Dormant hematopoietic stem cells (dHSCs) are atop the hematopoietic hierarchy. The molecular identity of dHSCs and the mechanisms regulating their maintenance or exit from dormancy remain uncertain. Here, we use single-cell RNA sequencing (RNA-seq) analysis to show that the transition from dormancy toward cell-cycle entry is a continuous developmental path associated with upregulation of biosynthetic processes rather than a stepwise progression. In addition, low Myc levels and high expression of a retinoic acid program are characteristic for dHSCs. To follow the behavior of dHSCs in situ, a Gprc5c-controlled reporter mouse was established. Treatment with all-trans retinoic acid antagonizes stress-induced activation of dHSCs by restricting protein translation and levels of reactive oxygen species (ROS) and Myc. Mice maintained on a vitamin A-free diet lose HSCs and show a disrupted re-entry into dormancy after exposure to inflammatory stress stimuli. Our results highlight the impact of dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity. VIDEO ABSTRACT.

Full-length RNA sequencing (RNA-Seq) has been applied to bulk tissue, cell lines and sorted cells to characterize transcriptomes 1-11 , but applying this technology to single cells has proven to be difficult, with less than ten single-cell transcriptomes having been analyzed thus far 12,13. Although single splicing events have been described for ≤200 single cells with statistical confidence 14,15 , full-length mRNA analyses for hundreds of cells have not been reported. Singlecell short-read 3′ sequencing enables the identification of cellular subtypes 16-21 , but full-length mRNA isoforms for these cell types cannot be profiled. We developed a method that starts with bulk tissue and identifies single-cell types and their full-length RNA isoforms without fluorescence-activated cell sorting. Using single-cell isoform RNA-Seq (ScISOr-Seq), we identified RNA isoforms in neurons, astrocytes, microglia, and cell subtypes such as Purkinje and Granule cells, and cell-typespecific combination patterns of distant splice sites 6-9,22,23. We used ScISOr-Seq to improve genome annotation in mouse Gencode version 10 by determining the cell-type-specific expression of 18,173 known and 16,872 novel isoforms. Unlike sorting-based methods (Supplementary Fig. 1a), ScISOr-Seq identifies isoforms in >1,000 single cells from bulk tissue without cell sorting by combining two technologies (Fig. 1a). We used microfluidics to amplify full-length cDNA from single cells in a sample. cDNA produced from each single cell was barcoded to enable cell-of-origin identification and then split into two pools, with one pool being used for short-read Illumina 3′ sequencing to measure gene expression and the other pool being used for long-read sequencing and isoform identification. Short-read 3′ sequencing provided molecular counts for each gene and cell, which enabled clustering of cells and cell type assignment using cell-type-specific markers. Long-read sequencing with Pacific Biosciences (PacBio) 1,2,4,5 or Oxford Nanopore 3 was used to identify full-length RNA isoforms. Single-cell barcodes were also present in long reads and could be used to determine the individual

A fundamental principle in biology is that the program for early development is established during oogenesis in the form of the maternal transcriptome1,2. How the maternal transcriptome acquires the appropriate content and dosage of transcripts is not fully understood. Here we show that TUT4/7-mediated mRNA 3′ terminal uridylation sculpts the mouse maternal transcriptome by eliminating transcripts during oocyte growth. TUT4/7-mediated uridylation is essential for both oocyte maturation and fertility. In comparison to somatic cells, the oocyte transcriptome displays shorter poly(A) tail length and a high relative proportion of terminal oligo-uridylation. TUT4/7 deletion leads to the accumulation of a cohort of transcripts with a high frequency of very short poly(A) tails and a loss of 3′ oligo-uridylation. In contrast, TUT4/7-deficiency does not alter gene expression in a variety of somatic cells. In summary, we show essential and specific functions for poly(A) tail length and 3′ terminal uridylation in sculpting a functional maternal transcriptome.

Splicing varies across brain regions, but the single-cell resolution of regional variation is unclear. We present a single-cell investigation of differential isoform expression (DIE) between brain regions using single-cell long-read sequencing in mouse hippocampus and prefrontal cortex in 45 cell types at postnatal day 7 (www.isoformAtlas.com). Isoform tests for DIE show better performance than exon tests. We detect hundreds of DIE events traceable to cell types, often corresponding to functionally distinct protein isoforms. Mostly, one cell type is responsible for brain-region specific DIE. However, for fewer genes, multiple cell types influence DIE. Thus, regional identity can, although rarely, override cell-type specificity. Cell types indigenous to one anatomic structure display distinctive DIE, e.g. the choroid plexus epithelium manifests distinct transcription-start-site usage. Spatial transcriptomics and long-read sequencing yield a spatially resolved splicing map. Our methods quantify isoform expression with cell-type and spatial resolution and it contributes to further our understanding of how the brain integrates molecular and cellular complexity.

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SignificanceMosquito blood cells are central players of immunity against the vector-borne pathogens that devastate the lives of millions of people worldwide. However, their molecular identity and classification remain controversial. By applying single-cell RNA sequencing and high-content imaging flow cytometry, we defined the molecular fingerprint of a subset of mosquito blood cells and characterized two transcriptionally distinct blood cell populations that resemble previously described cell types. Surprisingly, cell population analyses at a single-cell level uncovered an active molecular transfer between the two cell types that may contribute to cellular diversity and plasticity seen across biological systems.

Animal bodies comprise diverse arrays of cells. To characterize cellular identities across an entire body, we have compared the transcriptomes of single cells randomly picked from dissociated whole larvae of the marine annelid Platynereis dumerilii. We identify five transcriptionally distinct groups of differentiated cells, each expressing a unique set of transcription factors and effector genes that implement cellular phenotypes. Spatial mapping of cells into a cellular expression atlas, and wholemount in situ hybridization of group-specific genes reveals spatially coherent transcriptional domains in the larval body, comprising, for example, apical sensory-neurosecretory cells versus neural/epidermal surface cells. These domains represent new, basic subdivisions of the annelid body based entirely on differential gene expression, and are composed of multiple, transcriptionally similar cell types. They do not represent clonal domains, as revealed by developmental lineage analysis. We propose that the transcriptional domains that subdivide the annelid larval body represent families of related cell types that have arisen by evolutionary diversification. Their possible evolutionary conservation makes them a promising tool for evo–devo research.

Recent advances in single-cell transcriptomics techniques have opened the door to the study of gene regulatory networks (GRNs) at the single-cell level. Here, we studied the GRNs controlling the emergence of hematopoietic stem and progenitor cells from mouse embryonic endothelium using a combination of single-cell transcriptome assays. We found that a heptad of transcription factors (Runx1, Gata2, Tal1, Fli1, Lyl1, Erg and Lmo2) is specifically co-expressed in an intermediate population expressing both endothelial and hematopoietic markers. Within the heptad, we identified two sets of factors of opposing functions: one (Erg/Fli1) promoting the endothelial cell fate, the other (Runx1/Gata2) promoting the hematopoietic fate. Surprisingly, our data suggest that even though Fli1 initially supports the endothelial cell fate, it acquires a pro-hematopoietic role when co-expressed with Runx1. This work demonstrates the power of single-cell RNA-sequencing for characterizing complex transcription factor dynamics.