| 1. |
- Gyllborg, Daniel, et al.
(författare)
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Hybridization-based in situ sequencing (HybISS) for spatially resolved transcriptomics in human and mouse brain tissue
- 2020
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 48:19
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Tidskriftsartikel (refereegranskat)abstract
- Visualization of the transcriptome in situ has proven to be a valuable tool in exploring single-cell RNA-sequencing data, providing an additional spatial dimension to investigate multiplexed gene expression, cell types, disease architecture or even data driven discoveries. In situ sequencing (ISS) method based on padlock probes and rolling circle amplification has been used to spatially resolve gene transcripts in tissue sections of various origins. Here, we describe the next iteration of ISS, HybISS, hybridization-based in situ sequencing. Modifications in probe design allows for a new barcoding system via sequence-by-hybridization chemistry for improved spatial detection of RNA transcripts. Due to the amplification of probes, amplicons can be visualized with standard epifluorescence microscopes for high-throughput efficiency and the new sequencing chemistry removes limitations bound by sequence-by-ligation chemistry of ISS. HybISS design allows for increased flexibility and multiplexing, increased signal-to-noise, all without compromising throughput efficiency of imaging large fields of view. Moreover, the current protocol is demonstrated to work on human brain tissue samples, a source that has proven to be difficult to work with image-based spatial analysis techniques. Overall, HybISS technology works as a targeted amplification detection method for improved spatial transcriptomic visualization, and importantly, with an ease of implementation.
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| 2. |
- Gyllborg, Daniel
(författare)
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Molecular and cellular characterization of midbrain dopaminergic neuron development
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Midbrain dopaminergic (mDA) development is a complex yet highly controlled mechanistic process that is conserved across species. The understanding of these molecular details can open windows to new avenues of therapeutic medicine. Parkinson’s disease (PD) is a debilitating neurological disorder that to date has no cure or established cause. With various aspects of mDA development being revealed, the aim for a permanent treatment of PD itself is getting closer. Included within this thesis are four papers and two manuscripts covering diverse points of mDA neuron development and PD. In Paper I we explore the role of transcription factor Pbx1 to promote mDA differentiation through activation of Pitx3 and repression of Onecut2. Pbx1 is also involved in protection from oxidative stress through Nfe2l1, an important aspect of PD. In Paper II we explore the cellular diversity of the ventral midbrain through the use of single-cell RNA-sequencing. The cellular transcriptional profiles aid in revealing the mDA neuron lineage and a cross-species comparison of mouse and human. To conclude, we use molecular tools to evaluate stem-cell derived mDA preparations for cell replacement therapy (CRT) in PD. In Paper III we review the current knowledge of Wnt signaling related to mDA development and further investigate the human single-cell data set from Paper II for other possible Wnt components that have yet to be explored for their role in development. In Paper IV we explore the composition of the mDA cellular environment using RNA-sequencing data. Here we apply a novel approach to gain insight to specific contributions from various cell types to the extracellular matrix, its modulators, and signaling ligands. We find a transcription factor network centered around Arntl1 in radial glia type 1 cells, a putative progenitor to the neuronal lineage. In Paper V we investigate the matricellular protein R-spondin 2. As a Wnt signaling activator, we show R-spondin 2 has a role in mDA differentiation when applied to embryonic stem cell differentiation protocols. This has direct translational impact in CRT for PD. In Paper VI we explore the role of Wnt/planar cell polarity signaling in midbrain development. Specifically, we elucidate the roles of Ror2 and Vangl2 in mDA development and their participation in morphogenesis and neurogenesis. In conclusion, this thesis encompasses research on midbrain development from molecular details at a single-cell level to cellular components affecting global developmental processes. Here I present findings to be included towards a greater understanding of midbrain development and novel ideas relevant to translational research in CRT for PD.
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| 3. |
- hower, lee, et al.
(författare)
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investigating cell type changes in schizophrenia with spatially-resolved transcriptomics
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Annan publikation (populärvet., debatt m.m.)abstract
- Schizophrenia is a heritable and genetically complex disorder with poorly understood aetiology. Previous study from our lab has shown that this disorder could affect a limited number of cell types in the brain1. Here, we build on these findings and present the first spatially-resolved transcriptomics study of schizophrenia with a large number of samples (17 in total). By comparing spatial cell type distributions in diseased and healthy prefrontal cortices, we discover that certain non-neuronal cell types change their co-localization patterns
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| 4. |
- Kaiser, Karol, et al.
(författare)
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MEIS-WNT5A axis regulates development of fourth ventricle choroid plexus
- 2021
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Ingår i: Development. - : The Company of Biologists. - 0950-1991 .- 1477-9129. ; 148:10
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Tidskriftsartikel (refereegranskat)abstract
- The choroid plexus (ChP) produces cerebrospinal fluid and forms an essential brain barrier. ChP tissues form in each brain ventricle, each one adopting a distinct shape, but remarkably little is known about the mechanisms underlying ChP development. Here, we show that epithelial WNT5A is crucial for determining fourth ventricle (4V) ChP morphogenesis and size in mouse. Systemic Wnt5a knockout, or forced Wnt5a overexpression beginning at embryonic day 10.5, profoundly reduced ChP size and development. However, Wnt5a expression was enriched in Foxj1-positive epithelial cells of 4V ChP plexus, and its conditional deletion in these cells affected the branched, villous morphology of the 4V ChP. We found that WNT5A was enriched in epithelial cells localized to the distal tips of 4V ChP villi, where WNT5A acted locally to activate non-canonical WNT signaling via ROR1 and ROR2 receptors. During 4V ChP development, MEIS1 bound to the proximal Wnt5a promoter, and gain- and loss-of-function approaches demonstrated that MEIS1 regulated Wnt5a expression. Collectively, our findings demonstrate a dual function of WNT5A in ChP development and identify MEIS transcription factors as upstream regulators of Wnt5a in the 4V ChP epithelium.
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| 5. |
- Kaucka, Marketa, et al.
(författare)
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Analysis of neural crest-derived clones reveals novel aspects of facial development
- 2016
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Ingår i: Science Advances. - : American Association for the Advancement of Science. - 2375-2548. ; 2:8
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Tidskriftsartikel (refereegranskat)abstract
- Cranial neural crest cells populate the future facial region and produce ectomesenchyme-derived tissues, such as cartilage, bone, dermis, smooth muscle, adipocytes, and many others. However, the contribution of individual neural crest cells to certain facial locations and the general spatial clonal organization of the ectomesenchyme have not been determined. We investigated how neural crest cells give rise to clonally organized ectomesenchyme and how this early ectomesenchyme behaves during the developmental processes that shape the face. Using a combination of mouse and zebrafish models, we analyzed individual migration, cell crowd movement, oriented cell division, clonal spatial overlapping, and multilineage differentiation. The early face appears to be built from multiple spatially defined overlapping ectomesenchymal clones. During early face development, these clones remain oligopotent and generate various tissues in a given location. By combining clonal analysis, computer simulations, mouse mutants, and live imaging, we show that facial shaping results from an array of local cellular activities in the ectomesenchyme. These activities mostly involve oriented divisions and crowd movements of cells during morphogenetic events. Cellular behavior that can be recognized as individual cell migration is very limited and short-ranged and likely results from cellular mixing due to the proliferation activity of the tissue. These cellular mechanisms resemble the strategy behind limb bud morphogenesis, suggesting the possibility of common principles and deep homology between facial and limb outgrowth.
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| 6. |
- Kaucka, Marketa, et al.
(författare)
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Oriented clonal cell dynamics enables accurate growth and shaping of vertebrate cartilage
- 2017
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Ingår i: eLIFE. - : Elife Sciences Publications LTD. - 2050-084X. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Cartilaginous structures are at the core of embryo growth and shaping before the bone forms. Here we report a novel principle of vertebrate cartilage growth that is based on introducing transversally-oriented clones into pre-existing cartilage. This mechanism of growth uncouples the lateral expansion of curved cartilaginous sheets from the control of cartilage thickness, a process which might be the evolutionary mechanism underlying adaptations of facial shape. In rod-shaped cartilage structures (Meckel, ribs and skeletal elements in developing limbs), the transverse integration of clonal columns determines the well-defined diameter and resulting rod-like morphology. We were able to alter cartilage shape by experimentally manipulating clonal geometries. Using in silico modeling, we discovered that anisotropic proliferation might explain cartilage bending and groove formation at the macro-scale.
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| 7. |
- La Manno, Gioele, et al.
(författare)
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Molecular architecture of the developing mouse brain
- 2021
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 596:7870, s. 92-96
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Tidskriftsartikel (refereegranskat)abstract
- The mammalian brain develops through a complex interplay of spatial cues generated by diffusible morphogens, cell-cell interactions and intrinsic genetic programs that result in probably more than a thousand distinct cell types. A complete understanding of this process requires a systematic characterization of cell states over the entire spatiotemporal range of brain development. The ability of single-cell RNA sequencing and spatial transcriptomics to reveal the molecular heterogeneity of complex tissues has therefore been particularly powerful in the nervous system. Previous studies have explored development in specific brain regions(1-8), the whole adult brain(9) and even entire embryos(10). Here we report a comprehensive single-cell transcriptomic atlas of the embryonic mouse brain between gastrulation and birth. We identified almost eight hundred cellular states that describe a developmental program for the functional elements of the brain and its enclosing membranes, including the early neuroepithelium, region-specific secondary organizers, and both neurogenic and gliogenic progenitors. We also used in situ mRNA sequencing to map the spatial expression patterns of key developmental genes. Integrating the in situ data with our single-cell clusters revealed the precise spatial organization of neural progenitors during the patterning of the nervous system. A comprehensive single-cell transcriptomic atlas of the mouse brain between gastrulation and birth identifies hundreds of cellular states and reveals the spatiotemporal organization of brain development.
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| 8. |
- Langseth, Christoffer Mattsson, et al.
(författare)
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Comprehensive in situ mapping of human cortical transcriptomic cell types
- 2021
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 4:1
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Tidskriftsartikel (refereegranskat)abstract
- The ability to spatially resolve the cellular architecture of human cortical cell types over informative areas is essential to understanding brain function. We combined in situ sequencing gene expression data and single-nucleus RNA-sequencing cell type definitions to spatially map cells in sections of the human cortex via probabilistic cell typing. We mapped and classified a total of 59,816 cells into all 75 previously defined subtypes to create a first spatial atlas of human cortical cells in their native position, their abundances and genetic signatures. We also examined the precise within- and across-layer distributions of all the cell types and provide a resource for the cell atlas community. The abundances and locations presented here could serve as a reference for further studies, that include human brain tissues and disease applications at the cell type level.
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| 9. |
- Lee, Hower, et al.
(författare)
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Direct RNA targeted in situ sequencing for transcriptomic profiling in tissue
- 2022
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Li, Xiaofei, et al.
(författare)
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Profiling spatiotemporal gene expression of the developing human spinal cord and implications for ependymoma origin
- 2023
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Ingår i: Nature Neuroscience. - : Springer Nature. - 1097-6256 .- 1546-1726. ; 26:5, s. 891-901
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Tidskriftsartikel (refereegranskat)abstract
- The authors created a comprehensive developmental cell atlas for spatiotemporal gene expression of the human spinal cord, revealed species-specific regulation during development and used the atlas to infer novel markers for pediatric ependymomas. 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.
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