Systematic comparison of sequencing-based spatial transcriptomic methods.

Recent developments of sequencing-based spatial transcriptomics (sST) have catalyzed important advancements by facilitating transcriptome-scale spatial gene expression measurement. Despite this progress, efforts to comprehensively benchmark different platforms are currently lacking. The extant variability across technologies and datasets poses challenges in formulating standardized evaluation metrics. In this study, we established a collection of reference tissues and regions characterized by well-defined histological architectures, and used them to generate data to compare 11 sST methods. We highlighted molecular diffusion as a variable parameter across different methods and tissues, significantly affecting the effective resolutions. Furthermore, we observed that spatial transcriptomic data demonstrate unique attributes beyond merely adding a spatial axis to single-cell data, including an enhanced ability to capture patterned rare cell states along with specific markers, albeit being influenced by multiple factors including sequencing depth and resolution. Our study assists biologists in sST platform selection, and helps foster a consensus on evaluation standards and establish a framework for future benchmarking efforts that can be used as a gold standard for the development and benchmarking of computational tools for spatial transcriptomic analysis.

A spatiotemporal molecular atlas of mouse spinal cord injury identifies a distinct astrocyte subpopulation and therapeutic potential of IGFBP2.

Spinal cord injury (SCI) triggers a cascade of intricate molecular and cellular changes that determine the outcome. In this study, we resolve the spatiotemporal organization of the injured mouse spinal cord and quantitatively assess in situ cell-cell communication following SCI. By analyzing existing single-cell RNA sequencing datasets alongside our spatial data, we delineate a subpopulation of Igfbp2-expressing astrocytes that migrate from the white matter (WM) to gray matter (GM) and become reactive upon SCI, termed Astro-GMii. Further, Igfbp2 upregulation promotes astrocyte migration, proliferation, and reactivity, and the secreted IGFBP2 protein fosters neurite outgrowth. Finally, we show that IGFBP2 significantly reduces neuronal loss and remarkably improves the functional recovery in a mouse model of SCI in vivo. Together, this study not only provides a comprehensive molecular atlas of SCI but also exemplifies how this rich resource can be applied to endow cells and genes with functional insight and therapeutic potential.

Unveiling inflammatory and prehypertrophic cell populations as key contributors to knee cartilage degeneration in osteoarthritis using multi-omics data integration.

OBJECTIVES: Single-cell and spatial transcriptomics analysis of human knee articular cartilage tissue to present a comprehensive transcriptome landscape and osteoarthritis (OA)-critical cell populations. METHODS: Single-cell RNA sequencing and spatially resolved transcriptomic technology have been applied to characterise the cellular heterogeneity of human knee articular cartilage which were collected from 8 OA donors, and 3 non-OA control donors, and a total of 19 samples. The novel chondrocyte population and marker genes of interest were validated by immunohistochemistry staining, quantitative real-time PCR, etc. The OA-critical cell populations were validated through integrative analyses of publicly available bulk RNA sequencing data and large-scale genome-wide association studies. RESULTS: We identified 33 cell population-specific marker genes that define 11 chondrocyte populations, including 9 known populations and 2 new populations, that is, pre-inflammatory chondrocyte population (preInfC) and inflammatory chondrocyte population (InfC). The novel findings that make this an important addition to the literature include: (1) the novel InfC activates the mediator MIF-CD74; (2) the prehypertrophic chondrocyte (preHTC) and hypertrophic chondrocyte (HTC) are potentially OA-critical cell populations; (3) most OA-associated differentially expressed genes reside in the articular surface and superficial zone; (4) the prefibrocartilage chondrocyte (preFC) population is a major contributor to the stratification of patients with OA, resulting in both an inflammatory-related subtype and a non-inflammatory-related subtype. CONCLUSIONS: Our results highlight InfC, preHTC, preFC and HTC as potential cell populations to target for therapy. Also, we conclude that profiling of those cell populations in patients might be used to stratify patient populations for defining cohorts for clinical trials and precision medicine.

Time space and single-cell resolved tissue lineage trajectories and laterality of body plan at gastrulation.

Understanding of the molecular drivers of lineage diversification and tissue patterning during primary germ layer development requires in-depth knowledge of the dynamic molecular trajectories of cell lineages across a series of developmental stages of gastrulation. Through computational modeling, we constructed at single-cell resolution, a spatio-temporal transcriptome of cell populations in the germ-layers of gastrula-stage mouse embryos. This molecular atlas enables the inference of molecular network activity underpinning the specification and differentiation of the germ-layer tissue lineages. Heterogeneity analysis of cellular composition at defined positions in the epiblast revealed progressive diversification of cell types. The single-cell transcriptome revealed an enhanced BMP signaling activity in the right-side mesoderm of late-gastrulation embryo. Perturbation of asymmetric BMP signaling activity at late gastrulation led to randomization of left-right molecular asymmetry in the lateral mesoderm of early-somite-stage embryo. These findings indicate the asymmetric BMP activity during gastrulation may be critical for the symmetry breaking process.

Three-dimensional molecular architecture of mouse organogenesis.

Mammalian embryos exhibit sophisticated cellular patterning that is intricately orchestrated at both molecular and cellular level. It has recently become apparent that cells within the animal body display significant heterogeneity, both in terms of their cellular properties and spatial distributions. However, current spatial transcriptomic profiling either lacks three-dimensional representation or is limited in its ability to capture the complexity of embryonic tissues and organs. Here, we present a spatial transcriptomic atlas of all major organs at embryonic day 13.5 in the mouse embryo, and provide a three-dimensional rendering of molecular regulation for embryonic patterning with stacked sections. By integrating the spatial atlas with corresponding single-cell transcriptomic data, we offer a detailed molecular annotation of the dynamic nature of organ development, spatial cellular interactions, embryonic axes, and divergence of cell fates that underlie mammalian development, which would pave the way for precise organ engineering and stem cell-based regenerative medicine.

Spatial Reconstruction of Oligo and Single Cells by De Novo Coalescent Embedding of Transcriptomic Networks.

Single cell RNA-seq (scRNA-seq) profiles conceal temporal and spatial tissue developmental information. De novo reconstruction of single cell temporal trajectory has been fairly addressed, but reverse engineering single cell 3D spatial tissue organization is hitherto landmark based, and de novo spatial reconstruction is a compelling computational open problem. Here it is shown that a proposed algorithm for de novo coalescent embedding (D-CE) of oligo/single cell transcriptomic networks can help to address this problem. Relying on the spatial information encoded in the expression patterns of genes, it is found that D-CE of cell-cell association transcriptomic networks, by preserving mesoscale network organization, captures spatial domains, identifies spatially expressed genes, reconstructs cell samples' 3D spatial distribution, and uncovers spatial domains and markers necessary for understanding the design principles on spatial organization and pattern formation. Comparison to the novoSpaRC and CSOmap (the only available de novo 3D spatial reconstruction methods) on 14 datasets and 497 reconstructions, reveals a significantly superior performance of D-CE.

Simultaneous profiling of spatial gene expression and chromatin accessibility during mouse brain development.

The brain is a complex tissue whose function relies on coordinated anatomical and molecular features. However, the molecular annotation of the spatial organization of the brain is currently insufficient. Here, we describe microfluidic indexing-based spatial assay for transposase-accessible chromatin and RNA-sequencing (MISAR-seq), a method for spatially resolved joint profiling of chromatin accessibility and gene expression. By applying MISAR-seq to the developing mouse brain, we study tissue organization and spatiotemporal regulatory logics during mouse brain development.

Lung development and regeneration: newly defined cell types and progenitor status.

The lung is the most critical organ of the respiratory system supporting gas exchange. Constant interaction with the external environment makes the lung vulnerable to injury. Thus, a deeper understanding of cellular and molecular processes underlying lung development programs and evaluation of progenitor status within the lung is an essential part of lung regenerative medicine. In this review, we aim to discuss the current understanding of lung development process and regenerative capability. We highlight the advances brought by multi-omics approaches, single-cell transcriptome, in particular, that can help us further dissect the cellular player and molecular signaling underlying those processes.

Generation of functional posterior spinal motor neurons from hPSCs-derived human spinal cord neural progenitor cells.

Spinal motor neurons deficiency results in a series of devastating disorders such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA) and spinal cord injury (SCI). These disorders are currently incurable, while human pluripotent stem cells (hPSCs)-derived spinal motor neurons are promising but suffered from inappropriate regional identity and functional immaturity for the study and treatment of posterior spinal cord related injuries. In this study, we have established human spinal cord neural progenitor cells (hSCNPCs) via hPSCs differentiated neuromesodermal progenitors (NMPs) and demonstrated the hSCNPCs can be continuously expanded up to 40 passages. hSCNPCs can be rapidly differentiated into posterior spinal motor neurons with high efficiency. The functional maturity has been examined in detail. Moreover, a co-culture scheme which is compatible for both neural and muscular differentiation is developed to mimic the neuromuscular junction (NMJ) formation in vitro. Together, these studies highlight the potential avenues for generating clinically relevant spinal motor neurons and modeling neuromuscular diseases through our defined hSCNPCs.

Distinct reward processing by subregions of the nucleus accumbens.

The nucleus accumbens (NAc) plays an important role in motivation and reward processing. Recent studies suggest that different NAc subnuclei differentially contribute to reward-related behaviors. However, how reward is encoded in individual NAc neurons remains unclear. Using in vivo single-cell resolution calcium imaging, we find diverse patterns of reward encoding in the medial and lateral shell subdivision of the NAc (NAcMed and NAcLat, respectively). Reward consumption increases NAcLat activity but decreases NAcMed activity, albeit with high variability among neurons. The heterogeneity in reward encoding could be attributed to differences in their synaptic inputs and transcriptional profiles. Specific optogenetic activation of Nts-positive neurons in the NAcLat promotes positive reinforcement, while activation of Cartpt-positive neurons in the NAcMed induces behavior aversion. Collectively, our study shows the organizational and transcriptional differences in NAc subregions and provides a framework for future dissection of NAc subregions in physiological and pathological conditions.

Integration of Computational Analysis and Spatial Transcriptomics in Single-cell Studies.

Recent advances of single-cell transcriptomics technologies and allied computational methodologies have revolutionized molecular cell biology. Meanwhile, pioneering explorations in spatial transcriptomics have opened up avenues to address fundamental biological questions in health and diseases. Here, we review the technical attributes of single-cell RNA sequencing and spatial transcriptomics, and the core concepts of computational data analysis. We further highlight the challenges in the application of data integration methodologies and the interpretation of the biological context of the findings.

Single-cell technologies: From research to application.

In recent years, more and more single-cell technologies have been developed. A vast amount of single-cell omics data has been generated by large projects, such as the Human Cell Atlas, the Mouse Cell Atlas, the Mouse RNA Atlas, the Mouse ATAC Atlas, and the Plant Cell Atlas. Based on these single-cell big data, thousands of bioinformatics algorithms for quality control, clustering, cell-type annotation, developmental inference, cell-cell transition, cell-cell interaction, and spatial analysis are developed. With powerful experimental single-cell technology and state-of-the-art big data analysis methods based on artificial intelligence, the molecular landscape at the single-cell level can be revealed. With spatial transcriptomics and single-cell multi-omics, even the spatial dynamic multi-level regulatory mechanisms can be deciphered. Such single-cell technologies have many successful applications in oncology, assisted reproduction, embryonic development, and plant breeding. We not only review the experimental and bioinformatics methods for single-cell research, but also discuss their applications in various fields and forecast the future directions for single-cell technologies. We believe that spatial transcriptomics and single-cell multi-omics will become the next booming business for mechanism research and commercial industry.

Activation of Wnt/ß-catenin signaling by Zeb1 in endothelial progenitors induces vascular quiescence entry.

The establishment of a functional vasculature requires endothelial cells to enter quiescence during the completion of development, otherwise pathological overgrowth occurs. How such a transition is regulated remains unclear. Here, we uncover a role of Zeb1 in defining vascular quiescence entry. During quiescence acquisition, Zeb1 increases along with the progressive decline of endothelial progenitors' activities, with Zeb1 loss resulting in endothelial overgrowth and vascular deformities. RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin sequencing (ATAC-seq) analyses reveal that Zeb1 represses Wif1, thereby activating Wnt/ß-catenin signaling. Knockdown of Wif1 rescues the overgrowth induced by Zeb1 deletion. Importantly, local administration of surrogate Wnt molecules in the retina ameliorates the overgrowth defects of Zeb1 mutants. These findings show a mechanism by which Zeb1 induces quiescence of endothelial progenitors during the establishing of vascular homeostasis, providing molecular insight into the inherited neovascular pathologies associated with human ZEB1 mutations, suggesting pharmacological activation of Wnt/ß-catenin signaling as a potential therapeutical approach.

Spatial molecular anatomy of germ layers in the gastrulating cynomolgus monkey embryo.

During mammalian embryogenesis, spatial regulation of gene expression and cell signaling are functionally coupled with lineage specification, patterning of tissue progenitors, and germ layer morphogenesis. While the mouse model has been instrumental for understanding mammalian development, comparatively little is known about human and non-human primate gastrulation due to the restriction of both technical and ethical issues. Here, we present a spatial and temporal survey of the molecular dynamics of cell types populating the non-human primate embryos during gastrulation. We reconstructed three-dimensional digital models from serial sections of cynomolgus monkey (Macaca fascicularis) gastrulating embryos at 1-day temporal resolution from E17 to E21. Spatial transcriptomics identifies gene expression profiles unique to the germ layers. Cross-species comparison reveals a developmental coordinate of germ layer segregation between mouse and primates, and species-specific transcription programs during gastrulation. These findings offer insights into evolutionarily conserved and divergent processes during mammalian gastrulation.

Embryonic vascular establishment requires protein C receptor-expressing endothelial progenitors.

Vascular establishment is one of the early events in embryogenesis. It is believed that vessel-initiating endothelial progenitors cluster to form the first primitive vessel. Understanding the molecular identity of these progenitors is crucial in order to elucidate lineage hierarchy. In this study, we identify protein C receptor (Procr) as an endothelial progenitor marker and investigate the role of Procr+ progenitors during embryonic vascular development. Using a ProcrmGFP-2A-lacZ reporter, we reveal a much earlier Procr expression (embryonic day 7.5) than previously acknowledged (embryonic day 13.5). Genetic fate-mapping experiments using ProcrCre and ProcrCreER demonstrate that Procr+ cells give rise to blood vessels throughout the entire embryo proper. Single-cell RNA-sequencing analyses place Procr+ cells at the start of endothelial commitment and maturation. Furthermore, targeted ablation of Procr+ cells results in failure of vessel formation and early embryonic lethality. Notably, genetic fate mapping and scRNA-seq pseudotime analysis support the view that Procr+ progenitors can give rise to hemogenic endothelium. In this study, we establish a Procr expression timeline and identify Procr+ vessel-initiating progenitors, and demonstrate their indispensable role in establishment of the vasculature during embryo development.

Connecting past and present: single-cell lineage tracing.

Central to the core principle of cell theory, depicting cells' history, state and fate is a fundamental goal in modern biology. By leveraging clonal analysis and single-cell RNA-seq technologies, single-cell lineage tracing provides new opportunities to interrogate both cell states and lineage histories. During the past few years, many strategies to achieve lineage tracing at single-cell resolution have been developed, and three of them (integration barcodes, polylox barcodes, and CRISPR barcodes) are noteworthy as they are amenable in experimentally tractable systems. Although the above strategies have been demonstrated in animal development and stem cell research, much care and effort are still required to implement these methods. Here we review the development of single-cell lineage tracing, major characteristics of the cell barcoding strategies, applications, as well as technical considerations and limitations, providing a guide to choose or improve the single-cell barcoding lineage tracing.

Spatial transcriptomics: new dimension of understanding biological complexity.

Cells and tissues are exquisitely organized in a complex but ordered manner to form organs and bodies so that individuals can function properly. The spatial organization and tissue architecture represent a keynote property underneath all living organisms. Molecular architecture and cellular composition within intact tissues play a vital role in a variety of biological processes, such as forming the complicated tissue functionality, precise regulation of cell transition in all living activities, consolidation of central nervous system, cellular responses to immunological and pathological cues. To explore these biological events at a large scale and fine resolution, a genome-wide understanding of spatial cellular changes is essential. However, previous bulk RNA sequencing and single-cell RNA sequencing technologies could not obtain the important spatial information of tissues and cells, despite their ability to detect high content transcriptional changes. These limitations have prompted the development of numerous spatially resolved technologies which provide a new dimension to interrogate the regional gene expression, cellular microenvironment, anatomical heterogeneity and cell-cell interactions. Since the advent of spatial transcriptomics, related works that use these technologies have increased rapidly, and new methods with higher throughput and resolution have grown quickly, all of which hold great promise to accelerate new discoveries in understanding the biological complexity. In this review, we briefly discussed the historical evolution of spatially resolved transcriptome. We broadly surveyed the representative methods. Furthermore, we summarized the general computational analysis pipeline for the spatial gene expression data. Finally, we proposed perspectives for technological development of spatial multi-omics.

Base editing-mediated perturbation of endogenous PKM1/2 splicing facilitates isoform-specific functional analysis in vitro and in vivo.

OBJECTIVES: PKM1 and PKM2, which are generated from the alternative splicing of PKM gene, play important roles in tumourigenesis and embryonic development as rate-limiting enzymes in glycolytic pathway. However, because of the lack of appropriate techniques, the specific functions of the 2 PKM splicing isoforms have not been clarified endogenously yet. MATERIALS AND METHODS: In this study, we used CRISPR-based base editors to perturbate the endogenous alternative splicing of PKM by introducing mutations into the splicing junction sites in HCT116 cells and zebrafish embryos. Sanger sequencing, agarose gel electrophoresis and targeted deep sequencing assays were utilized for identifying mutation efficiencies and detecting PKM1/2 splicing isoforms. Cell proliferation assays and RNA-seq analysis were performed to describe the effects of perturbation of PKM1/2 splicing in tumour cell growth and zebrafish embryo development. RESULTS: The splicing sites of PKM, a 5' donor site of GT and a 3' acceptor site of AG, were efficiently mutated by cytosine base editor (CBE; BE4max) and adenine base editor (ABE; ABEmax-NG) with guide RNAs (gRNAs) targeting the splicing sites flanking exons 9 and 10 in HCT116 cells and/or zebrafish embryos. The mutations of the 5' donor sites of GT flanking exons 9 or 10 into GC resulted in specific loss of PKM1 or PKM2 expression as well as the increase in PKM2 or PKM1 respectively. Specific loss of PKM1 promoted cell proliferation of HCT116 cells and upregulated the expression of cell cycle regulators related to DNA replication and cell cycle phase transition. In contrast, specific loss of PKM2 suppressed cell growth of HCT116 cells and resulted in growth retardation of zebrafish. Meanwhile, we found that mutation of PKM1/2 splicing sites also perturbated the expression of non-canonical PKM isoforms and produced some novel splicing isoforms. CONCLUSIONS: This work proved that CRISPR-based base editing strategy can be used to disrupt the endogenous alternative splicing of genes of interest to study the function of specific splicing isoforms in vitro and in vivo. It also reminded us to notice some novel or undesirable splicing isoforms by targeting the splicing junction sites using base editors. In sum, we establish a platform to perturbate endogenous RNA splicing for functional investigation or genetic correction of abnormal splicing events in human diseases.

RNA helicase DDX5 acts as a critical regulator for survival of neonatal mouse gonocytes.

OBJECTIVES: Mammalian spermatogenesis is a biological process of male gamete formation. Gonocytes are the only precursors of spermatogonial stem cells (SSCs) which develop into mature spermatozoa. DDX5 is one of DEAD-box RNA helicases and expresses in male germ cells, suggesting that Ddx5 plays important functions during spermatogenesis. Here, we explore the functions of Ddx5 in regulating the specification of gonocytes. MATERIALS AND METHODS: Germ cell-specific Ddx5 knockout (Ddx5-/- ) mice were generated. The morphology of testes and epididymides and fertility in both wild-type and Ddx5-/- mice were analysed. Single-cell RNA sequencing (scRNA-seq) was used to profile the transcriptome in testes from wild-type and Ddx5-/- mice at postnatal day (P) 2. Dysregulated genes were validated by single-cell qRT-PCR and immunofluorescent staining. RESULTS: In male mice, Ddx5 was expressed in germ cells at different stages of development. Germ cell-specific Ddx5 knockout adult male mice were sterile due to completely devoid of germ cells. Male germ cells gradually disappeared in Ddx5-/- mice from E18.5 to P6. Single-cell transcriptome analysis showed that genes involved in cell cycle and glial cell line-derived neurotrophic factor (GDNF) pathway were significantly decreased in Ddx5-deficient gonocytes. Notably, Ddx5 ablation impeded the proliferation of gonocytes. CONCLUSIONS: Our study reveals the critical roles of Ddx5 in fate determination of gonocytes, offering a novel insight into the pathogenesis of male sterility.