FOXO1-mediated lipid metabolism maintains mammalian embryos in dormancy.

Mammalian developmental timing is adjustable in vivo by preserving pre-implantation embryos in a dormant state called diapause. Inhibition of the growth regulator mTOR (mTORi) pauses mouse development in vitro, yet how embryonic dormancy is maintained is not known. Here we show that mouse embryos in diapause are sustained by using lipids as primary energy source. In vitro, supplementation of embryos with the metabolite L-carnitine balances lipid consumption, puts the embryos in deeper dormancy and boosts embryo longevity. We identify FOXO1 as an essential regulator of the energy balance in dormant embryos and propose, through meta-analyses of dormant cell signatures, that it may be a common regulator of dormancy across adult tissues. Our results lift a constraint on in vitro embryo survival and suggest that lipid metabolism may be a critical metabolic transition relevant for longevity and stem cell function across tissues.

Enhanced cortical neural stem cell identity through short SMAD and WNT inhibition in human cerebral organoids facilitates emergence of outer radial glial cells.

Cerebral organoids exhibit broad regional heterogeneity accompanied by limited cortical cellular diversity despite the tremendous upsurge in derivation methods, suggesting inadequate patterning of early neural stem cells (NSCs). Here we show that a short and early Dual SMAD and WNT inhibition course is necessary and sufficient to establish robust and lasting cortical organoid NSC identity, efficiently suppressing non-cortical NSC fates, while other widely used methods are inconsistent in their cortical NSC-specification capacity. Accordingly, this method selectively enriches for outer radial glia NSCs, which cyto-architecturally demarcate well-defined outer sub-ventricular-like regions propagating from superiorly radially organized, apical cortical rosette NSCs. Finally, this method culminates in the emergence of molecularly distinct deep and upper cortical layer neurons, and reliably uncovers cortex-specific microcephaly defects. Thus, a short SMAD and WNT inhibition is critical for establishing a rich cortical cell repertoire that enables mirroring of fundamental molecular and cyto-architectural features of cortical development and meaningful disease modelling.

Visualizing Cluster-specific Genes from Single-cell Transcriptomics Data Using Association Plots.

Visualizing single-cell transcriptomics data in an informative way is a major challenge in biological data analysis. Clustering of cells is a prominent analysis step and the results are usually visualized in a planar embedding of the cells using methods like PCA, t-SNE, or UMAP. Given a cluster of cells, one frequently searches for the genes highly expressed specifically in that cluster. At this point, visualization is usually replaced by studying a list of differentially expressed genes. Association Plots are derived from correspondence analysis and constitute a planar visualization of the features which characterize a given cluster of observations. We have adapted Association Plots to address the challenge of visualizing cluster-specific genes in large single-cell data sets. Our method is made available as a free R package called APL. We demonstrate the application of APL and Association Plots to single-cell RNA-seq data on two example data sets. First, we present how to delineate novel marker genes using Association Plots with the example of Peripheral Blood Mononuclear Cell data. Second, we show how to apply Association Plots for annotating cell clusters to known cell types using Association Plots and a predefined list of marker genes. To do this we will use data from the human cell atlas of fetal gene expression. Results from Association Plots will also be compared to methods for deriving differentially expressed genes, and we will show the integration of APL with Gene Ontology Enrichment.

Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads.

Lumen morphogenesis results from the interplay between molecular pathways and mechanical forces. In several organs, epithelial cells share their apical surfaces to form a tubular lumen. In the liver, however, hepatocytes share the apical surface only between adjacent cells and form narrow lumina that grow anisotropically, generating a 3D network of bile canaliculi (BC). Here, by studying lumenogenesis in differentiating mouse hepatoblasts in vitro, we discovered that adjacent hepatocytes assemble a pattern of specific extensions of the apical membrane traversing the lumen and ensuring its anisotropic expansion. These previously unrecognized structures form a pattern, reminiscent of the bulkheads of boats, also present in the developing and adult liver. Silencing of Rab35 resulted in loss of apical bulkheads and lumen anisotropy, leading to cyst formation. Strikingly, we could reengineer hepatocyte polarity in embryonic liver tissue, converting BC into epithelial tubes. Our results suggest that apical bulkheads are cell-intrinsic anisotropic mechanical elements that determine the elongation of BC during liver tissue morphogenesis.