Barbara Treutlein

Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq

The mammalian lung is a highly branched network in which the distal regions of the bronchial tree transform during development into a densely packed honeycomb of alveolar air sacs that mediate gas exchange. Although this transformation has been studied by marker expression analysis and fate-mapping, the mechanisms that control the progression of lung progenitors along distinct lineages into mature alveolar cell types are still incompletely known, in part because of the limited number of lineage markers and the effects of ensemble averaging in conventional transcriptome analysis experiments on cell populations. Here we show that single-cell transcriptome analysis circumvents these problems and enables direct measurement of the various cell types and hierarchies in the developing lung. We used microfluidic single-cell RNA sequencing (RNA-seq) on 198 individual cells at four different stages encompassing alveolar differentiation to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We empirically classified cells into distinct groups by using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or the previous purification of cell populations. The results confirmed the basic outlines of the classical model of epithelial cell-type diversity in the distal lung and led to the discovery of many previously unknown cell-type markers, including transcriptional regulators that discriminate between the different populations. We reconstructed the molecular steps during maturation of bipotential progenitors along both alveolar lineages and elucidated the full life cycle of the alveolar type 2 cell lineage. This single-cell genomics approach is applicable to any developing or mature tissue to robustly delineate molecularly distinct cell types, define progenitors and lineage hierarchies, and identify lineage-specific regulatory factors.

Quantitative assessment of single-cell RNA-sequencing methods

Interest in single-cell whole-transcriptome analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. We compared commercially available single-cell RNA amplification methods with both microliter and nanoliter volumes, using sequence from bulk total RNA and multiplexed quantitative PCR as benchmarks to systematically evaluate the sensitivity and accuracy of various single-cell RNA-seq approaches. We show that single-cell RNA-seq can be used to perform accurate quantitative transcriptome measurement in individual cells with a relatively small number of sequencing reads and that sequencing large numbers of single cells can recapitulate bulk transcriptome complexity.

Lineage-negative progenitors mobilize to regenerate lung epithelium after major injury

Broadly, tissue regeneration is achieved in two ways: by proliferation of common differentiated cells and/or by deployment of specialized stem/progenitor cells. Which of these pathways applies is both organ- and injury-specific. Current models in the lung posit that epithelial repair can be attributed to cells expressing mature lineage markers. By contrast, here we define the regenerative role of previously uncharacterized, rare lineage-negative epithelial stem/progenitor (LNEP) cells present within normal distal lung. Quiescent LNEPs activate a ΔNp63 (a p63 splice variant) and cytokeratin 5 remodelling program after influenza or bleomycin injury in mice. Activated cells proliferate and migrate widely to occupy heavily injured areas depleted of mature lineages, at which point they differentiate towards mature epithelium. Lineage tracing revealed scant contribution of pre-existing mature epithelial cells in such repair, whereas orthotopic transplantation of LNEPs, isolated by a definitive surface profile identified through single-cell sequencing, directly demonstrated the proliferative capacity and multipotency of this population. LNEPs require Notch signalling to activate the ΔNp63 and cytokeratin 5 program, and subsequent Notch blockade promotes an alveolar cell fate. Persistent Notch signalling after injury led to parenchymal ‘micro-honeycombing’ (alveolar cysts), indicative of failed regeneration. Lungs from patients with fibrosis show analogous honeycomb cysts with evidence of hyperactive Notch signalling. Our findings indicate that distinct stem/progenitor cell pools repopulate injured tissue depending on the extent of the injury, and the outcomes of regeneration or fibrosis may depend in part on the dynamics of LNEP Notch signalling.

Human cerebral organoids recapitulate gene expression programs of fetal neocortex development

Significance We have used single-cell RNA sequencing to compare human cerebral organoids and fetal neocortex. We find that, with relatively few exceptions, cells in organoid cortex-like regions use genetic programs very similar to fetal tissue to generate a structured cerebral cortex. Our study is of interest, as it shows which genetic features underlying human cortical development can be accurately studied in organoid culture systems. This is important because although cerebral organoids have great promise for modeling human neurodevelopment, the extent to which organoids recapitulate neural progenitor proliferation and differentiation networks in vivo remained unclear. Cerebral organoids—3D cultures of human cerebral tissue derived from pluripotent stem cells—have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and previously unidentified interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single-cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.

Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq

Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states1–3. However, the intermediates through which individual cells progress are largely undefined. Here we used single-cell RNA-seq4–7 at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts (MEFs) to induced neuronal (iN) cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity, we find that the molecular reprogramming path is remarkably continuous. Overexpression of the proneural pioneer factor Ascl1 results in a well-defined initialization, causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. The initial transcriptional response is relatively homogeneous among fibroblasts suggesting the early steps are not limiting for productive reprogramming. Instead, the later emergence of a competing myogenic program and variable transgene dynamics over time appear to be the major efficiency limits of direct reprogramming. Moreover, a transcriptional state, distinct from donor and target cell programs, is transiently induced in cells undergoing productive reprogramming. Our data provide a high-resolution approach for understanding transcriptome states during lineage differentiation.

Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing

Significance Neurexins are presynaptic cell-adhesion molecules that are essential for synapse formation and synaptic transmission. Extensive alternative splicing of neurexin transcripts may generate thousands of isoforms, but it is unclear how many distinct neurexins are physiologically produced. We used unbiased long-read sequencing of full-length neurexin mRNAs to systematically assess the alternative splicing of neurexins in prefrontal cortex. We identified a novel, abundantly used alternatively spliced exon of neurexins, and found that the different events of alternative splicing of neurexins appear to be independent of each other. Our data suggest that thousands of neurexin isoforms are physiologically generated, consistent with the notion that neurexins represent transsynaptic protein-interaction scaffolds that mediate diverse functions and are regulated by alternative splicing at multiple independent sites. Neurexins are evolutionarily conserved presynaptic cell-adhesion molecules that are essential for normal synapse formation and synaptic transmission. Indirect evidence has indicated that extensive alternative splicing of neurexin mRNAs may produce hundreds if not thousands of neurexin isoforms, but no direct evidence for such diversity has been available. Here we use unbiased long-read sequencing of full-length neurexin (Nrxn)1α, Nrxn1β, Nrxn2β, Nrxn3α, and Nrxn3β mRNAs to systematically assess how many sites of alternative splicing are used in neurexins with a significant frequency, and whether alternative splicing events at these sites are independent of each other. In sequencing more than 25,000 full-length mRNAs, we identified a novel, abundantly used alternatively spliced exon of Nrxn1α and Nrxn3α (referred to as alternatively spliced sequence 6) that encodes a 9-residue insertion in the flexible hinge region between the fifth LNS (laminin-α, neurexin, sex hormone-binding globulin) domain and the third EGF-like sequence. In addition, we observed several larger-scale events of alternative splicing that deleted multiple domains and were much less frequent than the canonical six sites of alternative splicing in neurexins. All of the six canonical events of alternative splicing appear to be independent of each other, suggesting that neurexins may exhibit an even larger isoform diversity than previously envisioned and comprise thousands of variants. Our data are consistent with the notion that α-neurexins represent extracellular protein-interaction scaffolds in which different LNS and EGF domains mediate distinct interactions that affect diverse functions and are independently regulated by independent events of alternative splicing.

Multilineage communication regulates human liver bud development from pluripotency

Conventional two-dimensional differentiation from pluripotency fails to recapitulate cell interactions occurring during organogenesis. Three-dimensional organoids generate complex organ-like tissues; however, it is unclear how heterotypic interactions affect lineage identity. Here we use single-cell RNA sequencing to reconstruct hepatocyte-like lineage progression from pluripotency in two-dimensional culture. We then derive three-dimensional liver bud organoids by reconstituting hepatic, stromal, and endothelial interactions, and deconstruct heterogeneity during liver bud development. We find that liver bud hepatoblasts diverge from the two-dimensional lineage, and express epithelial migration signatures characteristic of organ budding. We benchmark three-dimensional liver buds against fetal and adult human liver single-cell RNA sequencing data, and find a striking correspondence between the three-dimensional liver bud and fetal liver cells. We use a receptor–ligand pairing analysis and a high-throughput inhibitor assay to interrogate signalling in liver buds, and show that vascular endothelial growth factor (VEGF) crosstalk potentiates endothelial network formation and hepatoblast differentiation. Our molecular dissection reveals interlineage communication regulating organoid development, and illuminates previously inaccessible aspects of human liver development.

Differences and similarities between human and chimpanzee neural progenitors during cerebral cortex development

Human neocortex expansion likely contributed to the remarkable cognitive abilities of humans. This expansion is thought to primarily reflect differences in proliferation versus differentiation of neural progenitors during cortical development. Here, we have searched for such differences by analysing cerebral organoids from human and chimpanzees using immunohistofluorescence, live imaging, and single-cell transcriptomics. We find that the cytoarchitecture, cell type composition, and neurogenic gene expression programs of humans and chimpanzees are remarkably similar. Notably, however, live imaging of apical progenitor mitosis uncovered a lengthening of prometaphase-metaphase in humans compared to chimpanzees that is specific to proliferating progenitors and not observed in non-neural cells. Consistent with this, the small set of genes more highly expressed in human apical progenitors points to increased proliferative capacity, and the proportion of neurogenic basal progenitors is lower in humans. These subtle differences in cortical progenitors between humans and chimpanzees may have consequences for human neocortex evolution. DOI: http://dx.doi.org/10.7554/eLife.18683.001

Structural reorganization of the chromatin remodeling enzyme Chd1 upon engagement with nucleosomes

The yeast Chd1 protein acts to position nucleosomes across genomes. Here, we model the structure of the Chd1 protein in solution and when bound to nucleosomes. In the apo state, the DNA-binding domain contacts the edge of the nucleosome while in the presence of the non-hydrolyzable ATP analog, ADP-beryllium fluoride, we observe additional interactions between the ATPase domain and the adjacent DNA gyre 1.5 helical turns from the dyad axis of symmetry. Binding in this conformation involves unravelling the outer turn of nucleosomal DNA and requires substantial reorientation of the DNA-binding domain with respect to the ATPase domains. The orientation of the DNA-binding domain is mediated by sequences in the N-terminus and mutations to this part of the protein have positive and negative effects on Chd1 activity. These observations indicate that the unfavorable alignment of C-terminal DNA-binding region in solution contributes to an auto-inhibited state. DOI: http://dx.doi.org/10.7554/eLife.22510.001

Mapping heterogeneity in patient-derived melanoma cultures by single-cell RNA-seq

Recent technological advances in single-cell genomics make it possible to analyze cellular heterogeneity of tumor samples. Here, we applied single-cell RNA-seq to measure the transcriptomes of 307 single cells cultured from three biopsies of three different patients with a BRAF/NRAS wild type, BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant melanoma metastasis, respectively. Analysis based on self-organizing maps identified sub-populations defined by multiple gene expression modules involved in proliferation, oxidative phosphorylation, pigmentation and cellular stroma. Gene expression modules had prognostic relevance when compared with gene expression data from published melanoma samples and patient survival data. We surveyed kinome expression patterns across sub-populations of the BRAF/NRAS wild type sample and found that CDK4 and CDK2 were consistently highly expressed in the majority of cells, suggesting that these kinases might be involved in melanoma progression. Treatment of cells with the CDK4 inhibitor palbociclib restricted cell proliferation to a similar, and in some cases greater, extent than MAPK inhibitors. Finally, we identified a low abundant sub-population in this sample that highly expressed a module containing ABC transporter ABCB5, surface markers CD271 and CD133, and multiple aldehyde dehydrogenases (ALDHs). Patient-derived cultures of the BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant metastases showed more homogeneous single-cell gene expression patterns with gene expression modules for proliferation and ABC transporters. Taken together, our results describe an intertumor and intratumor heterogeneity in melanoma short-term cultures which might be relevant for patient survival, and suggest promising targets for new treatment approaches in melanoma therapy.