In the enteric nervous system (ENS), glia outnumber neurons by 4-fold and form an extensive network throughout the gastrointestinal tract. Growing evidence for the essential role of enteric glia in bowel function makes it imperative to understand better their molecular marker expression and how they relate to glia in the rest of the nervous system. We analyzed expression of markers of astrocytes and oligodendrocytes in the ENS and found, unexpectedly, that proteolipid protein 1 (PLP1) is specifically expressed by glia in adult mouse intestine. PLP1 and S100β are the markers most widely expressed by enteric glia, while glial fibrillary acidic protein (GFAP) expression is more restricted. Marker expression in addition to cellular location and morphology distinguishes a specific subpopulation of intramuscular enteric glia, suggesting that a combinatorial code of molecular markers can be used to identify distinct subtypes. To assess the similarity between enteric and extra-enteric glia, we performed RNA sequencing analysis (RNA-Seq) on PLP1-expressing cells in the mouse intestine and compared their gene expression pattern to that of other types of glia. This analysis shows that enteric glia are transcriptionally unique and distinct from other cell types in the nervous system. Enteric glia express many genes characteristic of the myelinating glia, Schwann cells and oligodendrocytes, although there is no evidence of myelination in the murine ENS.

In multicellular organisms, the entry into meiosis is a complex process characterized by increasing meiotic specialization. Using single-cell RNA sequencing, we reconstructed the developmental program into maize male meiosis. A smooth continuum of expression stages before meiosis was followed by a two-step transcriptome reorganization in leptotene, during which 26.7% of transcripts changed in abundance by twofold or more. Analysis of cell-cycle gene expression indicated that nearly all pregerminal cells proliferate, eliminating a stem-cell model to generate meiotic cells. Mutants defective in somatic differentiation or meiotic commitment expressed transcripts normally present in early meiosis after a delay; thus, the germinal transcriptional program is cell autonomous and can proceed despite meiotic failure.

In multicellular organisms, the entry into meiosis is a complex process characterized by increasing levels of meiotic specialization. We used single-cell RNA-sequencing to reconstruct the developmental program into meiosis in maize. We observed a smooth continuum of expression stages leading up to meiosis, followed by a sharp reorganization of the transcriptome in early meiotic prophase. This latter transcriptional shift was dramatic, with 26.7% of expressed genes changing by 2 fold or more, and occurred just prior to a proposed cell cycle checkpoint. Changes in cell physiology accompanied the nuclear events of meiosis, including a decrease in protein translation capacity and increase in membrane-bound organelles. We further identified differences in gene expression between the mitotic and meiotic cell cycles. Our results uncover a multi-step pathway into meiosis and highlight the power of single cell RNA-seq to define developmental transitions.Meiosis is a pivotal event in the life cycle of sexually reproducing organisms, reducing the chromosome number in half and creating new allele combinations through recombination. The mechanisms that regulate meiotic entry in plants are not well understood 1,2 , but are of great importance to crop breeding and agricultural yield 3 .In maize anthers, archesporial (AR) cells are the first cell type in the lineage dedicated to a meiotic fate, acting as the developmental switch between somatic and germinal growth 4 . After a ~3 day period of transit amplifying mitotic divisions, AR cells become pollen mother cells (PMCs) and then enter meiotic prophase. There are substantial changes in cell morphology 4 and gene expression 5-8 during pre-meiotic and early meiotic development, but the cellular intermediates that arise during this process are not well-defined.Here, we applied single-cell RNA-sequencing (scRNA-seq) to characterize the developmental program leading up to meiosis in maize with high resolution. We introduce a quantitative framework, 'pseudotime velocity', to infer developmental transitions based on periods of relatively rapid gene expression change, and apply this framework to identify intermediates from our data. These results provide a road-map for reconstructing plant developmental pathways with scRNA-seq. Single-cell RNA-seq of pre-meiotic and early meiotic cellsMaize male germinal cells form within immature anthers, centrally located in each of four anther lobes (Fig. 1A). Anthers expand in size predictably during early development; consequently, anther length can be used as a reliable, continuous staging system that is correlated with both developmental events and organ age 4 . We established methods to isolate single pre-meiotic and meiotic cells from

Flowering plants alternate between multicellular haploid (gametophyte) and diploid (sporophyte) generations. Pollen actively transcribes its haploid genome, providing phenotypic diversity even among pollen grains from a single plant. In this study, we used allele-specific RNA sequencing of single pollen precursors to follow the shift to haploid expression in maize pollen. We observed widespread biallelic expression for 11 days after meiosis, indicating that transcripts synthesized by the diploid sporophyte persist long into the haploid phase. Subsequently, there was a rapid and global conversion to monoallelic expression at pollen mitosis I, driven by active new transcription from the haploid genome. Genes showed evidence of increased purifying selection if they were expressed after (but not before) pollen mitosis I. This work establishes the timing during which haploid selection may act in pollen.

We present a sensitive approach to predict genes expressed selectively in specific cell types, by searching publicly available expression data for genes with a similar expression profile to known cell-specific markers. Our method, CellMapper, strongly outperforms previous computational algorithms to predict cell type-specific expression, especially for rare and difficult-to-isolate cell types. Furthermore, CellMapper makes accurate predictions for human brain cell types that have never been isolated, and can be rapidly applied to diverse cell types from many tissues. We demonstrate a clinically relevant application to prioritize candidate genes in disease susceptibility loci identified by GWAS.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1062-5) contains supplementary material, which is available to authorized users.

Transcytosis plays an important role in establishing cell polarity and in mediating transport of large cargo across epithelial barriers, but its molecular basis is unclear. Nelms et al. present a new dataset of genes involved in receptor-mediated transcytosis and show that the apical and basolateral recycling and transcytotic pathways are genetically separable.

Flowering plants alternate between multicellular haploid (gametophyte) and diploid (sporophyte) generations. One consequence of this life cycle is that plants face substantial selection during the haploid phase (1-3). Pollen actively transcribes its haploid genome (4), providing phenotypic diversity even among pollen grains from a single plant. Currently, the timing that pollen precursors first establish this independence is unclear. Starting with an endowment of transcripts from the diploid parent, when do haploid cells generated by meiosis begin to express genes? Here, we follow the shift to haploid expression in maize pollen using allele-specific RNA-sequencing (RNA-Seq) of single pollen precursors. We observe widespread biallelic expression for 11 days after meiosis, indicating that transcripts synthesized by the diploid sporophyte persist long into the haploid phase. Subsequently, there was a rapid and global conversion to monoallelic expression at pollen mitosis I (PMI), driven by active new transcription from the haploid genome. Genes expressed during the haploid phase showed reduced rates of nonsynonymous relative to synonymous substitutions (dn/ds) if they were expressed after PMI, but not before, consistent with purifying selection acting on the haploid gametophyte. This work establishes the timing with which haploid selection may act in pollen and provides a detailed time-course of gene expression during pollen development.

No abstract