vacuole. Here we show that deletion of the carboxy-terminal transmembrane domain of Pep12p results in a temperature-conditional block in transport of CPY to the PVC. The PVC also receives traffic from the early endosome and the vacuole, and mutation in PEP12 also blocks these other trafficking pathways into the PVC. Therefore, Pep12p is a multifunctional syntaxin that is required for all known trafficking pathways into the yeast PVC. Finally, we found that the internalized pheromone receptor, Ste3p, can cycle out of the PVC in a VPS27-independent fashion.

At sites of ongoing inflammation, polymorphonuclear leukocytes (PMN, neutrophils) migrate across vascular endothelia, and such transmigration has the potential to disturb barrier properties and can result in intravascular fluid loss and edema. It was recently appreciated that endogenous pathways exist to dampen barrier disruption during such episodes and may provide an important anti-inflammatory link. For example, during transmigration, PMN-derived adenosine activates endothelial adenosine receptors and induces a cAMP-dependent resealing of endothelial barrier function. In our study reported here, we sought to understand the link between cyclic nucleotide elevation and increased endothelial barrier function. Initial studies revealed that adenosine-induced barrier function is tightly linked to activation of protein kinase A (PKA). Because PKA selectively phosphorylates serine and threonine residues, we screened zonula occludens-1 (ZO-1) immunoprecipitates for the existence of such phosphorylated proteins as targets for barrier regulation. This analysis revealed a dominantly phosphorylated band at 50 kDa. Microsequencing identified this protein as vasodilator-stimulated phosphoprotein (VASP), an actin binding protein with multiple serine/threonine phosphorylation sites. Immunofluorescent microscopy revealed that VASP localizes to endothelial junctional complexes and colocalizes with ZO-1, occludin, and junctional adhesion molecule-1 (JAM-1). To address the role of phospho-VASP in regulation of barrier function, we generated a phosphospecific VASP antibody targeting the Ser157 residue phosphorylation site, the site preferred by PKA. Immunolocalization studies with this antibody revealed that upon PKA activation, phospho-VASP appears at cell-cell junctions. Transient transfection of truncated VASP fragments revealed a parallel increase in barrier function. Taken together, these studies reveal a central role for phospho-VASP in the coordination of PKA-regulated barrier function, such as occurs during episodes of inflammation.

Stem cells are uniquely able to self-renew, to undergo multilineage differentiation, and to persist throughout life in a number of tissues. Stem cells are regulated by a combination of shared and tissue-specific mechanisms and are distinguished from restricted progenitors by differences in transcriptional and epigenetic regulation. Emerging evidence suggests that other aspects of cellular physiology, including mitosis, signal transduction, and metabolic regulation, also differ between stem cells and their progeny. These differences may allow stem cells to be regulated independently of differentiated cells in response to circadian rhythms, changes in metabolism, diet, exercise, mating, aging, infection, and disease. This allows stem cells to sustain homeostasis or to remodel relevant tissues in response to physiological change. Stem cells are therefore not only regulated by short-range signals that maintain homeostasis within their tissue of origin, but also by long-range signals that integrate stem cell function with systemic physiology.

Stem cells expand in number during development and persist throughout life by undergoing self-renewing divisions. The question of how stem cells self-renew throughout life is a fundamental problem in cell biology, with broad implications for understanding development, tissue regeneration, cancer, and aging. Recent insights demonstrate that self-renewal programs depend on key transcriptional regulators that are often shared among stem cells in different tissues but that often change between stem cells at different stages of life: Embryonic, fetal, young adult, and old adult stem cells are maintained by different self-renewal programs. Self-renewal programs change over time to contend with changes in tissue growth and repair demands as well as the increasing risk of malignant transformation during aging. The downstream mechanisms by which these programs regulate the cell cycle, developmental potential, and timing of differentiation are just starting to be elucidated. One key requirement for self-renewal is repression of the p16(Ink4a) and p19(Arf) tumor suppressors. This is accomplished by overlapping transcriptional regulators whose expression and function change with age, so as to maintain self-renewal potential throughout life while allowing increased expression of p16(Ink4a) and p19(Arf) in aging stem cells. This reduces stem cell function in aging tissues but also reduces cancer incidence.

Epithelial tubes that compose many organs are typically long lasting,except under specific developmental and physiological conditions when network remodeling occurs. Although there has been progress elucidating mechanisms of tube formation, little is known of the mechanisms that maintain tubes and destabilize them during network remodeling. Here, we describe Drosophila tendrils mutations that compromise maintenance of tracheal terminal branches, fine gauge tubes formed by tracheal terminal cells that ramify on and adhere tightly to tissues in order to supply them with oxygen. Homozygous tendrils terminal cell clones have fewer terminal branches than normal but individual branches contain multiple convoluted lumens. The phenotype arises late in development: terminal branches bud and form lumens normally early in development, but during larval life lumens become convoluted and mature branches degenerate. Their lumens, however, are retained in the remaining branches, resulting in the distinctive multi-lumen phenotype. Mapping and molecular studies demonstrate that tendrils is allelic to rhea, which encodes Drosophila talin, a large cytoskeletal protein that links integrins to the cytoskeleton. Terminal cells mutant for myospheroid, the major Drosophila β-integrin, or doubly mutant for multiple edematous wings and inflatedα-integrins, also show the tendrils phenotype, and localization of myospheroid β-integrin protein is disrupted in tendrils mutant terminal cells. The results provide evidence that integrin-talin adhesion complexes are necessary to maintain tracheal terminal branches and luminal organization. Similar complexes may stabilize other tubular networks and may be targeted for inactivation during network remodeling events.