Illuminating tissue organization by imaging the spatial transcriptome

Our bodies consist of a large collection of cells that each have their own function in the organ that they reside in. The cells are grouped by functionality in cell types that arise during development as the result of the gene regulatory network encoded in the genome. With the development of novel single cell technologies, we are starting to understand just how diverse our cells are. In the brain for instance there are at least 3,000 distinguishable types. However, we have little understanding of how all these cell types are spatially organized in the tissue, because conventional labeling and microscopy techniques are incapable of resolving such high complexity in a single experiment. In this thesis I present the development of two methods that can resolve the cellular complexity and spatial organization of mouse and (developmental) human brain samples. These methods are built upon the concept of cyclic RNA labeling with single molecule Fluorescent in situ Hybridization (smFISH) to detect hundreds of gene targets in tissue samples. The resulting RNA localizations can then be used to study spatial gene expression and to identify the cell type of each cell in the sample. The cellular identity and position can then be used to study spatial relationships between cells to understand the tissue architecture. To place the development of these two methods into context, I will first review the field of spatially resolved transcriptomics. I will discuss the methods that are based on microscopy and spatially tagged RNA sequencing, where I will compare their strengths and weaknesses. Then I will present the two projects: Paper I presents the development of a cyclic smFISH protocol called osmFISH that leverages the high detection efficiency of smFISH to measure the gene expression of 33 cell type marker genes in the mouse somatosensory cortex at single cell resolution. We developed the labeling technology, instrumentation and analysis software to enable the study of cellular organization at multiple length scales. Even though osmFISH and related microscopy-based methods generate high quality data they are limited by the spatial throughput so that only small tissue areas can be processed. In paper II I present another method called EEL FISH that uses electrophoresis to transfer the RNA from a 3D tissue section onto a flat surface. The collapsing of one dimension substantially reduces the time needed to image, while retaining the information, so that the complex spatial gene expression profiles of entire mouse brain sections, sub-structures of the human brain and human developmental tissues can be studied. Lastly, I will discuss these results and look at the future of the field of spatially resolved transcriptomics

Molecular Diversity of Midbrain Development in Mouse, Human, and Stem Cells.

Understanding human embryonic ventral midbrain is of major interest for Parkinson's disease. However, the cell types, their gene expression dynamics, and their relationship to commonly used rodent models remain to be defined. We performed single-cell RNA sequencing to examine ventral midbrain development in human and mouse. We found 25 molecularly defined human cell types, including five subtypes of radial glia-like cells and four progenitors. In the mouse, two mature fetal dopaminergic neuron subtypes diversified into five adult classes during postnatal development. Cell types and gene expression were generally conserved across species, but with clear differences in cell proliferation, developmental timing, and dopaminergic neuron development. Additionally, we developed a method to quantitatively assess the fidelity of dopaminergic neurons derived from human pluripotent stem cells, at a single-cell level. Thus, our study provides insight into the molecular programs controlling human midbrain development and provides a foundation for the development of cell replacement therapies.All authors were supported by EU FP7 grant DDPDGENES. S.L. was supported by European Research Council grant 261063 (BRAINCELL), Knut and Alice Wallenberg Foundation grant 2015.0041, Swedish Research Council (STARGET), and the Swedish Foundation for Strategic Research (RIF14-0057). A.Z. was supported by the Human Frontier Science Program. E.A. was supported by Swedish Research Council (VR projects: 2011-3116 and 2011-3318), Swedish Foundation for Strategic Research (SRL program), and Karolinska Institutet (SFO Thematic Center in Stem cells and Regenerative Medicine). E.A. and R.A.B. were supported by the EU FP7 grant NeuroStemcellRepair. R.A.B. was also supported by an NIHR Biomedical Research Centre award to the University of Cambridge/Addenbrookes Hospital. iCell dopaminergic neurons were a generous gift from Cellular Dynamics International. Single-cell RNA-seq servic0es were provided by the Eukaryotic Single-cell Genomics facility and the National Genomics Infrastructure at Science for Life Laboratory.This is the final version of the article. It first appeared from Elsevier via https://doi.org/10.1016/j.cell.2016.09.02

Genesis and spread of multiple reassortants during the 2016/2017 H5 avian influenza epidemic in Eurasia

Highly pathogenic avian influenza (HPAI) viruses of the H5 A/goose/Guangdong/1/96 lineage can cause severe disease in poultry and wild birds, and occasionally in humans. In recent years, H5 HPAI viruses of this lineage infecting poultry in Asia have spilled over into wild birds and spread via bird migration to countries in Europe, Africa, and North America. In 2016/2017, this spillover resulted in the largest HPAI epidemic on record in Europe and was associated with an unusually high frequency of reassortments between H5 HPAI viruses and cocirculating low-pathogenic avian influenza viruses. Here, we show that the seven main H5 reassortant viruses had various combinations of gene segments 1, 2, 3, 5, and 6. Using detailed time-resolved phylogenetic analysis, most of these gene segments likely originated from wild birds and at dates and locations that corresponded to their hosts' migratory cycles. However, some gene segments in two reassortant viruses likely originated from domestic anseriforms, either in spring 2016 in east China or in autumn 2016 in central Europe. Our results demonstrate that, in addition to domestic anseriforms in Asia, both migratory wild birds and domestic anseriforms in Europe are relevant sources of gene segments for recent reassortant H5 HPAI viruses. The ease with which these H5 HPAI viruses reassort, in combination with repeated spillovers of H5 HPAI viruses into wild birds, increases the risk of emergence of a reassortant virus that persists in wild bird populations yet remains highly pathogenic for poultry

The anticancer activity of lytic peptides is inhibited by heparan sulfate on the surface of the tumor cells

<p>Abstract</p> <p>Background</p> <p>Cationic antimicrobial peptides (CAPs) with antitumor activity constitute a promising group of novel anticancer agents. These peptides induce lysis of cancer cells through interactions with the plasma membrane. It is not known which cancer cell membrane components influence their susceptibility to CAPs. We have previously shown that CAPs interact with the two glycosaminoglycans (GAGs), heparan sulfate (HS) and chondroitin sulfate (CS), which are present on the surface of most cells. The purpose of this study was to investigate the role of the two GAGs in the cytotoxic activity of CAPs.</p> <p>Methods</p> <p>Various cell lines, expressing different levels of cell surface GAGs, were exposed to bovine lactoferricin (LfcinB) and the designer peptide, KW5. The cytotoxic effect of the peptides was investigated by use of the colorimetric MTT viability assay. The cytotoxic effect on wild type CHO cells, expressing normal amounts of GAGs on the cell surface, and the mutant pgsA-745, that has no expression of GAGs on the cell surface, was also investigated.</p> <p>Results</p> <p>We show that cells not expressing HS were more susceptible to CAPs than cells expressing HS at the cell surface. Further, exogenously added heparin inhibited the cytotoxic effect of the peptides. Chondroitin sulfate had no effect on the cytotoxic activity of KW5 and only minor effects on LfcinB cytotoxicity.</p> <p>Conclusion</p> <p>Our results show for the first time that negatively charged molecules at the surface of cancer cells inhibit the cytotoxic activity of CAPs. Our results indicate that HS at the surface of cancer cells sequesters CAPs away from the phospholipid bilayer and thereby impede their ability to induce cytolysis.</p

Spatial organization of the somatosensory cortex revealed by osmFISH

Global efforts to create a molecular census of the brain using single-cell transcriptomics are producing a large catalog of molecularly defined cell types. However, spatial information is lacking and new methods are needed to map a large number of cell type–specific markers simultaneously on large tissue areas. Here, we describe a cyclic single-molecule fluorescence in situ hybridization methodology and define the cellular organization of the somatosensory cortex

Cell segmentation-free inference of cell types from in situ transcriptomics data

Inaccurate cell segmentation has been the major problem for cell-type identification and tissue characterization of the in situ spatially resolved transcriptomics data. Here we show a robust cell segmentation-free computational framework (SSAM), for identifying cell types and tissue domains in 2D and 3D

Spatial tissue profiling by imaging-free molecular tomography

Several techniques are currently being developed for spatially resolved omics profiling, but each new method requires the setup of specific detection strategies or specialized instrumentation. Here we describe an imaging-free framework to localize high-throughput readouts within a tissue by cutting the sample into thin strips in a way that allows subsequent image reconstruction. We implemented this framework to transform a low-input RNA sequencing protocol into an imaging-free spatial transcriptomics technique (called STRP-seq) and validated it by profiling the spatial transcriptome of the mouse brain. We applied the technique to the brain of the Australian bearded dragon, Pogona vitticeps. Our results reveal the molecular anatomy of the telencephalon of this lizard, providing evidence for a marked regionalization of the reptilian pallium and subpallium. We expect that STRP-seq can be used to derive spatially resolved data from a range of other omics techniques. A refined spatial sampling technique transforms standard RNA sequencing into a spatial transcriptomics method

RNA velocity of single cells

RNA abundance is a powerful indicator of the state of individual cells. Single-cell RNA sequencing can reveal RNA abundance with high quantitative accuracy, sensitivity and throughput 1 . However, this approach captures only a static snapshot at a point in time, posing a challenge for the analysis of time-resolved phenomena such as embryogenesis or tissue regeneration. Here we show that RNA velocity—the time derivative of the gene expression state—can be directly estimated by distinguishing between unspliced and spliced mRNAs in common single-cell RNA sequencing protocols. RNA velocity is a high-dimensional vector that predicts the future state of individual cells on a timescale of hours. We validate its accuracy in the neural crest lineage, demonstrate its use on multiple published datasets and technical platforms, reveal the branching lineage tree of the developing mouse hippocampus, and examine the kinetics of transcription in human embryonic brain. We expect RNA velocity to greatly aid the analysis of developmental lineages and cellular dynamics, particularly in humans

Has Epizootic Become Enzootic? Evidence for a Fundamental Change in the Infection Dynamics of Highly Pathogenic Avian Influenza in Europe, 2021

Phylogenetic evidence from the recent resurgence of high-pathogenicity avian influenza (HPAI) virus subtype H5N1, clade 2.3.4.4b, observed in European wild birds and poultry since October 2021, suggests at least two different and distinct reservoirs. We propose contrasting hypotheses for this emergence: (i) resident viruses have been maintained, presumably in wild birds, in northern Europe throughout the summer of 2021 to cause some of the outbreaks that are part of the most recent autumn/winter 2021 epizootic, or (ii) further virus variants were reintroduced by migratory birds, and these two sources of reintroduction have driven the HPAI resurgence. Viruses from these two principal sources can be distinguished by their hemagglutinin genes, which segregate into two distinct sublineages (termed B1 and B2) within clade 2.3.4.4b, as well as their different internal gene compositions. The evidence of enzootic HPAI virus circulation during the summer of 2021 indicates a possible paradigm shift in the epidemiology of HPAI in Europe