Highlights d Heterogeneity and plasticity of non-parenchymal cells in healthy and NASH liver d Landscape of intrahepatic ligand-receptor signaling at single-cell resolution d Emergence of Trem2+ NASH-associated macrophages (NAMs) in mouse and human NASH d Stellakine secretion and contractile response to vasoactive hormones by HSCs

Cellular heterogeneity in growth and differentiation results in organ patterning. Single-cell transcriptomics allows characterization of gene expression heterogeneity in developing organs at unprecedented resolution. However, the original physical location of the cell is lost during this methodology. To recover the original location of cells in the developing organ is essential to link gene activity with cellular identity and function in plants. Here, we propose a method to reconstruct genome-wide gene expression patterns of individual cells in a 3D flower meristem by combining single-nuclei RNA-seq with microcopy-based 3D spatial reconstruction. By this, gene expression differences among meristematic domains giving rise to different tissue and organ types can be determined. As a proof of principle, the method is used to trace the initiation of vascular identity within the floral meristem. Our work demonstrates the power of spatially reconstructed single cell transcriptome atlases to understand plant morphogenesis. The floral meristem 3D gene expression atlas can be accessed at http://threed-flower-meristem.herokuapp.com.

Multicellular organisms display a fascinating complexity of cellular identities and patterns of diversification. The concept of ‘‘cell type’’ aims to describe and categorize this complexity. In this review, we discuss the traditional concept of cell types and highlight the impact of single-cell technologies and spatial omics on the understanding of cellular differentiation in plants. We summarize and compare position-based and lineage-based mechanisms of cell identity specification using flower development as a model system. More than understanding ontogenetic origins of differentiated cells, an important question in plant science is to understand their position- and developmental stage-specific heterogeneity. Combinatorial action and crosstalk of external and internal signals is the key to cellular heterogeneity, often converging on transcription factors that orchestrate gene expression programs.

Single-cell genomics provides unprecedented potential for research on plant development and environmental responses. Here, we introduce a generic procedure for plant nucleus isolation combined with nanowellbased library preparation. Our method enables the transcriptome analysis of thousands of individual plant nuclei. It serves as an alternative to the use of protoplast isolation, which is currently a standard methodology for plant single-cell genomics, although it can be challenging for some plant tissues. We show the applicability of our nucleus isolation method by using different plant materials from different species. The potential of our single-nucleus RNA sequencing method is shown through the characterization of transcriptomes of seedlings and developing flowers from Arabidopsis thaliana. We evaluated the transcriptome dynamics during the early stages of anther development, identified stage-specific activities of transcription factors regulating this process, and predicted potential target genes of these transcription factors. Our nucleus isolation procedure can be applied in different plant species and tissues, thus expanding the toolkit for plant single-cell genomics experiments.

Single-cell genomics has slowly advanced in plant research. Here, we introduce a generic procedure for plant nuclei isolation and nanowell-based library preparation for short-read sequencing. This plant-nuclei sequencing (PN-seq) method allows for analyzing several thousands of genes in thousands of individual plant cells. In this way, we expand the toolset for single-cell genomics in the field of plant biology to generate plant transcriptome atlases in development and environmental response.