RESUMO
The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.
Assuntos
Envelhecimento , Sistema Nervoso Entérico/citologia , Feto/citologia , Saúde , Intestinos/citologia , Intestinos/crescimento & desenvolvimento , Linfonodos/citologia , Linfonodos/crescimento & desenvolvimento , Adulto , Animais , Criança , Doença de Crohn/patologia , Conjuntos de Dados como Assunto , Sistema Nervoso Entérico/anatomia & histologia , Sistema Nervoso Entérico/embriologia , Sistema Nervoso Entérico/crescimento & desenvolvimento , Células Epiteliais/citologia , Feminino , Feto/anatomia & histologia , Feto/embriologia , Humanos , Intestinos/embriologia , Intestinos/inervação , Linfonodos/embriologia , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Organogênese , Receptores de IgG/metabolismo , Transdução de Sinais , Análise Espaço-Temporal , Fatores de TempoRESUMO
Cerebellar malformations are diverse congenital anomalies frequently associated with developmental disability. Although genetic and prenatal non-genetic causes have been described, no systematic analysis has been performed. Here, we present a large-exome sequencing study of Dandy-Walker malformation (DWM) and cerebellar hypoplasia (CBLH). We performed exome sequencing in 282 individuals from 100 families with DWM or CBLH, and we established a molecular diagnosis in 36 of 100 families, with a significantly higher yield for CBLH (51%) than for DWM (16%). The 41 variants impact 27 neurodevelopmental-disorder-associated genes, thus demonstrating that CBLH and DWM are often features of monogenic neurodevelopmental disorders. Though only seven monogenic causes (19%) were identified in more than one individual, neuroimaging review of 131 additional individuals confirmed cerebellar abnormalities in 23 of 27 genetic disorders (85%). Prenatal risk factors were frequently found among individuals without a genetic diagnosis (30 of 64 individuals [47%]). Single-cell RNA sequencing of prenatal human cerebellar tissue revealed gene enrichment in neuronal and vascular cell types; this suggests that defective vasculogenesis may disrupt cerebellar development. Further, de novo gain-of-function variants in PDGFRB, a tyrosine kinase receptor essential for vascular progenitor signaling, were associated with CBLH, and this discovery links genetic and non-genetic etiologies. Our results suggest that genetic defects impact specific cerebellar cell types and implicate abnormal vascular development as a mechanism for cerebellar malformations. We also confirmed a major contribution for non-genetic prenatal factors in individuals with cerebellar abnormalities, substantially influencing diagnostic evaluation and counseling regarding recurrence risk and prognosis.
Assuntos
Cerebelo/anormalidades , Cerebelo/diagnóstico por imagem , Estudos de Coortes , Feminino , Humanos , Masculino , GravidezRESUMO
In human telencephalon at 8-12 postconceptional weeks, ribonucleic acid quantitative sequencing and immunohistochemistry revealed cortical chicken ovalbumin upstream promotor-transcription factor 1 (COUP-TFI) expression in a high ventro-posterior to low anterior gradient except for raised immunoreactivity in the anterior ventral pallium. Unlike in mouse, COUP-TFI and SP8 were extensively co-expressed in dorsal sensory neocortex and dorsal hippocampus whereas COUPTFI/COUPTFII co-expression defined ventral temporal cortex and ventral hippocampus. In the ganglionic eminences (GEs) COUP-TFI immunoreactivity demarcated the proliferative zones of caudal GE (CGE), dorsal medial GE (MGE), MGE/lateral GE (LGE) boundary, and ventral LGE whereas COUP-TFII was limited to ventral CGE and the MGE/LGE boundary. Co-labeling with gamma amino butyric acidergic interneuron markers revealed that COUP-TFI was expressed in subpopulations of either MGE-derived (SOX6+) or CGE-derived (calretinin+/SP8+) interneurons. COUP-TFII was mainly confined to CGE-derived interneurons. Twice as many GAD67+ cortical cells co-labeled for COUP-TFI than for COUP-TFII. A fifth of COUP-TFI cells also co-expressed COUP-TFII, and cells expressing either transcription factor followed posterior or anterio-lateral pathways into the cortex, therefore, a segregation of migration pathways according to COUP-TF expression as proposed in mouse was not observed. In cultures differentiated from isolated human cortical progenitors, many cells expressed either COUP-TF and 30% also co-expressed GABA, however no cells expressed NKX2.1. This suggests interneurons could be generated intracortically from progenitors expressing either COUP-TF.
Assuntos
Fator II de Transcrição COUP/metabolismo , Fator I de Transcrição COUP/metabolismo , Neurônios GABAérgicos/metabolismo , Interneurônios/metabolismo , Telencéfalo/crescimento & desenvolvimento , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hipocampo/metabolismo , Humanos , Imuno-Histoquímica/métodos , Neocórtex/crescimento & desenvolvimento , Neocórtex/metabolismoRESUMO
Brain development and function depend on the precise regulation of gene expression. However, our understanding of the complexity and dynamics of the transcriptome of the human brain is incomplete. Here we report the generation and analysis of exon-level transcriptome and associated genotyping data, representing males and females of different ethnicities, from multiple brain regions and neocortical areas of developing and adult post-mortem human brains. We found that 86 per cent of the genes analysed were expressed, and that 90 per cent of these were differentially regulated at the whole-transcript or exon level across brain regions and/or time. The majority of these spatio-temporal differences were detected before birth, with subsequent increases in the similarity among regional transcriptomes. The transcriptome is organized into distinct co-expression networks, and shows sex-biased gene expression and exon usage. We also profiled trajectories of genes associated with neurobiological categories and diseases, and identified associations between single nucleotide polymorphisms and gene expression. This study provides a comprehensive data set on the human brain transcriptome and insights into the transcriptional foundations of human neurodevelopment.
Assuntos
Envelhecimento/genética , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Transcriptoma/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Encéfalo/embriologia , Criança , Pré-Escolar , Éxons/genética , Feminino , Feto/metabolismo , Redes Reguladoras de Genes/genética , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Controle de Qualidade , Locos de Características Quantitativas/genética , Caracteres Sexuais , Fatores de Tempo , Adulto JovemRESUMO
The human neonatal cerebellum is one-fourth of its adult size yet contains the blueprint required to integrate environmental cues with developing motor, cognitive and emotional skills into adulthood. Although mature cerebellar neuroanatomy is well studied, understanding of its developmental origins is limited. In this study, we systematically mapped the molecular, cellular and spatial composition of human fetal cerebellum by combining laser capture microscopy and SPLiT-seq single-nucleus transcriptomics. We profiled functionally distinct regions and gene expression dynamics within cell types and across development. The resulting cell atlas demonstrates that the molecular organization of the cerebellar anlage recapitulates cytoarchitecturally distinct regions and developmentally transient cell types that are distinct from the mouse cerebellum. By mapping genes dominant for pediatric and adult neurological disorders onto our dataset, we identify relevant cell types underlying disease mechanisms. These data provide a resource for probing the cellular basis of human cerebellar development and disease.
Assuntos
Cerebelo/embriologia , Neurogênese , Feto , Humanos , Microdissecção e Captura a Laser , Análise de Célula Única , TranscriptomaRESUMO
We present histological and molecular analyses of the developing human cerebellum from 30 days after conception to 9 months after birth. Differences in developmental patterns between humans and mice include spatiotemporal expansion of both ventricular and rhombic lip primary progenitor zones to include subventricular zones containing basal progenitors. The human rhombic lip persists longer through cerebellar development than in the mouse and undergoes morphological changes to form a progenitor pool in the posterior lobule, which is not seen in other organisms, not even in the nonhuman primate the macaque. Disruptions in human rhombic lip development are associated with posterior cerebellar vermis hypoplasia and Dandy-Walker malformation. The presence of these species-specific neural progenitor populations refines our insight into human cerebellar developmental disorders.
Assuntos
Cerebelo/embriologia , Cerebelo/crescimento & desenvolvimento , Células-Tronco/citologia , Animais , Síndrome de Dandy-Walker , Humanos , Camundongos , Malformações do Sistema Nervoso , Análise Espaço-Temporal , Especificidade da Espécie , TranscriptomaRESUMO
Left-right asymmetry is subtle but pervasive in the human central nervous system. This asymmetry is initiated early during development, but its mechanisms are poorly known. Forebrains and midbrains were dissected from six human embryos at Carnegie stages 15 or 16, one of which was female. The structures were divided into left and right sides, and RNA was isolated. RNA was sequenced with 100 base-pair paired ends using Illumina Hiseq 4000. After quality control, five paired brain sides were available for midbrain and forebrain. A paired analysis between left- and right sides of a given brain structure across the embryos identified left-right differences. The dataset, consisting of Fastq files and a read count table, can be further used to study early development of the human brain.
Assuntos
Mesencéfalo/fisiologia , Prosencéfalo/fisiologia , Transcriptoma , Padronização Corporal , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Mesencéfalo/embriologia , Prosencéfalo/embriologia , Análise de Sequência de RNARESUMO
Left-right laterality is an important aspect of human -and in fact all vertebrate- brain organization for which the genetic basis is poorly understood. Using RNA sequencing data we contrasted gene expression in left- and right-sided samples from several structures of the anterior central nervous systems of post mortem human embryos and foetuses. While few individual genes stood out as significantly lateralized, most structures showed evidence of laterality of their overall transcriptomic profiles. These left-right differences showed overlap with age-dependent changes in expression, indicating lateralized maturation rates, but not consistently in left-right orientation over all structures. Brain asymmetry may therefore originate in multiple locations, or if there is a single origin, it is earlier than 5 weeks post conception, with structure-specific lateralized processes already underway by this age. This pattern is broadly consistent with the weak correlations reported between various aspects of adult brain laterality, such as language dominance and handedness.
Assuntos
Encéfalo/embriologia , Feto/fisiologia , Lateralidade Funcional , Transcriptoma , Animais , Padronização Corporal , Encéfalo/fisiologia , Feto/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Peixe-ZebraRESUMO
Joubert syndrome (JS) is a genetically heterogeneous autosomal recessive ciliopathy with 22 genes implicated to date, including a small, ciliary GTPase, ARL13B. ARL13B is required for cilia formation in vertebrates. JS patients display multiple symptoms characterized by ataxia due to the cerebellar vermis hypoplasia, and that can also include ocular abnormalities, renal cysts, liver fibrosis or polydactyly. These symptoms are shared with other ciliopathies, some of which display additional phenotypes, such as obesity. Here we identified a novel homozygous missense variant in ARL13B/JBTS8 in a JS patient who displayed retinal defects and obesity. We demonstrate the variant disrupts ARL13B function, as its expression did not rescue the mutant phenotype either in Arl13b(scorpion) zebrafish or in Arl13b(hennin) mouse embryonic fibroblasts, while the wild-type ARL13B did. Finally, we show that ARL13B is localized within the primary cilia of neonatal mouse hypothalamic neurons consistent with the known link between hypothalamic ciliary function and obesity. Thus our data identify a novel ARL13B variant that causes JS and retinopathy and suggest an extension of the phenotypic spectrum of ARL13B mutations to obesity.
Assuntos
Fatores de Ribosilação do ADP/genética , Doenças Cerebelares/diagnóstico , Doenças Cerebelares/genética , Oftalmopatias Hereditárias/diagnóstico , Oftalmopatias Hereditárias/genética , Mutação , Obesidade/genética , Fenótipo , Doenças Retinianas/genética , Fatores de Ribosilação do ADP/química , Fatores de Ribosilação do ADP/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/patologia , Biologia Computacional , Consanguinidade , Modelos Animais de Doenças , Ligação Genética , Homozigoto , Humanos , Imuno-Histoquímica , Lactente , Imageamento por Ressonância Magnética , Masculino , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Obesidade/diagnóstico , Linhagem , Conformação Proteica , Doenças Retinianas/diagnóstico , Alinhamento de Sequência , Análise de Sequência de DNA , Peixe-ZebraRESUMO
During brain development, neural stem cells (NSCs) receive on-or-off signals important for regulating their amplification and reaching adequate neuron density. However, how a coordinated regulation of intracellular pathways and genetic programs is achieved has remained elusive. Here, we found that the embryonic (e) CSF contains 10¹² nanoparticles/ml (77 nm diameter), some of which were identified as exosome nanovesicles that contain evolutionarily conserved molecules important for coordinating intracellular pathways. eCSF nanovesicles collected from rodent and human embryos encapsulate protein and microRNA components of the insulin-like growth factor (IGF) signaling pathway. Supplementation of eCSF nanovesicles to a mixed culture containing eNSCs activated the IGF-mammalian target of rapamycin complex 1 (mTORC1) pathway in eNSCs and expanded the pool of proliferative eNSCs. These data show that the eCSF serves as a medium for the distribution of nanovesicles, including exosomes, and the coordinated transfer of evolutionary conserved molecules that regulate eNSC amplification during corticogenesis.
Assuntos
Líquido Cefalorraquidiano/citologia , Embrião de Mamíferos/citologia , Evolução Molecular , Nanopartículas , Células-Tronco Neurais/citologia , Animais , Proliferação de Células , Exossomos/metabolismo , Feminino , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Complexos Multiproteicos/metabolismo , Gravidez , Ratos , Transdução de Sinais , Somatomedinas/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
The human neocortex has numerous specialized functional areas whose formation is poorly understood. Here, we describe a 15-base pair deletion mutation in a regulatory element of GPR56 that selectively disrupts human cortex surrounding the Sylvian fissure bilaterally including "Broca's area," the primary language area, by disrupting regional GPR56 expression and blocking RFX transcription factor binding. GPR56 encodes a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor required for normal cortical development and is expressed in cortical progenitor cells. GPR56 expression levels regulate progenitor proliferation. GPR56 splice forms are highly variable between mice and humans, and the regulatory element of gyrencephalic mammals directs restricted lateral cortical expression. Our data reveal a mechanism by which control of GPR56 expression pattern by multiple alternative promoters can influence stem cell proliferation, gyral patterning, and, potentially, neocortex evolution.
Assuntos
Processamento Alternativo , Padronização Corporal/genética , Córtex Cerebral/embriologia , Células-Tronco Neurais/fisiologia , Receptores Acoplados a Proteínas G/genética , Animais , Sequência de Bases , Evolução Biológica , Gatos , Proliferação de Células , Córtex Cerebral/anatomia & histologia , Córtex Cerebral/citologia , Códon sem Sentido , Lobo Frontal/anatomia & histologia , Lobo Frontal/citologia , Lobo Frontal/embriologia , Variação Genética , Haplótipos , Humanos , Camundongos , Dados de Sequência Molecular , Células-Tronco Neurais/citologia , Linhagem , Regiões Promotoras Genéticas/genética , Deleção de SequênciaRESUMO
During vertebrate central nervous system development, the apical neuroepithelium is bathed with embryonic Cerebrospinal Fluid (e-CSF) which plays regulatory roles in cortical cell proliferation and maintenance. Here, we report the first proteomic analysis of human e-CSF and compare it to an extensive proteomic analysis of rat e-CSF. As expected, we identified a large collection of protease inhibitors, extracellular matrix proteins, and transport proteins in CSF. However, we also found a surprising suite of signaling and intracellular proteins not predicted by previous proteomic analysis. Some of the intracellular proteins are likely to represent the contents of microvesicles recently described within the CSF (Marzesco, A. M., et al. J. Cell Sci. 2005, 118 (Pt. 13), 2849-2858). Defining the rich composition of e-CSF will enable a greater understanding of its concerted actions during critical stages of brain development.