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Cultured human pluripotent stem cells can be differentiated to immature pancreatic beta cells, but no one has yet succeeded in maturing these cells in vitro . Blum et al . define markers of beta-cell maturation that can be used to screen conditions for generating fully functional beta cells. Insulin-expressing cells that have been differentiated from human pluripotent stem cells in vitro lack the glucose responsiveness characteristic of mature beta cells. Beta-cell maturation in mice was studied to find genetic markers that enable screens for factors that induce bona fide beta cells in vitro . We find that functional beta-cell maturation is marked by an increase in the glucose threshold for insulin secretion and by expression of the gene urocortin 3.

Human pluripotent stem cells (hPSCs) have the potential to generate any human cell type, and one widely recognized goal is to make pancreatic β cells. To this end, comparisons between differentiated cell types produced in vitro and their in vivo counterparts are essential to validate hPSC-derived cells. Genome-wide transcriptional analysis of sorted insulin-expressing (INS ⁺) cells derived from three independent hPSC lines, human fetal pancreata, and adult human islets points to two major conclusions: (i) Different hPSC lines produce highly similar INS ⁺ cells and (ii) hPSC-derived INS ⁺ (hPSC-INS ⁺) cells more closely resemble human fetal β cells than adult β cells. This study provides a direct comparison of transcriptional programs between pure hPSC-INS ⁺ cells and true β cells and provides a catalog of genes whose manipulation may convert hPSC-INS ⁺ cells into functional β cells.

Neurons in the posterior parietal cortex contribute to the execution of goal-directed navigation 1 and other decision-making tasks 2 – 4 . Although molecular studies have catalogued more than 50 cortical cell types 5 , it remains unclear what distinct functions they have in this area. Here we identified a molecularly defined subset of somatostatin (Sst) inhibitory neurons that, in the mouse posterior parietal cortex, carry a cell-type-specific error-correction signal for navigation. We obtained repeatable experimental access to these cells using an adeno-associated virus in which gene expression is driven by an enhancer that functions specifically in a subset of Sst cells 6 . We found that during goal-directed navigation in a virtual environment, this subset of Sst neurons activates in a synchronous pattern that is distinct from the activity of surrounding neurons, including other Sst neurons. Using in vivo two-photon photostimulation and ex vivo paired patch-clamp recordings, we show that nearby cells of this Sst subtype excite each other through gap junctions, revealing a self-excitation circuit motif that contributes to the synchronous activity of this cell type. These cells selectively activate as mice execute course corrections for deviations in their virtual heading during navigation towards a reward location, for both self-induced and experimentally induced deviations. We propose that this subtype of Sst neurons provides a self-reinforcing and cell-type-specific error-correction signal in the posterior parietal cortex that may help with the execution and learning of accurate goal-directed navigation trajectories. A molecularly defined subset of somatostatin-positive inhibitory neurons in the mouse posterior parietal cortex carries a cell-type-specific error-correction signal for navigation.

Transcriptional profiling is a key technique in the study of cell biology that is limited by the availability of reagents to uniquely identify specific cell types and isolate high quality RNA from them. We report a Method for Analyzing RNA following Intracellular Sorting (MARIS) that generates high quality RNA for transcriptome profiling following cellular fixation, intracellular immunofluorescent staining and FACS. MARIS can therefore be used to isolate high quality RNA from many otherwise inaccessible cell types simply based on immunofluorescent tagging of unique intracellular proteins. As proof of principle, we isolate RNA from sorted human embryonic stem cell-derived insulin-expressing cells as well as adult human β cells. MARIS is a basic molecular biology technique that could be used across several biological disciplines.

Kutznerides, actinomycete-derived cyclic depsipetides, consist of six nonproteinogenic residues, including a highly oxygenated tricyclic hexahydropyrroloindole, a chlorinated piperazic acid, 2-(1-methylcyclopropyl)-glycine, a β-branched-hydroxy acid, and 3-hydroxy glutamic acid, for which biosynthetic logic has not been elucidated. Herein we describe the biosynthetic gene cluster for the kutzneride family, identified by degenerate primer PCR for halogenating enzymes postulated to be involved in biosyntheses of these unusual monomers. The 56-kb gene cluster encodes a series of six nonribosomal peptide synthetase (NRPS) modules distributed over three proteins and a variety of tailoring enzymes, including both mononuclear nonheme iron and two flavin-dependent halogenases, and an array of oxygen transfer catalysts. The sequence and organization of NRPS genes support incorporation of the unusual monomer units into the densely functionalized scaffold of kutznerides. Our work provides insight into the formation of this intriguing class of compounds and provides a foundation for elucidating the timing and mechanisms of their biosynthesis.

Enhancers are the primary DNA regulatory elements that confer cell type specificity of gene expression. Recent studies characterizing individual enhancers have revealed their potential to direct heterologous gene expression in a highly cell-type-specific manner. However, it has not yet been possible to systematically identify and test the function of enhancers for each of the many cell types in an organism. We have developed PESCA, a scalable and generalizable method that leverages ATAC- and single-cell RNA-sequencing protocols, to characterize cell-type-specific enhancers that should enable genetic access and perturbation of gene function across mammalian cell types. Focusing on the highly heterogeneous mammalian cerebral cortex, we apply PESCA to find enhancers and generate viral reagents capable of accessing and manipulating a subset of somatostatin-expressing cortical interneurons with high specificity. This study demonstrates the utility of this platform for developing new cell-type-specific viral reagents, with significant implications for both basic and translational research.

In females with X-linked genetic disorders, wild-type and mutant cells coexist within brain tissue because of X-chromosome inactivation, posing challenges for interpreting the effects of X-linked mutant alleles on gene expression. We present a single-nucleus RNA sequencing approach that resolves mosaicism by using single-nucleotide polymorphisms in genes expressed in cis with the X-linked mutation to determine which nuclei express the mutant allele even when the mutant gene is not detected. This approach enables gene expression comparisons between mutant and wild-type cells within the same individual, eliminating variability introduced by comparisons to controls with different genetic backgrounds. We apply this approach to mosaic female mouse models and humans with Rett syndrome, an X-linked neurodevelopmental disorder caused by mutations in the gene encoding the methyl-DNA-binding protein MECP2, and observe that cell-type-specific DNA methylation predicts the degree of gene upregulation in MECP2-mutant neurons. This approach can be broadly applied to study gene expression in mosaic X-linked disorders.

Neuronal activity-dependent gene expression is essential for brain development. Although transcriptional and epigenetic effects of neuronal activity have been explored in mice, such an investigation is lacking in humans. Because alterations in GABAergic neuronal circuits are implicated in neurological disorders, we conducted a comprehensive activity-dependent transcriptional and epigenetic profiling of human induced pluripotent stem cell-derived GABAergic neurons similar to those of the early developing striatum. We identified genes whose expression is inducible after membrane depolarization, some of which have specifically evolved in primates and/or are associated with neurological diseases, including schizophrenia and autism spectrum disorder (ASD). We define the genome-wide profile of human neuronal activity-dependent enhancers, promoters and the transcription factors CREB and CRTC1. We found significant heritability enrichment for ASD in the inducible promoters. Our results suggest that sequence variation within activity-inducible promoters of developing human forebrain GABAergic neurons contributes to ASD risk. Boulting et al. profile activity-dependent gene expression and regulatory elements in human induced pluripotent stem cell-derived GABAergic neurons and uncover a possible role for calcium-responsive gene promoters of these neurons in autism risk.

The septum is a ventral forebrain structure known to regulate innate behaviors. During embryonic development, septal neurons are produced in multiple proliferative areas from neural progenitors following transcriptional programs that are still largely unknown. Here, we use a combination of single-cell RNA sequencing, histology, and genetic models to address how septal neuron diversity is established during neurogenesis. We find that the transcriptional profiles of septal progenitors change along neurogenesis, coinciding with the generation of distinct neuron types. We characterize the septal eminence, an anatomically distinct and transient proliferative zone composed of progenitors with distinctive molecular profiles, proliferative capacity, and fate potential compared to the rostral septal progenitor zone. We show that Nkx2.1 -expressing septal eminence progenitors give rise to neurons belonging to at least three morphological classes, born in temporal cohorts that are distributed across different septal nuclei in a sequential fountain-like pattern. Our study provides insight into the molecular programs that control the sequential production of different neuronal types in the septum, a structure with important roles in regulating mood and motivation.

Mutations in the GJB2 gene cause DFNB1, the most common hereditary hearing loss. GJB2 is expressed by cochlear epithelial cells and fibrocytes, but not by sensory hair cells or neurons. Attempts to treat DFNB1 mouse models with gene therapy have not substantially restored function, as inappropriate expression in hair cells and neurons might compromise their electrical activity. Here, we use ATAC-seq to identify candidate gene regulatory elements (GREs) that can drive cell-type-specific expression of GJB2 . HA-tagged GJB2, delivered to a conditional knockout mouse with AAV vectors carrying GREs, is expressed by the appropriate cells, prevents degeneration, and rescues hearing by only 10–20 dB. In a Gjb2 partial knockdown model, a vector lacking HA prevents degeneration and completely restores hearing. In cynomolgus monkey cochleas, human GJB2.HA delivered with similar vectors is located in the appropriate cell types and causes little or no compromise of hearing sensitivity. Together, these findings suggest that GRE-mediated expression of GJB2 can prevent hearing loss in DFNB1 patients. Mutations in GJB2 cause DFNB1, the most common hereditary deafness. ATAC-seq identification of gene regulatory elements enables targeted GJB2 delivery to cochlear cells, preventing hearing loss in mouse models.