Biology subjects

Castelo-Branco, G.

Publications and source records attributed to Castelo-Branco, G..

31 records · Page 2Linked to original sources

bioRxiv2021

Developmental landscape of human forebrain at a single-cell level unveils early waves of oligodendrogenesis

Oligodendrogenesis in the human central nervous system has been mainly observed at the second trimester of gestation, a much later developmental stage compared to mouse. Here we characterize the transcriptomic neural diversity in the human forebrain at post conceptual weeks (PCW) 8 to 10, using single-cell RNA-Seq. 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 with 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 fifty key genes using 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 mouse and human and describe regulatory mechanisms required for human OPC specification.

neuroscience↗

bioRxiv2021

Spatial-ATAC-seq: spatially resolved chromatin accessibility profiling of tissues at genome scale and cellular level

Cellular function in tissue is dependent upon the local environment, requiring new methods for spatial mapping of biomolecules and cells in the tissue context. The emergence of spatial transcriptomics has enabled genome-scale gene expression mapping, but it remains elusive to capture spatial epigenetic information of tissue at cellular level and genome scale. Here we report on spatial-ATAC-seq: spatially resolved chromatin accessibility profiling of tissue section via next-generation sequencing by combining in situ Tn5 transposition chemistry and microfluidic deterministic barcoding. Spatial chromatin accessibility profiling of mouse embryos delineated tissue region-specific epigenetic landscapes and identified gene regulators implicated in the central nerve system development. Mapping the accessible genome in human tonsil tissue with 20m pixel size revealed spatially distinct organization of immune cell types and states in lymphoid follicles and extrafollicular zones. This technology takes spatial biology to a new realm by enabling spatially resolved epigenomics to improve our understanding of cell identity, state, and fate decision in relation to epigenetic underpinnings in development and disease.

genomics↗

bioRxiv2021

Spatial cell type mapping of the oligodendrocyte lineage in the mouse juvenile and adult CNS with in situ sequencing

Oligodendrocytes show transcriptional heterogeneity but the regional and functional implications of this heterogeneity are less clear. Here, we apply in situ sequencing (ISS) to simultaneously probe the expression of 124 marker genes of distinct oligodendrocyte populations, providing comprehensive maps of corpus callosum, cingulate, motor and somatosensory cortex in the brain, as well as gray (GM) and white matter (WM) regions in the spinal cord, at juvenile and adult stages. We systematically compare abundances of these populations and investigate the neighboring preference of distinct oligodendrocyte populations. As previously described, we observed that oligodendrocyte lineage progression is more advanced in the juvenile spinal cord compared to the brain. Additionally, myelination is ongoing in the adult corpus callosum while it is mostly completed in the cortex. Interestingly, we found a medial-to-lateral gradient of oligodendrocyte lineage progression in the juvenile cortex, which could be linked to arealization, as well as a deep-to-superficial gradient with mature oligodendrocytes preferentially accumulating in the deeper layers of the cortex. We observed differences in abundances and population dynamics over time between GM and WM regions in the brain and spinal cord, indicating regional differences within GM and WM. We also found that oligodendroglia populations neighboring preferences are altered from the juvenile to the adult CNS. Thus, our ISS dataset reveals spatial heterogeneity of the oligodendrocyte lineage progression in the brain and spinal cord, which could be relevant to further investigate functional heterogeneity of oligodendroglia.

neuroscience↗

bioRxiv2020

Single-cell profiling of histone modifications in the mouse brain

The development of the mouse central nervous system (CNS) involves coordinated execution of transcriptional and epigenetic programs. These programs have been extensively studied through single-cell technologies in a pursuit to characterize the underlying cell heterogeneity. However, histone modifications pose additional layers of both positive and negative regulation that defines cellular identity. Here we show that the Cut&Tag technology can be coupled with a droplet-based single cell library preparation platform to produce high quality chromatin modifications data at a single cell resolution in tens of thousands of cells. We apply single-cell Cut&Tag (scC&T) to probe histone modifications characteristic of active promoters (H3K4me3), active promoters and enhancers (H3K27ac), active gene bodies (H3K36me3) and inactive regions (H3K27me3) and generate scC&T profiles for almost 50,000 cells. scC&T profiles of each of these histone modifications were sufficient to determine cell identity and deconvolute at single cell level regulatory principles such as promoter bivalency, spreading of H3K4me3 and promoter-enhancer connectivity. Moreover, we used scC&T to investigate the single-cell chromatin occupancy of transcription factor Olig2 and the cohesin complex component Rad21. Our results indicate that analysis of histone modifications and transcription factor occupancy at a single cell resolution can provide unique insights of epigenomic landscapes in the CNS. We also provide an online resource that can be used to interactively explore the data at https://castelobranco.shinyapps.io/BrainCutAndTag2020/.

neuroscience↗

bioRxiv2020

A primed immune transcriptional program is activated in oligodendroglia in multiple sclerosis

Multiple sclerosis (MS) is a disease characterized by a targeted immune attack on myelin in the central nervous system (CNS). We have previously shown that oligodendrocytes (OLs), myelin producing cells in the CNS, and their precursors (OPCs), ac-quire disease-specific transcriptional states in MS 1,2. To under-stand how these alternative transcriptional programs are acti-vated in disease, we performed single-cell assay for transposase accessible chromatin using sequencing (scATAC-seq) on the OL lineage in the experimental autoimmune encephalomyeli-tis (EAE) mouse model of MS. We identified regulatory regions with increased accessibility in oligodendroglia (OLG) in EAE, some of which in the proximity of immune genes. A similar re-modeling of chromatin accessibility was observed upon treat-ment of postnatal OPCs with interferon-gamma (IFN{gamma}), but not with dexamethasone. These changes in accessibility were not exclusive to distal enhancers, but also occurred at promoter re-gions, suggesting a role for promoters in mediating cell-state transitions. Notably, we found that a subset of immune genes al-ready exhibited chromatin accessibility in OPCs ex vivo and in vivo, suggesting a primed chromatin state in OLG compatible with rapid transitions to an immune-competent state. Several primed genes presented bivalency of H3K4me3 and H3K27me3 at promoters in OPCs, with loss of H3K27me3 upon IFN{gamma} treatment. Inhibition of JMJD3/Kdm6b, mediating removal of H3K27me3, led to the inability to activate these genes upon IFN{gamma} treatment. Importantly, OLGs from the adult human brain showed chromatin accessibility at immune gene loci, par-ticularly at MHC-I pathway genes. A subset of single-nucleotide polymorphisms (SNPs) associated with MS susceptibility over-lapped with these primed regulatory regions in OLG from both mouse and human CNS. Our data suggest that susceptibility for MS may involve activation of immune gene programs in OLG. These programs are under tight control at the chromatin level in OLG and may therefore constitute novel targets for immunological-based therapies for MS.

neuroscience↗

bioRxiv2020

Cell-type specialization in the brain is encoded by specific long-range chromatin topologies

Neurons and oligodendrocytes are terminally differentiated cells that sustain cascades of gene activation and repression to execute highly specialized functions, while retaining homeostatic control. To study long-range chromatin folding without disturbing the native tissue environment, we developed Genome Architecture Mapping in combination with immunoselection (immunoGAM), and applied it to three cell types from the adult murine brain: dopaminergic neurons (DNs) from the midbrain, pyramidal glutamatergic neurons (PGNs) from the hippocampus, and oligodendroglia (OLGs) from the cortex. We find cell-type specific 3D chromatin structures that relate with patterns of gene expression at multiple genomic scales, including extensive reorganization of topological domains (TADs) and chromatin compartments. We discover the loss of TAD insulation, or TAD melting, at long genes (>400 kb) when they are highly transcribed. We find many neuron-specific contacts which contain accessible chromatin regions enriched for putative binding sites for multiple neuronal transcription factors, and which connect cell-type specific genes that are associated with neurodegenerative disorders such as Parkinsons disease, or specialized functions such as synaptic plasticity and memory. Lastly, sensory receptor genes exhibit increased membership in heterochromatic compartments that establish strong contacts in brain cells. However, their silencing is compromised in a subpopulation of PGNs with molecular signatures of long-term potentiation. Overall, our work shows that the 3D organization of the genome is highly cell-type specific, and essential to better understand mechanisms of gene regulation in highly specialized tissues such as the brain.

molecular biology↗

bioRxiv2019

Functionally Distinct Subgroups of Oligodendrocyte Precursor Cells Integrate Neural Activity and Execute Myelin Formation

Recent reports revealed heterogeneity of oligodendrocyte precursor cells (OPCs). It remains unclear if heterogeneity reflects different types of cells with distinct functions, or rather transiently acquired states of cells with the same function. By integrating lineage formation of individual OPC clones, single-cell transcriptomics, calcium imaging and manipulation of neural activity, we show that OPCs in the zebrafish spinal cord can be divided into two functionally distinct entities. One subgroup forms elaborate networks of processes and exhibits a high degree of calcium signalling, but infrequently differentiates, despite contact to permissive axons. Instead, these OPCs divide in an activity and calcium dependent manner to produce another subgroup with higher process motility and less calcium signaling, which readily differentiates. Our data show that OPC subgroups are functionally diverse in responding to neurons and reveal that activity regulates proliferation of a subset of OPCs that is distinct from the cells that generate differentiated oligodendrocytes.

neuroscience↗

bioRxiv2019

RADICL-seq identifies general and cell type-specific principles of genome-wide RNA-chromatin interactions

Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression and chromatin remodelling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed. However, they still present some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA-chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared to existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type-specific RNA-chromatin interactions, and emphasizes the role of transcription in the establishment of chromatin structure.

genomics↗

bioRxiv2019

Specific oligodendrocyte populations have differential spatial distribution and susceptibility to injury

Oligodendrocytes (OLs), the myelinating cells of the central nervous system, are transcriptionally heterogeneous,1 the origin and functional consequences of which are unknown. Functional heterogeneity of MOLs might correlate with the local environment or their interactions with different neuron types.2 Here, we show that distinct MOL populations have spatial preference in the mammalian central nervous system and differential susceptibility to traumatic spinal cord injury. We also show that the generation of distinct MOL populations is independent of the OPC developmental origin. We found that OPCs originating from the previously described developmental waves3-5 have comparable potential to differentiate into the main MOL populations. Furthermore, we found that MOL type 2 (MOL2) is enriched in the spinal cord and almost absent in the brain, while MOL5/6 is enriched with age in all analyzed regions. MOL2 and MOL5/6 also have differential preference for motor and sensory tracts in the spinal cord. In the context of disease, we found that MOL2 and MOL5/6 have differential susceptibility to traumatic spinal cord injury, where MOL2 are lost and MOL5/6 increased their contribution to the OL lineage. Importantly, MOL2 susceptibility is disease specific, as we found MOL2 is not lost in a mouse model of multiple sclerosis. Our results demonstrate that the MOL populations, previously described by single-cell transcriptomics,1 have distinct spatial preference and responses. We anticipate our study to pave the way for a better understanding of the MOL populations-specific functional roles in development, health, and disease, allowing for better targeting of the OL subtypes important for the regeneration and repair of the central nervous system.

neuroscience↗

bioRxiv2019

Interaction of Sox2 with RNA binding proteins in mouse embryonic stem cells

Sox2 is a master transcriptional regulator of embryonic development. In this study, we determined the protein interactome of Sox2 in the chromatin and nucleoplasm of mouse embryonic stem (mES) cells. Apart from canonical interactions with pluripotency-regulating transcription factors, we identified interactions with several chromatin modulators, including members of the heterochromatin protein 1 (HP1) family, suggesting a role of Sox2 in chromatin-mediated transcriptional repression. Sox2 was also found to interact with RNA binding proteins (RBPs), including proteins involved in RNA processing. RNA immunoprecipitation followed by sequencing revealed that Sox2 associates with different messenger RNAs, as well as small nucleolar RNA Snord34 and the non-coding RNA 7SK. 7SK has been shown to regulate transcription at regulatory regions, which could suggest a functional interaction with Sox2 for chromatin recruitment. Nevertheless, we found no evidence of Sox2 modulating recruitment of 7SK to chromatin when examining 7SK chromatin occupancy by Chromatin Isolation by RNA Purification (ChIRP) in Sox2 depleted mES cells. In addition, knockdown of 7SK in mES cells did not lead to any change in Sox2 occupancy at 7SK-regulated genes. Thus, our results show that Sox2 extensively interact with RBPs, and suggest that Sox2 and 7SK co-exist in a ribonucleoprotein complex whose function is not to regulate chromatin recruitment, but might rather regulate other processes in the nucleoplasm. Summary blurbSox2 interacts with RNA-binding proteins and diverse RNAs

molecular biology↗

bioRxiv2018

PADI2-mediated citrullination is required for efficient oligodendrocyte differentiation and myelination

Citrullination, the deimination of arginine residues into citrulline, has been implicated in the aetiology of several diseases. In multiple sclerosis (MS), citrullination is thought to be a major driver of pathology, through hypercitrullination and destabilization of myelin. As such, inhibition of citrullination has been suggested as a therapeutic strategy for MS. Here, in contrast, we show citrullination by peptidylarginine deiminase 2 (PADI2) is required for normal oligodendrocyte differentiation, myelination and motor function. We identify several targets for PADI2, including myelin-related proteins and chromatin-associated proteins, implicating PADI2 in epigenetic regulation. Accordingly, we observe that PADI2 inhibition and its knockdown affect chromatin accessibility and prevent the upregulation of oligodendrocyte differentiation genes. Moreover, mice lacking PADI2, display motor dysfunction and decreased number of myelinated axons in the corpus callosum. We conclude that citrullination is required for oligodendrocyte lineage progression and myelination and suggest its targeted activation in the oligodendrocyte lineage might be beneficial in the context of remyelination.

neuroscience↗

bioRxiv2017

RNA velocity in single cells

RNA abundance is a powerful indicator of the state of individual cells, but does not directly reveal dynamic processes such as cellular differentiation. Here we show that RNA velocity--the time derivative of RNA abundance--can be estimated by distinguishing unspliced and spliced mRNAs in standard single-cell RNA sequencing protocols. We show that RNA velocity is a vector that predicts the future state of individual cells on a timescale of hours. We validate the accuracy of RNA velocity in the neural crest lineage, demonstrate its use on multiple technical platforms, reconstruct the branching lineage tree of the mouse hippocampus, and measure RNA kinetics in human embryonic brain. We expect RNA velocity to greatly aid the analysis of developmental lineages and cellular dynamics, particularly in humans.

genomics↗

bioRxiv2017

Single-cell transcriptomic profiling of progenitors of the oligodendrocyte lineage reveals transcriptional convergence during development

Pdgfra+ oligodendrocyte precursor cells (OPCs) arise in distinct specification waves during embryogenesis in the central nervous system (CNS). It is unclear whether there is a correlation between these waves and different transcriptional oligodendrocyte (OL) states at adult stages. Here we present a bulk and single-cell transcriptomics resource providing insights on how transitions between these states occur. We show that E13.5 Pdgfra+ populations are not OPCs, exhibiting instead hallmarks of neural progenitors. A subset of these progenitors, which we refer as pre-OPCs, rewires their transcriptional landscape, converging into indistinguishable OPC states at E17.5 and post-natal stages. P7 brain and spinal cord OPCs present similar transcriptional profiles at the single-cell level, indicating that OPC states are not region-specific. Postnatal OPC progeny of E13.5 Pdgfra+ have electrophysiological and transcriptional profiles similar to OPCs derived from subsequent specification waves. In addition, lineage tracing indicates that a subset of E13.5 Pdgfra+ cells also originate cells of the pericyte lineage. In summary, our results indicate that embryonic Pdgfra+ cells are diverse and give rise at post-natal stages to distinct cell lineages, including OPCs with convergent transcriptional profiles in different CNS regions.

neuroscience↗