RESUMO
Little is known about the origin of germ cells in humans. We previously leveraged post-zygotic mutations to reconstruct zygote-rooted cell lineage ancestry trees in a phenotypically normal woman, termed NC0. Here, by sequencing the genome of her children and their father, we analyzed the transmission of early pregastrulation lineages and corresponding mutations across human generations. We found that the germline in NC0 is polyclonal and is founded by at least two cells likely descending from the two blastomeres arising from the first zygotic cleavage. Analyses of public data from several multi-children families and from 1,934 familial quads confirmed this finding in larger cohorts, revealing that known imbalances of up to 90:10 in early lineages allocation in somatic tissues are not reflected in transmission to offspring, establishing a fundamental difference in lineage allocation between the soma and the germline. Analyses of all the data consistently suggest that germline has a balanced 50:50 lineage allocation from the first two blastomeres.
RESUMO
Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) is characterized by the abrupt onset of significant obsessive-compulsive symptoms (OCS) and/or severe food restriction, together with other neuropsychiatric manifestations. An autoimmune pathogenesis triggered by infection has been proposed for at least a subset of PANS. The older diagnosis of Pediatric Autoimmune Neuropsychiatric Disorder Associated with Streptococcus (PANDAS) describes rapid onset of OCD and/or tics associated with infection with Group A Streptococcus. The pathophysiology of PANS and PANDAS remains incompletely understood. We recently found serum antibodies from children with rigorously defined PANDAS to selectively bind to cholinergic interneurons (CINs) in the striatum. Here we examine this binding in children with relapsing and remitting PANS, a more heterogeneous condition, collected in a distinct clinical context from those examined in our previous work, from children with a clinical history of Streptococcus infection. IgG from PANS cases showed elevated binding to striatal CINs in both mouse and human brain. Patient plasma collected during symptom flare decreased a molecular marker of CIN activity, phospho-riboprotein S6, in ex vivo brain slices; control plasma did not. Neither elevated antibody binding to CINs nor diminished CIN activity was seen with plasma collected from the same children during remission. These findings replicate what we have seen previously in PANDAS and support the hypothesis that at least a subset of PANS cases have a neuroimmune pathogenesis. Given the critical role of CINs in modulating basal ganglia function, these findings confirm striatal CINs as a locus of interest in the pathophysiology of both PANS and PANDAS.
RESUMO
The implications of the early phases of human telencephalic development, involving neural stem cells (NSCs), in the etiology of cortical disorders remain elusive. Here, we explored the expression dynamics of cortical and neuropsychiatric disorder-associated genes in datasets generated from human NSCs across telencephalic fate transitions in vitro and in vivo. We identified risk genes expressed in brain organizers and sequential gene regulatory networks across corticogenesis revealing disease-specific critical phases, when NSCs are more vulnerable to gene dysfunctions, and converging signaling across multiple diseases. Moreover, we simulated the impact of risk transcription factor (TF) depletions on different neural cell types spanning the developing human neocortex and observed a spatiotemporal-dependent effect for each perturbation. Finally, single-cell transcriptomics of newly generated autism-affected patient-derived NSCs in vitro revealed recurrent alterations of TFs orchestrating brain patterning and NSC lineage commitment. This work opens new perspectives to explore human brain dysfunctions at the early phases of development.
RESUMO
The repertory of neurons generated by progenitor cells depends on their location along antero-posterior and dorso-ventral axes of the neural tube. To understand if recreating those axes was sufficient to specify human brain neuronal diversity, we designed a mesofluidic device termed Duo-MAPS to expose induced pluripotent stem cells (iPSC) to concomitant orthogonal gradients of a posteriorizing and a ventralizing morphogen, activating WNT and SHH signaling, respectively. Comparison of single cell transcriptomes with fetal human brain revealed that Duo-MAPS-patterned organoids generated the major neuronal lineages of the forebrain, midbrain, and hindbrain. Morphogens crosstalk translated into early patterns of gene expression programs predicting the generation of specific brain lineages. Human iPSC lines from six different genetic backgrounds showed substantial differences in response to morphogens, suggesting that interindividual genomic and epigenomic variations could impact brain lineages formation. Morphogen gradients promise to be a key approach to model the brain in its entirety.
RESUMO
Nucleotide changes in gene regulatory elements are important determinants of neuronal development and diseases. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated the cis-regulatory activity of 102,767 open chromatin regions, including thousands of sequences with cell type-specific accessibility and variants associated with brain gene regulation. In primary cells, we identified 46,802 active enhancer sequences and 164 variants that alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development.
Assuntos
Córtex Cerebral , Neurogênese , Organoides , Humanos , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Cromatina/metabolismo , Cromatina/genética , Aprendizado Profundo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese/genética , Neurônios/metabolismo , Organoides/metabolismo , Sequências Reguladoras de Ácido Nucleico , Regiões Promotoras Genéticas , Elementos Reguladores de TranscriçãoRESUMO
Sample-wise deconvolution methods estimate cell-type proportions and gene expressions in bulk tissue samples, yet their performance and biological applications remain unexplored, particularly in human brain transcriptomic data. Here, nine deconvolution methods were evaluated with sample-matched data from bulk tissue RNA sequencing (RNA-seq), single-cell/nuclei (sc/sn) RNA-seq, and immunohistochemistry. A total of 1,130,767 nuclei per cells from 149 adult postmortem brains and 72 organoid samples were used. The results showed the best performance of dtangle for estimating cell proportions and bMIND for estimating sample-wise cell-type gene expressions. For eight brain cell types, 25,273 cell-type eQTLs were identified with deconvoluted expressions (decon-eQTLs). The results showed that decon-eQTLs explained more schizophrenia GWAS heritability than bulk tissue or single-cell eQTLs did alone. Differential gene expressions associated with Alzheimer's disease, schizophrenia, and brain development were also examined using the deconvoluted data. Our findings, which were replicated in bulk tissue and single-cell data, provided insights into the biological applications of deconvoluted data in multiple brain disorders.
Assuntos
Encéfalo , Análise de Célula Única , Transcriptoma , Humanos , Encéfalo/metabolismo , Análise de Célula Única/métodos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Perfilação da Expressão Gênica/métodos , Esquizofrenia/genética , Esquizofrenia/metabolismo , Esquizofrenia/patologia , Estudo de Associação Genômica Ampla/métodos , Análise de Sequência de RNA/métodos , AdultoRESUMO
Tourette syndrome (TS) is a disorder of high-order integration of sensory, motor, and cognitive functions afflicting as many as 1 in 150 children and characterized by motor hyperactivity and tics. Despite high familial recurrence rates, a few risk genes and no biomarkers have emerged as causative or predisposing factors. The syndrome is believed to originate in basal ganglia, where patterns of motor programs are encoded. Postmortem immunocytochemical analyses of brains with severe TS revealed decreases in cholinergic, fast-spiking parvalbumin, and somatostatin interneurons within the striatum (caudate and putamen nuclei). Here, we performed single cell transcriptomic and chromatin accessibility analyses of the caudate nucleus from 6 adult TS and 6 control post-mortem brains. The data reproduced the known cellular composition of the adult human striatum, including a majority of medium spiny neurons (MSN) and small populations of GABAergic and cholinergic interneurons. Comparative analysis revealed that interneurons were decreased by roughly 50% in TS brains, while no difference was observed for other cell types. Differential gene expression analysis suggested that mitochondrial function, and specifically oxidative metabolism, in MSN and synaptic function in interneurons are both impaired in TS subjects. Furthermore, such an impairment was coupled with activation of immune response pathways in microglia. Also, our data explicitly link gene expression changes to changes in cis-regulatory activity in the corresponding cell types, suggesting de-regulation as a factor for the etiology of TS. These findings expand on previous research and suggest that impaired modulation of striatal function by interneurons may be the origin of TS symptoms.
RESUMO
Regulation of gene expression through enhancers is one of the major processes shaping the structure and function of the human brain during development. High-throughput assays have predicted thousands of enhancers involved in neurodevelopment, and confirming their activity through orthogonal functional assays is crucial. Here, we utilized Massively Parallel Reporter Assays (MPRAs) in stem cells and forebrain organoids to evaluate the activity of ~ 7000 gene-linked enhancers previously identified in human fetal tissues and brain organoids. We used a Gaussian mixture model to evaluate the contribution of background noise in the measured activity signal to confirm the activity of ~ 35% of the tested enhancers, with most showing temporal-specific activity, suggesting their evolving role in neurodevelopment. The temporal specificity was further supported by the correlation of activity with gene expression. Our findings provide a valuable gene regulatory resource to the scientific community.
Assuntos
Regulação da Expressão Gênica , Sequências Reguladoras de Ácido Nucleico , Humanos , Organoides , Prosencéfalo , Elementos Facilitadores GenéticosRESUMO
Somatic mosaicism is defined as an occurrence of two or more populations of cells having genomic sequences differing at given loci in an individual who is derived from a single zygote. It is a characteristic of multicellular organisms that plays a crucial role in normal development and disease. To study the nature and extent of somatic mosaicism in autism spectrum disorder, bipolar disorder, focal cortical dysplasia, schizophrenia, and Tourette syndrome, a multi-institutional consortium called the Brain Somatic Mosaicism Network (BSMN) was formed through the National Institute of Mental Health (NIMH). In addition to genomic data of affected and neurotypical brains, the BSMN also developed and validated a best practices somatic single nucleotide variant calling workflow through the analysis of reference brain tissue. These resources, which include >400 terabytes of data from 1087 subjects, are now available to the research community via the NIMH Data Archive (NDA) and are described here.
Assuntos
Transtornos Mentais , Humanos , Transtorno do Espectro Autista/genética , Encéfalo , Genômica , Mosaicismo , Genoma Humano , Transtornos Mentais/genéticaRESUMO
Idiopathic autism spectrum disorder (ASD) is highly heterogeneous, and it remains unclear how convergent biological processes in affected individuals may give rise to symptoms. Here, using cortical organoids and single-cell transcriptomics, we modeled alterations in the forebrain development between boys with idiopathic ASD and their unaffected fathers in 13 families. Transcriptomic changes suggest that ASD pathogenesis in macrocephalic and normocephalic probands involves an opposite disruption of the balance between excitatory neurons of the dorsal cortical plate and other lineages such as early-generated neurons from the putative preplate. The imbalance stemmed from divergent expression of transcription factors driving cell fate during early cortical development. While we did not find genomic variants in probands that explained the observed transcriptomic alterations, a significant overlap between altered transcripts and reported ASD risk genes affected by rare variants suggests a degree of gene convergence between rare forms of ASD and the developmental transcriptome in idiopathic ASD.
Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Masculino , Humanos , Transtorno Autístico/genética , Transtorno do Espectro Autista/patologia , Neurônios/metabolismo , Neurogênese , Prosencéfalo/metabolismo , Organoides/metabolismoRESUMO
Regulation of gene expression through enhancers is one of the major processes shaping the structure and function of the human brain during development. High-throughput assays have predicted thousands of enhancers involved in neurodevelopment, and confirming their activity through orthogonal functional assays is crucial. Here, we utilized Massively Parallel Reporter Assays (MPRAs) in stem cells and forebrain organoids to evaluate the activity of ~7,000 gene-linked enhancers previously identified in human fetal tissues and brain organoids. We used a Gaussian mixture model to evaluate the contribution of background noise in the measured activity signal to confirm the activity of ~35% of the tested enhancers, with most showing temporal-specific activity, suggesting their evolving role in neurodevelopment. The temporal specificity was further supported by the correlation of activity with gene expression. Our findings provide a valuable gene regulatory resource to the scientific community.
RESUMO
The CRISPR-Cas9 system has enabled researchers to precisely modify/edit the sequence of a genome. A typical editing experiment consists of two steps: (1) editing cultured cells; (2) cell cloning and selection of clones with and without intended edit, presumed to be isogenic. The application of CRISPR-Cas9 system may result in off-target edits, whereas cloning will reveal culture-acquired mutations. We analyzed the extent of the former and the latter by whole genome sequencing in three experiments involving separate genomic loci and conducted by three independent laboratories. In all experiments we hardly found any off-target edits, whereas detecting hundreds to thousands of single nucleotide mutations unique to each clone after relatively short culture of 10-20 passages. Notably, clones also differed in copy number alterations (CNAs) that were several kb to several mb in size and represented the largest source of genomic divergence among clones. We suggest that screening of clones for mutations and CNAs acquired in culture is a necessary step to allow correct interpretation of DNA editing experiments. Furthermore, since culture associated mutations are inevitable, we propose that experiments involving derivation of clonal lines should compare a mix of multiple unedited lines and a mix of multiple edited lines.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Mutação , DNARESUMO
Mosaic mutations can be used to track cell ancestries and reconstruct high-resolution lineage trees during cancer progression and during development, starting from the first cell divisions of the zygote. However, this approach requires sampling and analyzing the genomes of multiple cells, which can be redundant in lineage representation, limiting the scalability of the approach. We describe a strategy for cost- and time-efficient lineage reconstruction using clonal induced pluripotent stem cell lines from human skin fibroblasts. The approach leverages shallow sequencing coverage to assess the clonality of the lines, clusters redundant lines and sums their coverage to accurately discover mutations in the corresponding lineages. Only a fraction of lines needs to be sequenced to high coverage. We demonstrate the effectiveness of this approach for reconstructing lineage trees during development and in hematologic malignancies. We discuss and propose an optimal experimental design for reconstructing lineage trees.
Assuntos
Linhagem da Célula , Neoplasias , Software , Humanos , Células Germinativas , Mutação , Neoplasias/patologiaRESUMO
Fibroblast growth factor receptor 2 (FGFR2) is almost exclusively expressed in glial cells in postnatal mouse brain, but its impact in glia for brain behavioral functioning is poorly understood. We compared behavioral effects from FGFR2 loss in both neurons and astroglial cells and from FGFR2 loss in astroglial cells by using either the pluripotent progenitor-driven hGFAP-cre or the tamoxifen-inducible astrocyte-driven GFAP-creERT2 in Fgfr2 floxed mice. When FGFR2 was eliminated in embryonic pluripotent precursors or in early postnatal astroglia, mice were hyperactive, and had small changes in working memory, sociability, and anxiety-like behavior. In contrast, FGFR2 loss in astrocytes starting at 8 weeks of age resulted only in reduced anxiety-like behavior. Therefore, early postnatal loss of FGFR2 in astroglia is critical for broad behavioral dysregulation. Neurobiological assessments demonstrated that astrocyte-neuron membrane contact was reduced and glial glutamine synthetase expression increased only by early postnatal FGFR2 loss. We conclude that altered astroglial cell function dependent on FGFR2 in the early postnatal period may result in impaired synaptic development and behavioral regulation, modeling childhood behavioral deficits like attention deficit hyperactivity disorder (ADHD).
Assuntos
Astrócitos , Memória de Curto Prazo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Animais , Camundongos , Astrócitos/metabolismo , Locomoção , Neuroglia/metabolismo , Neurônios/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
Sample-wise deconvolution methods have been developed to estimate cell-type proportions and gene expressions in bulk-tissue samples. However, the performance of these methods and their biological applications has not been evaluated, particularly on human brain transcriptomic data. Here, nine deconvolution methods were evaluated with sample-matched data from bulk-tissue RNAseq, single-cell/nuclei (sc/sn) RNAseq, and immunohistochemistry. A total of 1,130,767 nuclei/cells from 149 adult postmortem brains and 72 organoid samples were used. The results showed the best performance of dtangle for estimating cell proportions and bMIND for estimating sample-wise cell-type gene expression. For eight brain cell types, 25,273 cell-type eQTLs were identified with deconvoluted expressions (decon-eQTLs). The results showed that decon-eQTLs explained more schizophrenia GWAS heritability than bulk-tissue or single-cell eQTLs alone. Differential gene expression associated with multiple phenotypes were also examined using the deconvoluted data. Our findings, which were replicated in bulk-tissue RNAseq and sc/snRNAseq data, provided new insights into the biological applications of deconvoluted data.
RESUMO
Nucleotide changes in gene regulatory elements are important determinants of neuronal development and disease. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated the cis-regulatory activity of 102,767 sequences, including differentially accessible cell-type specific regions in the developing cortex and single-nucleotide variants associated with psychiatric disorders. In primary cells, we identified 46,802 active enhancer sequences and 164 disorder-associated variants that significantly alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning, we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development.
RESUMO
BACKGROUND: Recent research implicates fibroblast growth factor 2 (FGF2) in anxiety and depressive symptoms of childhood. This study is the first to examine an intergenerational pathway linking FGF2 levels in mothers to FGF2 levels in children, and to the children's anxiety and depressive symptoms. METHODS: We assayed serum FGF2 in 259 mothers and their children, with a range of anxiety and depressive symptoms: 194 were mothers of clinic-referred anxious and depressed children; 65 were mothers of non-referred children. We examined associations between FGF2 levels in mothers and children, and anxiety and depression symptoms. We used structural equation modeling (SEM) to examine associations between maternal and child FGF2 levels, and between maternal and child FGF2 levels and symptoms of anxiety and depression in and children. RESULTS: FGF2 levels in mothers and children were significantly positively correlated. Children's FGF2 levels were significantly negatively correlated with their ratings of anxiety and depression. Results of the SEM model showed that increases in maternal FGF2 levels were significantly associated with increases in child FGF2, which in turn was associated with decreases in child anxiety and child depression, controlling for maternal anxiety and depression. LIMITATIONS: We relied on self-reported ratings of anxiety and depression, and on a single measurement of FGF2 levels for each participant. CONCLUSIONS: Our results point to a role for FGF2 in the intergenerational transmission of risk for, and resilience to, anxiety and depression in youth.