ABSTRACT
The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data4-8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb.
Subject(s)
Animals, Newborn , Embryo, Mammalian , Embryonic Development , Gastrula , Single-Cell Analysis , Time-Lapse Imaging , Animals , Female , Mice , Pregnancy , Animals, Newborn/embryology , Animals, Newborn/genetics , Cell Differentiation/genetics , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Embryonic Development/genetics , Gastrula/cytology , Gastrula/embryology , Gastrulation/genetics , Kidney/cytology , Kidney/embryology , Mesoderm/cytology , Mesoderm/enzymology , Neurons/cytology , Neurons/metabolism , Retina/cytology , Retina/embryology , Somites/cytology , Somites/embryology , Time Factors , Transcription Factors/genetics , Transcription, Genetic , Organ Specificity/geneticsABSTRACT
Cerebellar dysplasia with cysts (CDC) is an imaging finding typically seen in combination with cobblestone cortex and congenital muscular dystrophy in individuals with dystroglycanopathies. More recently, CDC was reported in seven children without neuromuscular involvement (Poretti-Boltshauser syndrome). Using a combination of homozygosity mapping and whole-exome sequencing, we identified biallelic mutations in LAMA1 as the cause of CDC in seven affected individuals (from five families) independent from those included in the phenotypic description of Poretti-Boltshauser syndrome. Most of these individuals also have high myopia, and some have retinal dystrophy and patchy increased T2-weighted fluid-attenuated inversion recovery (T2/FLAIR) signal in cortical white matter. In one additional family, we identified two siblings who have truncating LAMA1 mutations in combination with retinal dystrophy and mild cerebellar dysplasia without cysts, indicating that cysts are not an obligate feature associated with loss of LAMA1 function. This work expands the phenotypic spectrum associated with the lamininopathy disorders and highlights the tissue-specific roles played by different laminin-encoding genes.
Subject(s)
Cerebellar Cortex/abnormalities , Cerebellar Diseases/genetics , Cysts/genetics , Laminin/genetics , Retinal Dystrophies/genetics , Adult , Alleles , Base Sequence , Child , Child, Preschool , Exome/genetics , Female , Humans , Male , Muscular Dystrophies/genetics , Sequence Analysis, DNA , Young AdultABSTRACT
Joubert syndrome (JBTS) is a recessive ciliopathy in which a subset of affected individuals also have the skeletal dysplasia Jeune asphyxiating thoracic dystrophy (JATD). Here, we have identified biallelic truncating CSPP1 (centrosome and spindle pole associated protein 1) mutations in 19 JBTS-affected individuals, four of whom also have features of JATD. CSPP1 mutations explain â¼5% of JBTS in our cohort, and despite truncating mutations in all affected individuals, the range of phenotypic severity is broad. Morpholino knockdown of cspp1 in zebrafish caused phenotypes reported in other zebrafish models of JBTS (curved body shape, pronephric cysts, and cerebellar abnormalities) and reduced ciliary localization of Arl13b, further supporting loss of CSPP1 function as a cause of JBTS. Fibroblasts from affected individuals with CSPP1 mutations showed reduced numbers of primary cilia and/or short primary cilia, as well as reduced axonemal localization of ciliary proteins ARL13B and adenylyl cyclase III. In summary, CSPP1 mutations are a major cause of the Joubert-Jeune phenotype in humans; however, the mechanism by which these mutations lead to both JBTS and JATD remains unknown.
Subject(s)
Cell Cycle Proteins/genetics , Cerebellar Diseases/genetics , Cilia/genetics , Ellis-Van Creveld Syndrome/genetics , Eye Abnormalities/genetics , Kidney Diseases, Cystic/genetics , Microtubule-Associated Proteins/genetics , Mutation , Retina/abnormalities , Abnormalities, Multiple , Adolescent , Animals , Cerebellum/abnormalities , Child , Child, Preschool , Cilia/pathology , Exons , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Knockdown Techniques , Humans , Infant , Male , Phenotype , Sequence Analysis, DNA , Young Adult , Zebrafish/geneticsABSTRACT
BACKGROUND: Joubert syndrome (JS) is a ciliopathy characterised by a distinctive brain malformation (the 'molar tooth sign'), developmental delay, abnormal eye movements and abnormal breathing pattern. Retinal dystrophy, cystic kidney disease, liver fibrosis and polydactyly are variably present, resulting in significant phenotypic heterogeneity and overlap with other ciliopathies. JS is also genetically heterogeneous, resulting from mutations in 13 genes. These factors render clinical/molecular diagnosis and management challenging. CC2D2A mutations are a relatively common cause of JS and also cause Meckel syndrome. The clinical consequences of CC2D2A mutations in patients with JS have been incompletely reported. METHODS: Subjects with JS from 209 families were evaluated to identify mutations in CC2D2A. Clinical and imaging features in subjects with CC2D2A mutations were compared with those in subjects without CC2D2A mutations and reports in the literature. RESULTS: 10 novel CC2D2A mutations in 20 subjects were identified; a summary is provided of all published CC2D2A mutations. Subjects with CC2D2A-related JS were more likely to have ventriculomegaly (p<0.0001) and seizures (p=0.024) than subjects without CC2D2A mutations. No mutation-specific genotype-phenotype correlations could be identified, but the findings confirm the observation that mutations that cause CC2D2A-related JS are predicted to be less deleterious than mutations that cause CC2D2A-related Meckel syndrome. Missense variants in the coiled-coil and C2 domains, as well as the C-terminal region, identify these regions as important for the biological mechanisms underlying JS. CONCLUSIONS: CC2D2A testing should be prioritised in patients with JS and ventriculomegaly and/or seizures. Patients with CC2D2A-related JS should be monitored for hydrocephalus and seizures.
Subject(s)
Cerebellar Diseases/genetics , Eye Abnormalities/genetics , Genetic Association Studies , Hydrocephalus/genetics , Kidney Diseases, Cystic/genetics , Proteins/genetics , Seizures/genetics , Abnormalities, Multiple , Adolescent , Adult , Alleles , Cerebellar Diseases/diagnosis , Cerebellar Diseases/epidemiology , Cerebellum/abnormalities , Child , Child, Preschool , Cytoskeletal Proteins , Eye Abnormalities/diagnosis , Eye Abnormalities/epidemiology , Genotype , Humans , Hydrocephalus/diagnosis , Infant , Kidney Diseases, Cystic/diagnosis , Kidney Diseases, Cystic/epidemiology , Magnetic Resonance Imaging , Neuroimaging , Phenotype , Prevalence , Retina/abnormalities , Young AdultABSTRACT
Cross-species comparison and prediction of gene expression profiles are important to understand regulatory changes during evolution and to transfer knowledge learned from model organisms to humans. Single-cell RNA-seq (scRNA-seq) profiles enable us to capture gene expression profiles with respect to variations among individual cells; however, cross-species comparison of scRNA-seq profiles is challenging because of data sparsity, batch effects, and the lack of one-to-one cell matching across species. Moreover, single-cell profiles are challenging to obtain in certain biological contexts, limiting the scope of hypothesis generation. Here we developed Icebear, a neural network framework that decomposes single-cell measurements into factors representing cell identity, species, and batch factors. Icebear enables accurate prediction of single-cell gene expression profiles across species, thereby providing high-resolution cell type and disease profiles in under-characterized contexts. Icebear also facilitates direct cross-species comparison of single-cell expression profiles for conserved genes that are located on the X chromosome in eutherian mammals but on autosomes in chicken. This comparison, for the first time, revealed evolutionary and diverse adaptations of X-chromosome upregulation in mammals.
ABSTRACT
Cataloging the diverse cellular architecture of the primate brain is crucial for understanding cognition, behavior, and disease in humans. Here, we generated a brain-wide single-cell multimodal molecular atlas of the rhesus macaque brain. Together, we profiled 2.58 M transcriptomes and 1.59 M epigenomes from single nuclei sampled from 30 regions across the adult brain. Cell composition differed extensively across the brain, revealing cellular signatures of region-specific functions. We also identified 1.19 M candidate regulatory elements, many previously unidentified, allowing us to explore the landscape of cis-regulatory grammar and neurological disease risk in a cell type-specific manner. Altogether, this multi-omic atlas provides an open resource for investigating the evolution of the human brain and identifying novel targets for disease interventions.
Subject(s)
Brain , Multiomics , Animals , Macaca mulatta/genetics , TranscriptomeABSTRACT
The house mouse, Mus musculus, is an exceptional model system, combining genetic tractability with close homology to human biology. Gestation in mouse development lasts just under three weeks, a period during which its genome orchestrates the astonishing transformation of a single cell zygote into a free-living pup composed of >500 million cells. Towards a global framework for exploring mammalian development, we applied single cell combinatorial indexing (sci-*) to profile the transcriptional states of 12.4 million nuclei from 83 precisely staged embryos spanning late gastrulation (embryonic day 8 or E8) to birth (postnatal day 0 or P0), with 2-hr temporal resolution during somitogenesis, 6-hr resolution through to birth, and 20-min resolution during the immediate postpartum period. From these data (E8 to P0), we annotate dozens of trajectories and hundreds of cell types and perform deeper analyses of the unfolding of the posterior embryo during somitogenesis as well as the ontogenesis of the kidney, mesenchyme, retina, and early neurons. Finally, we leverage the depth and temporal resolution of these whole embryo snapshots, together with other published data, to construct and curate a rooted tree of cell type relationships that spans mouse development from zygote to pup. Throughout this tree, we systematically nominate sets of transcription factors (TFs) and other genes as candidate drivers of the in vivo differentiation of hundreds of mammalian cell types. Remarkably, the most dramatic shifts in transcriptional state are observed in a restricted set of cell types in the hours immediately following birth, and presumably underlie the massive changes in physiology that must accompany the successful transition of a placental mammal to extrauterine life.
ABSTRACT
Tooth enamel secreted by ameloblasts (AMs) is the hardest material in the human body, acting as a shield to protect the teeth. However, the enamel is gradually damaged or partially lost in over 90% of adults and cannot be regenerated due to a lack of ameloblasts in erupted teeth. Here, we use single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) to establish a spatiotemporal single-cell census for the developing human tooth and identify regulatory mechanisms controlling the differentiation process of human ameloblasts. We identify key signaling pathways involved between the support cells and ameloblasts during fetal development and recapitulate those findings in human ameloblast in vitro differentiation from induced pluripotent stem cells (iPSCs). We furthermore develop a disease model of amelogenesis imperfecta in a three-dimensional (3D) organoid system and show AM maturation to mineralized structure in vivo. These studies pave the way for future regenerative dentistry.
Subject(s)
Dental Enamel , Odontogenesis , Tooth , Humans , Ameloblasts/metabolism , Amelogenesis/geneticsABSTRACT
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.
Subject(s)
Cerebellum/embryology , Neurogenesis , Fetus , Humans , Laser Capture Microdissection , Single-Cell Analysis , TranscriptomeABSTRACT
The chromatin landscape underlying the specification of human cell types is of fundamental interest. We generated human cell atlases of chromatin accessibility and gene expression in fetal tissues. For chromatin accessibility, we devised a three-level combinatorial indexing assay and applied it to 53 samples representing 15 organs, profiling ~800,000 single cells. We leveraged cell types defined by gene expression to annotate these data and cataloged hundreds of thousands of candidate regulatory elements that exhibit cell type-specific chromatin accessibility. We investigated the properties of lineage-specific transcription factors (such as POU2F1 in neurons), organ-specific specializations of broadly distributed cell types (such as blood and endothelial), and cell type-specific enrichments of complex trait heritability. These data represent a rich resource for the exploration of in vivo human gene regulation in diverse tissues and cell types.
Subject(s)
Chromatin/metabolism , Fetus/cytology , Fetus/metabolism , Gene Expression Profiling , Gene Expression Regulation, Developmental , Single-Cell Analysis , Atlases as Topic , Humans , Neurons/metabolism , Transcription Factors/metabolismABSTRACT
The gene expression program underlying the specification of human cell types is of fundamental interest. We generated human cell atlases of gene expression and chromatin accessibility in fetal tissues. For gene expression, we applied three-level combinatorial indexing to >110 samples representing 15 organs, ultimately profiling ~4 million single cells. We leveraged the literature and other atlases to identify and annotate hundreds of cell types and subtypes, both within and across tissues. Our analyses focused on organ-specific specializations of broadly distributed cell types (such as blood, endothelial, and epithelial), sites of fetal erythropoiesis (which notably included the adrenal gland), and integration with mouse developmental atlases (such as conserved specification of blood cells). These data represent a rich resource for the exploration of in vivo human gene expression in diverse tissues and cell types.
Subject(s)
Chromatin/metabolism , Fetus/cytology , Fetus/metabolism , Gene Expression Profiling , Gene Expression Regulation, Developmental , Single-Cell Analysis , Atlases as Topic , Humans , Neurons/metabolism , Transcription Factors/metabolismABSTRACT
Gene expression levels vary across developmental stage, cell type, and region in the brain. Genomic variants also contribute to the variation in expression, and some neuropsychiatric disorder loci may exert their effects through this mechanism. To investigate these relationships, we present BrainVar, a unique resource of paired whole-genome and bulk tissue RNA sequencing from the dorsolateral prefrontal cortex of 176 individuals across prenatal and postnatal development. Here we identify common variants that alter gene expression (expression quantitative trait loci [eQTLs]) constantly across development or predominantly during prenatal or postnatal stages. Both "constant" and "temporal-predominant" eQTLs are enriched for loci associated with neuropsychiatric traits and disorders and colocalize with specific variants. Expression levels of more than 12,000 genes rise or fall in a concerted late-fetal transition, with the transitional genes enriched for cell-type-specific genes and neuropsychiatric risk loci, underscoring the importance of cataloging developmental trajectories in understanding cortical physiology and pathology.
Subject(s)
Brain/embryology , Computational Biology/methods , Prefrontal Cortex/metabolism , Base Sequence/genetics , Brain/growth & development , Brain/metabolism , Databases, Genetic , Genetic Predisposition to Disease/genetics , Genetic Variation/genetics , Genome-Wide Association Study/methods , Genomics/methods , Humans , Phenotype , Polymorphism, Single Nucleotide/genetics , Quantitative Trait Loci/genetics , Sequence Analysis, RNA/methods , Transcriptome/genetics , Exome Sequencing/methods , Whole Genome Sequencing/methodsABSTRACT
Dickkopf-like1 (Dkkl1) encodes a glycoprotein secreted by postmeiotic male germ cells. We report here that adult Dkkl1-deficient males have elevated sperm counts caused by a decrease in postpubertal spermatocyte apoptosis and display, upon aging, increased local production of testosterone. Molecular analyses identified the Fas death ligand (FasL) as a target for Dkkl1 pro-apoptotic activity in adult mice. Accordingly, adult FasL-deficient gld mice display an increased sperm count and decreased spermatocyte apoptosis phenotype similar to the one observed in Dkkl1-deficient mice. We also show that the elevated testosterone level observed in aging Dkkl1-deficient males is secondary to increased expression in Leydig cells of CYP11A and CYP17, two genes implicated in steroidogenesis. Furthermore, treatment of Leydig cells with Dkkl1 decreases DNA binding and transcriptional activity of steroidogenic factor 1, a pivotal regulator of gene expression in testis. Thus, this study establishes Dkkl1 as a negative regulator of adult testis homeostasis and identifies a novel, Dkkl1/FasL-dependent, regulation that specifically controls the number of postpubertal spermatocytes.
Subject(s)
Apoptosis/physiology , Intercellular Signaling Peptides and Proteins/physiology , Spermatocytes/physiology , Testosterone/biosynthesis , Animals , Fas Ligand Protein/physiology , Intercellular Signaling Peptides and Proteins/deficiency , Intercellular Signaling Peptides and Proteins/genetics , Male , Mice , Mice, Knockout , Polymerase Chain Reaction , RNA/genetics , RNA/isolation & purification , Sperm Count , Spermatocytes/cytology , TransfectionABSTRACT
Marijuana is one of the most abused drugs among pregnant women leading to maternal and fetal abnormalities. Cannabinoids are the active ingredients of marijuana, which interact with cannabinoid receptors such as CNR1 and CNR2 to activate cellular signaling pathways. Human endometrium and placenta are known to express CNR1 and CNR2 and can respond to cannabinoid signaling. In this study, we show that marijuana use significantly increases mRNA or protein expression of CNR1 and CNR2 in human endometrium from the first and early second trimester pregnancies, with minor effects on placental expression of CNRs.
Subject(s)
Endometrium/metabolism , Marijuana Use/adverse effects , Placenta/metabolism , Receptor, Cannabinoid, CB1/genetics , Receptor, Cannabinoid, CB1/metabolism , Receptor, Cannabinoid, CB2/genetics , Receptor, Cannabinoid, CB2/metabolism , Adolescent , Adult , Alcohol Drinking/adverse effects , Alcohol Drinking/genetics , Alcohol Drinking/metabolism , Case-Control Studies , Female , Humans , Marijuana Use/genetics , Marijuana Use/metabolism , Pregnancy , Pregnancy Trimester, First , Pregnancy Trimester, Second , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction , Smoking/adverse effects , Smoking/genetics , Smoking/metabolism , Young AdultABSTRACT
Despite frequent herpes simplex virus (HSV) reactivation, peripheral nerve destruction and sensory anesthesia are rare. We discovered that skin biopsies obtained during asymptomatic human HSV-2 reactivation exhibit a higher density of nerve fibers relative to biopsies during virological and clinical quiescence. We evaluated the effects of HSV infection on keratinocytes, the initial target of HSV replication, to better understand this observation. Keratinocytes produced IL-17c during HSV-2 reactivation, and IL-17RE, an IL-17c-specific receptor, was expressed on nerve fibers in human skin and sensory neurons in dorsal root ganglia. In ex vivo experiments, exogenous human IL-17c provided directional guidance and promoted neurite growth and branching in microfluidic devices. Exogenous murine IL-17c pretreatment reduced apoptosis in HSV-2-infected primary neurons. These results suggest that IL-17c is a neurotrophic cytokine that protects peripheral nerve systems during HSV reactivation. This mechanism could explain the lack of nerve damage from recurrent HSV infection and may provide insight to understanding and treating sensory peripheral neuropathies.
Subject(s)
Herpes Genitalis/physiopathology , Herpesvirus 2, Human/physiology , Interleukin-17/physiology , Keratinocytes/metabolism , Peripheral Nervous System/virology , Animals , Herpes Genitalis/virology , Humans , Keratinocytes/virology , Neurites/physiology , Neuroblastoma/physiopathology , Peripheral Nervous System/physiopathology , Virus Activation/physiologyABSTRACT
BACKGROUND: The discovery of disease pathogenesis requires systematic agnostic screening of multiple homeostatic processes that may become deregulated. We illustrate this principle in the evaluation and diagnosis of a 5-year-old boy with Joubert syndrome type 10 (JBTS10). He carried the OFD1 mutation p.Gln886Lysfs*2 (NM_003611.2: c.2656del) and manifested features of Joubert syndrome. METHODS: We integrated exome sequencing, MALDI-TOF mass spectrometry analyses of plasma and cultured dermal fibroblasts glycomes, and full clinical evaluation of the proband. Analyses of cilia formation and lectin staining were performed by immunofluorescence. Measurement of cellular nucleotide sugar levels was performed with high-performance anion-exchange chromatography with pulsed amperometric detection. Statistical analyses utilized the Student's and Fisher's exact t tests. RESULTS: Glycome analyses of plasma and cultured dermal fibroblasts identified abnormal N- and O-linked glycosylation profiles. These findings replicated in two unrelated males with OFD1 mutations. Cultured fibroblasts from affected individuals had a defect in ciliogenesis. The proband's fibroblasts also had an abnormally elevated nuclear sialylation signature and increased total cellular levels of CMP-sialic acid. Ciliogenesis and each glycosylation anomaly were rescued by expression of wild-type OFD1. CONCLUSIONS: The rescue of ciliogenesis and glycosylation upon reintroduction of WT OFD1 suggests that both contribute to the pathogenesis of JBTS10.
ABSTRACT
Common polymorphisms in the first intron of FTO are associated with increased body weight in adults. Previous studies have suggested that a CUX1-regulatory element within the implicated FTO region controls expression of FTO and the nearby ciliary gene, RPGRIP1L. Given the role of ciliary genes in energy homeostasis, we hypothesized that mice hypomorphic for Rpgrip1l would display increased adiposity. We find that Rpgrip1lâº/â» mice are hyperphagic and fatter, and display diminished suppression of food intake in response to leptin administration. In the hypothalamus of Rpgrip1lâº/â» mice, and in human fibroblasts with hypomorphic mutations in RPGRIP1L, the number of AcIII-positive cilia is diminished, accompanied by impaired convening of the leptin receptor to the vicinity of the cilium, and diminished pStat3 in response to leptin. These findings suggest that RPGRIP1L may be partly or exclusively responsible for the obesity susceptibility signal at the FTO locus.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Adiposity/genetics , Cilia/genetics , Polymorphism, Genetic/genetics , Proteins/genetics , Alpha-Ketoglutarate-Dependent Dioxygenase FTO , Animals , Cells, Cultured , Eating/genetics , Female , Fibroblasts/metabolism , Humans , Hypothalamus , Introns , Leptin/genetics , Mice , Mice, Inbred C57BL , Receptors, Leptin/genetics , STAT3 Transcription Factor/genetics , Weight Gain/geneticsABSTRACT
Exome sequencing studies of autism spectrum disorders (ASDs) have identified many de novo mutations but few recurrently disrupted genes. We therefore developed a modified molecular inversion probe method enabling ultra-low-cost candidate gene resequencing in very large cohorts. To demonstrate the power of this approach, we captured and sequenced 44 candidate genes in 2446 ASD probands. We discovered 27 de novo events in 16 genes, 59% of which are predicted to truncate proteins or disrupt splicing. We estimate that recurrent disruptive mutations in six genes-CHD8, DYRK1A, GRIN2B, TBR1, PTEN, and TBL1XR1-may contribute to 1% of sporadic ASDs. Our data support associations between specific genes and reciprocal subphenotypes (CHD8-macrocephaly and DYRK1A-microcephaly) and replicate the importance of a ß-catenin-chromatin-remodeling network to ASD etiology.