SUMMARY The structural organization of neural circuits is strongly influenced by experience, but the underlying mechanisms are incompletely understood. We found that, in the developing dentate gyrus (DG), excitatory drive promotes the somatic innervation of principal granule cells (GCs) by parvalbumin (PV)-positive basket cells. By contrast, presynaptic differentiation of GCs and interneuron sub-types that inhibit GC dendrites is largely resistant to loss of glutamatergic neurotransmission. The networks of PV basket cells in the DG are regulated by vesicular release from projection entorhinal cortical neurons and, at least in part, by NMDA receptors in interneurons. Finally, we present evidence that glutamatergic inputs and NMDA receptors regulate these networks through a presynaptic mechanism that appears to control the branching of interneuron axons. Our results provide insights into how cortical activity tunes the inhibition in a subcortical circuit, and reveal new principles of interneuron plasticity.

The rise in throughput and quality of long-read sequencing should allow unambiguous identification of full-length transcript isoforms. However, its application to single-cell RNA-seq has been limited by throughput and expense. Here we develop and characterize long-read Split-seq (LR-Split-seq), which uses combinatorial barcoding to sequence single cells with long reads. Applied to the C2C12 myogenic system, LR-split-seq associates isoforms to cell types with relative economy and design flexibility. We find widespread evidence of changing isoform expression during differentiation including alternative transcription start sites (TSS) and/or alternative internal exon usage. LR-Split-seq provides an affordable method for identifying cluster-specific isoforms in single cells.

Multiple mouse models have been generated that strive to recapitulate human Alzheimer′s disease (AD) pathological features to investigate disease mechanisms and potential treatments. The 3xTg-AD mouse presents the two major hallmarks of AD, which are plaques and tangles that increase during aging. While behavioral changes and the accumulation of plaques and tangles have been well described in the 3xTg-AD mice, the subpopulations of neurons and glial cells present throughout disease progression have not been characterized. Here, we used single-cell RNA-seq to investigate changes in subpopulations of microglia, and single-nucleus RNA-seq to explore subpopulations of neurons, astrocytes, and oligodendrocytes in the hippocampus and cortex of aging 3xTg-AD as well as 5xFAD mice for comparison. We recovered a common path of age-associated astrocyte activation between the 3xTg-AD and the 5xFAD models and found that 3xTg-AD-derived astrocytes seem to be less activated. We identified multiple subtypes of microglia, including a subpopulation with a distinct transcription factor expression profile that showed an early increase in Csf1 expression before the switch to disease associated microglia (DAM). We used bulk RNA-seq in the hippocampus of 3xTg-AD mice across their lifespan to identify distinct modules of genes whose expression increases with aging and worsening pathology. Finally, scATAC-seq revealed multiple subpopulations of cells with accessible chromatin in regions around genes associated with glial activation. Overall, differences between the main glial groups point to a slower activation process in the 3xTg-AD model when compared to the 5xFAD. Our study contributes to the identification of progressive transcriptional changes of glial cells in a mouse model that has plaques and tangles, thus providing information to aid in targeted AD therapeutics that could translate into positive clinical outcomes.

The majority of mammalian genes encode multiple transcript isoforms that result from differential promoter use, changes in exonic splicing, and alternative 3′ end choice. Detecting and quantifying transcript isoforms across tissues, cell types, and species has been extremely challenging because transcripts are much longer than the short reads normally used for RNA-seq. By contrast, long-read RNA-seq (LR-RNA-seq) gives the complete structure of most transcripts. We sequenced 264 LR-RNA-seq PacBio libraries totaling over 1 billion circular consensus reads (CCS) for 81 unique human and mouse samples. We detect at least one full-length transcript from 87.7% of annotated human protein coding genes and a total of ~200,000 full-length transcripts, ~40% of which have novel exon junction chains. To capture and compute on the three sources of transcript structure diversity, we introduce a gene and transcript annotation framework that uses triplets representing the transcript start site, exon junction chain, and transcript end site of each transcript. Using triplets in a simplex representation demonstrates how promoter selection, splice pattern, and 3′ processing are deployed across human tissues, with nearly half of multitranscript protein coding genes showing a clear bias toward one of the three diversity mechanisms. Evaluated across samples, the predominantly expressed transcript changes for 74% of protein coding genes. In evolution, the human and mouse transcriptomes are globally similar in types of transcript structure diversity, yet among individual orthologous gene pairs, more than half (57.8%) show substantial differences in mechanism of diversification in matching tissues. This initial large-scale survey of human and mouse long-read transcriptomes provides a foundation for further analyses of alternative transcript usage, and is complemented by short-read and microRNA data on the same samples and by epigenome data elsewhere in the ENCODE4 collection.

SummaryGene regulatory networks (GRNs) provide a powerful framework for studying cellular differentiation. However, it is less clear how GRNs encode cellular responses to everyday microenvironmental cues. Macrophages can be polarized and potentially repolarized based on environmental signaling. In order to identify the GRNs that drive macrophage polarization and the heterogeneous single-cell subpopulations that are present in the process, we used a high-resolution time course of bulk and single-cell RNA-seq and ATAC-seq assays of HL-60-derived macrophages polarized towards M1 or M2 over 24 hours. We identified transient M1 and M2 markers, including the main transcription factors that underlie polarization, and subpopulations of naive, transitional, and terminally polarized macrophages. We built bulk and single-cell polarization GRNs to compare the recovered interactions and found that each technology recovered only a subset of known interactions. Our data provide a resource to study the GRN of cellular maturation in response to microenvironmental stimuli in a variety of contexts in homeostasis and disease.