RESUMO
Multiplexed spatial profiling of mRNAs has recently gained traction as a tool to explore the cellular diversity and the architecture of tissues. We propose a sensitive, open-source, simple and flexible method for the generation of in-situ expression maps of hundreds of genes. We exploit direct ligation of padlock probes on mRNAs, coupled with rolling circle amplification and hybridization-based in situ combinatorial barcoding, to achieve high detection efficiency, high throughput and large multiplexing. We validate the method across a number of species, and show its use in combination with orthogonal methods such as antibody staining, highlighting its potential value for developmental and tissue biology studies. Finally, we provide an end-to-end computational workflow that covers the steps of probe design, image processing, data extraction, cell segmentation, clustering and annotation of cell types. By enabling easier access to high-throughput spatially resolved transcriptomics, we hope to encourage a diversity of applications and the exploration of a wide range of biological questions.
RESUMO
We present a spatial omics approach that combines histology, mass spectrometry imaging and spatial transcriptomics to facilitate precise measurements of mRNA transcripts and low-molecular-weight metabolites across tissue regions. The workflow is compatible with commercially available Visium glass slides. We demonstrate the potential of our method using mouse and human brain samples in the context of dopamine and Parkinson's disease.
RESUMO
The spatiotemporal regulation of cell fate specification in the human developing spinal cord remains largely unknown. In this study, by performing integrated analysis of single-cell and spatial multi-omics data, we used 16 prenatal human samples to create a comprehensive developmental cell atlas of the spinal cord during post-conceptional weeks 5-12. This revealed how the cell fate commitment of neural progenitor cells and their spatial positioning are spatiotemporally regulated by specific gene sets. We identified unique events in human spinal cord development relative to rodents, including earlier quiescence of active neural stem cells, differential regulation of cell differentiation and distinct spatiotemporal genetic regulation of cell fate choices. In addition, by integrating our atlas with pediatric ependymomas data, we identified specific molecular signatures and lineage-specific genes of cancer stem cells during progression. Thus, we delineate spatiotemporal genetic regulation of human spinal cord development and leverage these data to gain disease insight.
Assuntos
Ependimoma , Células-Tronco Neurais , Criança , Feminino , Gravidez , Humanos , Medula Espinal , Ependimoma/genética , Ependimoma/metabolismo , Diferenciação Celular/genética , Células-Tronco Neurais/fisiologia , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genéticaRESUMO
Highly multiplexed spatial mapping of transcripts within tissues allows for investigation of the transcriptomic and cellular diversity of mammalian organs previously unseen. Here we explore a direct RNA (dRNA) detection approach incorporating the use of padlock probes and rolling circle amplification in combination with hybridization-based in situ sequencing chemistry. We benchmark a High Sensitivity Library Preparation Kit from CARTANA that circumvents the reverse transcription needed for cDNA-based in situ sequencing (ISS) via direct RNA detection. We found a fivefold increase in transcript detection efficiency when compared to cDNA-based ISS and also validated its multiplexing capability by targeting a curated panel of 50 genes from previous publications on mouse brain sections, leading to additional data interpretation such as de novo cell clustering. With this increased efficiency, we also found to maintain specificity, multiplexing capabilities and ease of implementation. Overall, the dRNA chemistry shows significant improvements in target detection efficiency, closing the gap to other fluorescent in situ hybridization-based technologies and opens up possibilities to explore new biological questions previously not possible with cDNA-based ISS.
Assuntos
RNA , Transcriptoma , Animais , DNA Complementar/genética , Hibridização in Situ Fluorescente , Mamíferos , Camundongos , Hibridização de Ácido Nucleico , Transcriptoma/genéticaRESUMO
Oligodendrogenesis in the human central nervous system has been observed mainly at the second trimester of gestation, a much later developmental stage compared to oligodendrogenesis in mice. Here, we characterize the transcriptomic neural diversity in the human forebrain at post-conception weeks (PCW) 8-10. Using single-cell RNA sequencing, we find evidence of the emergence of a first wave of oligodendrocyte lineage cells as early as PCW 8, which we also confirm at the epigenomic level through the use of single-cell ATAC-seq. Using regulatory network inference, we predict key transcriptional events leading to the specification of oligodendrocyte precursor cells (OPCs). Moreover, by profiling the spatial expression of 50 key genes through the use of in situ sequencing (ISS), we identify regions in the human ventral fetal forebrain where oligodendrogenesis first occurs. Our results indicate evolutionary conservation of the first wave of oligodendrogenesis between mice and humans and describe regulatory mechanisms involved in human OPC specification.