RESUMO
The vertebrate Dlx gene family encode homeobox transcription factors that are related to the Drosophila Distal-less (Dll) gene and are crucial for development. Over the last â¼35â years detailed information has accrued about the redundant and unique expression and function of the six mammalian Dlx family genes. DLX proteins interact with general transcriptional regulators, and co-bind with other transcription factors to enhancer elements with highly specific activity in the developing forebrain. Integration of the genetic and biochemical data has yielded a foundation for a gene regulatory network governing the differentiation of forebrain GABAergic neurons. In this Primer, we describe the discovery of vertebrate Dlx genes and their crucial roles in embryonic development. We largely focus on the role of Dlx family genes in mammalian forebrain development revealed through studies in mice. Finally, we highlight questions that remain unanswered regarding vertebrate Dlx genes despite over 30â years of research.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio , Prosencéfalo , Fatores de Transcrição , Animais , Prosencéfalo/metabolismo , Prosencéfalo/embriologia , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Humanos , Mamíferos/genética , CamundongosRESUMO
OBJECTIVE: Holoprosencephaly (HPE) is the most common aberration of forebrain development, and it leads to a wide spectrum of developmental and craniofacial anomalies. HPE etiology is highly heterogeneous and includes both chromosomal abnormalities and single-gene defects. METHODS: Here, we report an FGFR1 heterozygous variant detected by prenatal exome sequencing and inherited from the asymptomatic mother, in association with recurrent neurological abnormalities in the HPE spectrum in two consecutive pregnancies. RESULTS: Individuals with germline pathogenic variants in FGFR1 (MIM: 136350) show extensive phenotypic variability, which ranges from asymptomatic carriers to hypogonadotropic hypogonadism, arhinencephaly, Kallmann's syndrome with associated features such as cleft lip and palate, skeletal anomalies, isolated HPE, and Hartsfield syndrome. CONCLUSION: The presented case supports the role of exome sequencing in prenatal diagnosis when fetal midline structural anomalies are suggestive of a genetic etiology, as early as the first trimester of gestation. The profound heterogeneity of FGFR1 allelic disorders needs to be considered when planning prenatal screening even in asymptomatic carriers.
Assuntos
Holoprosencefalia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Humanos , Feminino , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Gravidez , Holoprosencefalia/genética , Holoprosencefalia/diagnóstico , Adulto , Diagnóstico Pré-Natal/métodos , Sequenciamento do Exoma , Ultrassonografia Pré-Natal , Prosencéfalo/anormalidades , Prosencéfalo/embriologia , HeterozigotoRESUMO
Interspecies blastocyst complementation (IBC) provides a unique platform to study development and holds the potential to overcome worldwide organ shortages. Despite recent successes, brain tissue has not been achieved through IBC. Here, we developed an optimized IBC strategy based on C-CRISPR, which facilitated rapid screening of candidate genes and identified that Hesx1 deficiency supported the generation of rat forebrain tissue in mice via IBC. Xenogeneic rat forebrain tissues in adult mice were structurally and functionally intact. Cross-species comparative analyses revealed that rat forebrain tissues developed at the same pace as the mouse host but maintained rat-like transcriptome profiles. The chimeric rate of rat cells gradually decreased as development progressed, suggesting xenogeneic barriers during mid-to-late pre-natal development. Interspecies forebrain complementation opens the door for studying evolutionarily conserved and divergent mechanisms underlying brain development and cognitive function. The C-CRISPR-based IBC strategy holds great potential to broaden the study and application of interspecies organogenesis.
Assuntos
Prosencéfalo , Animais , Prosencéfalo/metabolismo , Prosencéfalo/embriologia , Camundongos , Ratos , Blastocisto/metabolismo , Feminino , Sistemas CRISPR-Cas/genética , Transcriptoma , Organogênese , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Masculino , Camundongos Endogâmicos C57BLRESUMO
The human nervous system is a highly complex but organized organ. The foundation of its complexity and organization is laid down during regional patterning of the neural tube, the embryonic precursor to the human nervous system. Historically, studies of neural tube patterning have relied on animal models to uncover underlying principles. Recently, models of neurodevelopment based on human pluripotent stem cells, including neural organoids1-5 and bioengineered neural tube development models6-10, have emerged. However, such models fail to recapitulate neural patterning along both rostral-caudal and dorsal-ventral axes in a three-dimensional tubular geometry, a hallmark of neural tube development. Here we report a human pluripotent stem cell-based, microfluidic neural tube-like structure, the development of which recapitulates several crucial aspects of neural patterning in brain and spinal cord regions and along rostral-caudal and dorsal-ventral axes. This structure was utilized for studying neuronal lineage development, which revealed pre-patterning of axial identities of neural crest progenitors and functional roles of neuromesodermal progenitors and the caudal gene CDX2 in spinal cord and trunk neural crest development. We further developed dorsal-ventral patterned microfluidic forebrain-like structures with spatially segregated dorsal and ventral regions and layered apicobasal cellular organizations that mimic development of the human forebrain pallium and subpallium, respectively. Together, these microfluidics-based neurodevelopment models provide three-dimensional lumenal tissue architectures with in vivo-like spatiotemporal cell differentiation and organization, which will facilitate the study of human neurodevelopment and disease.
Assuntos
Padronização Corporal , Microfluídica , Tubo Neural , Humanos , Técnicas de Cultura de Células em Três Dimensões , Diferenciação Celular , Crista Neural/citologia , Crista Neural/embriologia , Tubo Neural/citologia , Tubo Neural/embriologia , Células-Tronco Pluripotentes/citologia , Prosencéfalo/citologia , Prosencéfalo/embriologia , Medula Espinal/citologia , Medula Espinal/embriologiaRESUMO
The assembly of cortical circuits involves the generation and migration of interneurons from the ventral to the dorsal forebrain1-3, which has been challenging to study at inaccessible stages of late gestation and early postnatal human development4. Autism spectrum disorder and other neurodevelopmental disorders (NDDs) have been associated with abnormal cortical interneuron development5, but which of these NDD genes affect interneuron generation and migration, and how they mediate these effects remains unknown. We previously developed a platform to study interneuron development and migration in subpallial organoids and forebrain assembloids6. Here we integrate assembloids with CRISPR screening to investigate the involvement of 425 NDD genes in human interneuron development. The first screen aimed at interneuron generation revealed 13 candidate genes, including CSDE1 and SMAD4. We subsequently conducted an interneuron migration screen in more than 1,000 forebrain assembloids that identified 33 candidate genes, including cytoskeleton-related genes and the endoplasmic reticulum-related gene LNPK. We discovered that, during interneuron migration, the endoplasmic reticulum is displaced along the leading neuronal branch before nuclear translocation. LNPK deletion interfered with this endoplasmic reticulum displacement and resulted in abnormal migration. These results highlight the power of this CRISPR-assembloid platform to systematically map NDD genes onto human development and reveal disease mechanisms.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Transtornos do Neurodesenvolvimento , Feminino , Humanos , Recém-Nascido , Gravidez , Movimento Celular/genética , Sistemas CRISPR-Cas/genética , Interneurônios/citologia , Interneurônios/metabolismo , Interneurônios/patologia , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Organoides/citologia , Organoides/embriologia , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Organoides/patologia , Retículo Endoplasmático/metabolismo , Prosencéfalo/citologia , Prosencéfalo/embriologia , Prosencéfalo/crescimento & desenvolvimento , Prosencéfalo/metabolismo , Prosencéfalo/patologia , Transporte Ativo do Núcleo CelularRESUMO
During embryonic development, the forebrain roof plate undergoes invagination, leading to separation of the cerebral hemispheres. Any defects in this process, in humans, lead to middle interhemispheric holoprosencephaly (MIH-HPE). In this study, we have identified a previously unreported downstream mediator of retinoic acid (RA) signaling, CNKSR2, which is expressed in the forebrain roof plate in the chick embryo. Knockdown of CNKSR2 affects invagination, cell proliferation and patterning of the roof plate, similar to the phenotypes observed upon inhibition of RA signaling. We further demonstrate that CNKSR2 functions by modulating the Ras/Raf/MEK signaling. This appears to be crucial for patterning of the forebrain roof plate and its subsequent invagination, leading to the formation of the cerebral hemispheres. Thus, a set of novel molecular players have been identified that regulate the morphogenesis of the avian forebrain.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Holoprosencefalia , Prosencéfalo , Tretinoína , Animais , Embrião de Galinha , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Holoprosencefalia/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Prosencéfalo/embriologia , Tretinoína/metabolismoRESUMO
Cell migration from the olfactory placode (OP) is a well-known phenomenon wherein various cell types, such as gonadotropin-releasing hormone (GnRH)-producing neurons, migrate toward the telencephalon (TEL) during early embryonic development. However, the spatial relationship between early migratory cells and the forebrain is unclear. We examined the early development of whole-mount chick embryos to observe the three-dimensional spatial relationship among OP-derived migratory neurons, olfactory nerve (ON), and TEL. Migratory neurons that express highly polysialylated neural cell adhesion molecule (PSA-NCAM) emerge from the OP and spread over a relatively wide TEL area at the Hamburger and Hamilton (HH) stage 17. Most migratory neurons form a cellular cord between the olfactory pit and rostral TEL within HH18-20. The earliest axons from the olfactory epithelium (OE) were detected along this neuronal cord using DiI-labeling at HH21. Furthermore, a few PSA-NCAM-positive neurons were dispersed around the cellular cord and over the lateral TEL at HH18. A long cellular cord branch extending to the lateral TEL was transiently observed within HH18-24. These results suggest a novel migratory route of OP-derived neurons during the early developmental stages. Following GFP vector introduction into the OP of HH13-15 embryos, labeled neurons were detected around and within the lateral TEL at HH23 and HH27. At HH36, labeled cells were observed in the rostral-lateral TEL, including the olfactory bulb (OB) region. GFP-labeled and calretinin-positive neurons were detected in the OB, suggesting that early OP-derived neurons enter the forebrain and function as interneurons in the OB.
Assuntos
Neurônios , Bulbo Olfatório , Telencéfalo , Animais , Embrião de Galinha , Axônios , Movimento Celular , Neurônios/metabolismo , Bulbo Olfatório/embriologia , Nervo Olfatório/embriologia , Prosencéfalo/embriologia , Telencéfalo/embriologiaRESUMO
Embryonic stem (ES) cells can undergo many aspects of mammalian embryogenesis in vitro1-5, but their developmental potential is substantially extended by interactions with extraembryonic stem cells, including trophoblast stem (TS) cells, extraembryonic endoderm stem (XEN) cells and inducible XEN (iXEN) cells6-11. Here we assembled stem cell-derived embryos in vitro from mouse ES cells, TS cells and iXEN cells and showed that they recapitulate the development of whole natural mouse embryo in utero up to day 8.5 post-fertilization. Our embryo model displays headfolds with defined forebrain and midbrain regions and develops a beating heart-like structure, a trunk comprising a neural tube and somites, a tail bud containing neuromesodermal progenitors, a gut tube, and primordial germ cells. This complete embryo model develops within an extraembryonic yolk sac that initiates blood island development. Notably, we demonstrate that the neurulating embryo model assembled from Pax6-knockout ES cells aggregated with wild-type TS cells and iXEN cells recapitulates the ventral domain expansion of the neural tube that occurs in natural, ubiquitous Pax6-knockout embryos. Thus, these complete embryoids are a powerful in vitro model for dissecting the roles of diverse cell lineages and genes in development. Our results demonstrate the self-organization ability of ES cells and two types of extraembryonic stem cells to reconstitute mammalian development through and beyond gastrulation to neurulation and early organogenesis.
Assuntos
Embrião de Mamíferos , Gastrulação , Modelos Biológicos , Neurulação , Organogênese , Animais , Linhagem da Célula , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Células-Tronco Embrionárias/citologia , Endoderma/citologia , Endoderma/embriologia , Coração/embriologia , Mesencéfalo/embriologia , Camundongos , Tubo Neural/embriologia , Fator de Transcrição PAX6/deficiência , Fator de Transcrição PAX6/genética , Prosencéfalo/embriologia , Somitos/embriologiaRESUMO
BACKGROUND: Fragile X syndrome, the major cause of inherited intellectual disability among men, is due to deficiency of the synaptic functional regulator FMR1 protein (FMRP), encoded by the FMRP translational regulator 1 (FMR1) gene. FMR1 alternative splicing produces distinct transcripts that may consequently impact FMRP functional roles. In transcripts without exon 14 the translational reading frame is shifted. For deepening current knowledge of the differential expression of Fmr1 exon 14 along the rat nervous system development, we conducted a descriptive study employing quantitative RT-PCR and BLAST of RNA-Seq datasets. RESULTS: We observed in the rat forebrain progressive decline of total Fmr1 mRNA from E11 to P112 albeit an elevation on P3; and exon-14 skipping in E17-E20 with downregulation of the resulting mRNA. We tested if the reduced detection of messages without exon 14 could be explained by nonsense-mediated mRNA decay (NMD) vulnerability, but knocking down UPF1, a major component of this pathway, did not increase their quantities. Conversely, it significantly decreased FMR1 mRNA having exon 13 joined with either exon 14 or exon 15 site A. CONCLUSIONS: The forebrain in the third embryonic week of the rat development is a period with significant skipping of Fmr1 exon 14. This alternative splicing event chronologically precedes a reduction of total Fmr1 mRNA, suggesting that it may be part of combinatorial mechanisms downregulating the gene's expression in the late embryonic period. The decay of FMR1 mRNA without exon 14 should be mediated by a pathway different from NMD. Finally, we provide evidence of FMR1 mRNA stabilization by UPF1, likely depending on FMRP.
Assuntos
Processamento Alternativo , Proteína do X Frágil da Deficiência Intelectual , Prosencéfalo , Processamento Alternativo/genética , Animais , Desenvolvimento Embrionário , Éxons/genética , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Prosencéfalo/embriologia , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Ratos , Transativadores/genética , Transativadores/metabolismoRESUMO
The human cortex contains inhibitory interneurons derived from the medial ganglionic eminence (MGE), a germinal zone in the embryonic ventral forebrain. How this germinal zone generates sufficient interneurons for the human brain remains unclear. We found that the human MGE (hMGE) contains nests of proliferative neuroblasts with ultrastructural and transcriptomic features that distinguish them from other progenitors in the hMGE. When dissociated hMGE cells are transplanted into the neonatal mouse brain, they reform into nests containing proliferating neuroblasts that generate young neurons that migrate extensively into the mouse forebrain and mature into different subtypes of functional interneurons. Together, these results indicate that the nest organization and sustained proliferation of neuroblasts in the hMGE provide a mechanism for the extended production of interneurons for the human forebrain.
Assuntos
Interneurônios/fisiologia , Eminência Mediana/embriologia , Células-Tronco Neurais/fisiologia , Neurogênese , Prosencéfalo/citologia , Animais , Animais Recém-Nascidos , Movimento Celular , Proliferação de Células , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/fisiologia , Perfilação da Expressão Gênica , Idade Gestacional , Humanos , Interneurônios/citologia , Eminência Mediana/citologia , Eminência Mediana/crescimento & desenvolvimento , Camundongos , Células-Tronco Neurais/transplante , Prosencéfalo/embriologia , Prosencéfalo/crescimento & desenvolvimento , Transplante HeterólogoRESUMO
GABA is a major neurotransmitter in the mammalian central nervous system. Glutamate decarboxylase (GAD) synthesizes GABA from glutamate, and two isoforms of GAD, GAD65, and GAD67, are separately encoded by the Gad2 and Gad1 genes, respectively. The phenotypes differ in severity between GAD single isoform-deficient mice and rats. For example, GAD67 deficiency causes cleft palate and/or omphalocele in mice but not in rats. In this study, to further investigate the functional roles of GAD65 and/or GAD67 and to determine the contribution of these isoforms to GABA synthesis during development, we generated various kinds of GAD isoform(s)-deficient rats and characterized their phenotypes. The age of death was different among Gad mutant rat genotypes. In particular, all Gad1-/- ; Gad2-/- rats died at postnatal day 0 and showed little alveolar space in their lungs, suggesting that the cause of their death was respiratory failure. All Gad1-/- ; Gad2-/- rats and 18% of Gad1-/- ; Gad2+/- rats showed cleft palate. In contrast, none of the Gad mutant rats including Gad1-/- ; Gad2-/- rats, showed omphalocele. These results suggest that both rat GAD65 and GAD67 are involved in palate formation, while neither isoform is critical for abdominal wall formation. The GABA content in Gad1-/- ; Gad2-/- rat forebrains and retinas at embryonic day 20 was extremely low, indicating that almost all GABA was synthesized from glutamate by GADs in the perinatal period. The present study shows that Gad mutant rats are a good model for further defining the role of GABA during development.
Assuntos
Glutamato Descarboxilase/deficiência , Palato/embriologia , Prosencéfalo/embriologia , Retina/embriologia , Animais , Glutamato Descarboxilase/metabolismo , Ratos , Ratos MutantesRESUMO
Organizing centers secrete morphogens that specify the emergence of germ layers and the establishment of the body's axes during embryogenesis. While traditional experimental embryology tools have been instrumental in dissecting the molecular aspects of organizers in model systems, they are impractical in human in-vitro model systems to dissect the relationships between signaling and fate along embryonic coordinates. To systematically study human embryonic organizer centers, we devised a collection of optogenetic ePiggyBac vectors to express a photoactivatable Cre-loxP recombinase, that allows the systematic induction of organizer structures by shining blue-light on human embryonic stem cells (hESCs). We used a light stimulus to geometrically confine SHH expression in neuralizing hESCs. This led to the self-organization of mediolateral neural patterns. scRNA-seq analysis established that these structures represent the dorsal-ventral forebrain, at the end of the first month of development. Here, we show that morphogen light-stimulation is a scalable tool that induces self-organizing centers.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Hedgehog/metabolismo , Células-Tronco Embrionárias Humanas/fisiologia , Prosencéfalo/embriologia , Linhagem da Célula/fisiologia , Embriologia/métodos , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Vetores Genéticos/genética , Humanos , Integrases/genética , Luz , Optogenética/métodos , Prosencéfalo/metabolismo , RNA-Seq , Transdução de Sinais/fisiologia , Transdução de Sinais/efeitos da radiação , Análise de Célula ÚnicaRESUMO
Growth arrest-specific 1 (GAS1) acts as a co-receptor to patched 1, promoting sonic hedgehog (SHH) signaling in the developing nervous system. GAS1 mutations in humans and animal models result in forebrain and craniofacial malformations, defects ascribed to a function for GAS1 in SHH signaling during early neurulation. Here, we confirm loss of SHH activity in the forebrain neuroepithelium in GAS1-deficient mice and in induced pluripotent stem cell-derived cell models of human neuroepithelial differentiation. However, our studies document that this defect can be attributed, at least in part, to a novel role for GAS1 in facilitating NOTCH signaling, which is essential to sustain a persistent SHH activity domain in the forebrain neuroepithelium. GAS1 directly binds NOTCH1, enhancing ligand-induced processing of the NOTCH1 intracellular domain, which drives NOTCH pathway activity in the developing forebrain. Our findings identify a unique role for GAS1 in integrating NOTCH and SHH signal reception in neuroepithelial cells, and they suggest that loss of GAS1-dependent NOTCH1 activation contributes to forebrain malformations in individuals carrying GAS1 mutations.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Hedgehog/metabolismo , Prosencéfalo/metabolismo , Receptor Notch1/metabolismo , Animais , Proteínas de Ciclo Celular/deficiência , Diferenciação Celular , Embrião de Mamíferos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/metabolismo , Humanos , Camundongos , Mutação , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Receptor Patched-1/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Prosencéfalo/citologia , Prosencéfalo/embriologia , Transdução de SinaisRESUMO
The members of the IgLON superfamily of cell adhesion molecules facilitate fundamental cellular communication during brain development, maintain functional brain circuitry, and are associated with several neuropsychiatric disorders such as depression, autism, schizophrenia, and intellectual disabilities. Usage of alternative promoter-specific 1a and 1b mRNA isoforms in Lsamp, Opcml, Ntm, and the single promoter of Negr1 in the mouse and human brain has been previously described. To determine the precise spatiotemporal expression dynamics of Lsamp, Opcml, Ntm isoforms, and Negr1, in the developing brain, we generated isoform-specific RNA probes and carried out in situ hybridization in the developing (embryonic, E10.5, E11.5, 13.5, 17; postnatal, P0) and adult mouse brains. We show that promoter-specific expression of IgLONs is established early during pallial development (at E10.5), where it remains throughout its differentiation through adulthood. In the diencephalon, midbrain, and hindbrain, strong expression patterns are initiated a few days later and begin fading after birth, being only faintly expressed during adulthood. Thus, the expression of specific IgLONs in the developing brain may provide the means for regionally specific functionality as well as for specific regional vulnerabilities. The current study will therefore improve the understanding of how IgLON genes are implicated in the development of neuropsychiatric disorders.
Assuntos
Encéfalo/embriologia , Moléculas de Adesão Celular/metabolismo , Regiões Promotoras Genéticas/genética , Animais , Encéfalo/metabolismo , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Hipocampo/embriologia , Hipocampo/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Masculino , Mesencéfalo/embriologia , Mesencéfalo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Prosencéfalo/embriologia , Prosencéfalo/metabolismo , Medula Espinal/embriologia , Medula Espinal/metabolismoRESUMO
5-hydroxymethylcytosine (5hmC) undergoes dynamic changes during mammalian brain development, and its dysregulation is associated with Alzheimer's disease (AD). The dynamics of 5hmC during early human brain development and how they contribute to AD pathologies remain largely unexplored. We generate 5hmC and transcriptome profiles encompassing several developmental time points of healthy forebrain organoids and organoids derived from several familial AD patients. Stage-specific differentially hydroxymethylated regions demonstrate an acquisition or depletion of 5hmC modifications across developmental stages. Additionally, genes concomitantly increasing or decreasing in 5hmC and gene expression are enriched in neurobiological or early developmental processes, respectively. Importantly, our AD organoids corroborate cellular and molecular phenotypes previously observed in human AD brains. 5hmC is significantly altered in developmentally programmed 5hmC intragenic regions in defined fetal histone marks and enhancers in AD organoids. These data suggest a highly coordinated molecular system that may be dysregulated in these early developing AD organoids.
Assuntos
5-Metilcitosina/análogos & derivados , Doença de Alzheimer/genética , Neurogênese/genética , Organoides/embriologia , Prosencéfalo/embriologia , 5-Metilcitosina/metabolismo , Animais , Humanos , CamundongosRESUMO
BACKGROUND: Fetal alcohol syndrome (FAS) due to gestational alcohol exposure represents one of the most common causes of nonheritable lifelong disability worldwide. In vitro and in vivo models have successfully recapitulated multiple facets of the disorder, including morphological and behavioral deficits, but far less is understood regarding the molecular and genetic mechanisms underlying FAS. METHODS: In this study, we utilized an in vitro human pluripotent stem cell-based (hPSC) model of corticogenesis to probe the effects of early, chronic intermittent alcohol exposure on the transcriptome of first trimester-equivalent cortical neurons. RESULTS: We used RNA sequencing of developing hPSC-derived neurons treated for 50 days with 50 mM ethanol and identified a relatively small number of biological pathways significantly altered by alcohol exposure. These included cell-type specification, axon guidance, synaptic function, and regional patterning, with a notable upregulation of WNT signaling-associated transcripts observed in alcohol-exposed cultures relative to alcohol-naïve controls. Importantly, this effect paralleled a shift in gene expression of transcripts associated with regional patterning, such that caudal forebrain-related transcripts were upregulated at the expense of more anterior ones. Results from H9 embryonic stem cells were largely replicated in an induced pluripotent stem cell line (IMR90-4), indicating that these patterning alterations are not cell line-specific. CONCLUSIONS: We found that a major effect of chronic intermittent alcohol on the developing cerebral cortex is an overall imbalance in regionalization, with enrichment of gene expression related to the production of posterodorsal progenitors and a diminution of anteroventral progenitors. This finding parallels behavioral and morphological phenotypes observed in animal models of high-dose prenatal alcohol exposure, as well as patients with FAS.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Depressores do Sistema Nervoso Central/farmacologia , Córtex Cerebral/efeitos dos fármacos , Etanol/farmacologia , Transtornos do Espectro Alcoólico Fetal/genética , Expressão Gênica/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Orientação de Axônios/efeitos dos fármacos , Orientação de Axônios/genética , Diferenciação Celular/genética , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Humanos , Técnicas In Vitro , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Células-Tronco Pluripotentes , Prosencéfalo/efeitos dos fármacos , Prosencéfalo/embriologia , Prosencéfalo/metabolismo , RNA-Seq , Via de Sinalização Wnt/genéticaRESUMO
Although cell lineage information is fundamental to understanding organismal development, very little direct information is available for humans. We performed high-depth (250×) whole-genome sequencing of multiple tissues from three individuals to identify hundreds of somatic single-nucleotide variants (sSNVs). Using these variants as "endogenous barcodes" in single cells, we reconstructed early embryonic cell divisions. Targeted sequencing of clonal sSNVs in different organs (about 25,000×) and in more than 1000 cortical single cells, as well as single-nucleus RNA sequencing and single-nucleus assay for transposase-accessible chromatin sequencing of ~100,000 cortical single cells, demonstrated asymmetric contributions of early progenitors to extraembryonic tissues, distinct germ layers, and organs. Our data suggest onset of gastrulation at an effective progenitor pool of about 170 cells and about 50 to 100 founders for the forebrain. Thus, mosaic mutations provide a permanent record of human embryonic development at very high resolution.
Assuntos
Linhagem da Célula , Gastrulação , Mutação , Células-Tronco Neurais/citologia , Prosencéfalo/citologia , Adolescente , Adulto , Divisão Celular , Células Clonais/citologia , Desenvolvimento Embrionário/genética , Feminino , Gástrula/citologia , Variação Genética , Camadas Germinativas/citologia , Humanos , Masculino , Neurônios/citologia , Organogênese , Polimorfismo de Nucleotídeo Único , Prosencéfalo/embriologia , Análise de Célula Única , Sequenciamento Completo do GenomaRESUMO
The Evf2 long non-coding RNA directs Dlx5/6 ultraconserved enhancer(UCE)-intrachromosomal interactions, regulating genes across a 27 Mb region on chromosome 6 in mouse developing forebrain. Here, we show that Evf2 long-range gene repression occurs through multi-step mechanisms involving the transcription factor Sox2. Evf2 directly interacts with Sox2, antagonizing Sox2 activation of Dlx5/6UCE, and recruits Sox2 to the Dlx5/6eii shadow enhancer and key Dlx5/6UCE interaction sites. Sox2 directly interacts with Dlx1 and Smarca4, as part of the Evf2 ribonucleoprotein complex, forming spherical subnuclear domains (protein pools, PPs). Evf2 targets Sox2 PPs to one long-range repressed target gene (Rbm28), at the expense of another (Akr1b8). Evf2 and Sox2 shift Dlx5/6UCE interactions towards Rbm28, linking Evf2/Sox2 co-regulated topological control and gene repression. We propose a model that distinguishes Evf2 gene repression mechanisms at Rbm28 (Dlx5/6UCE position) and Akr1b8 (limited Sox2 availability). Genome-wide control of RNPs (Sox2, Dlx and Smarca4) shows that co-recruitment influences Sox2 DNA binding. Together, these data suggest that Evf2 organizes a Sox2 PP subnuclear domain and, through Sox2-RNP sequestration and recruitment, regulates chromosome 6 long-range UCE targeting and activity with genome-wide consequences.
Assuntos
Cromossomos de Mamíferos/genética , Regulação da Expressão Gênica no Desenvolvimento , Prosencéfalo/metabolismo , RNA Longo não Codificante/genética , Fatores de Transcrição SOXB1/genética , Animais , DNA Helicases/genética , DNA Helicases/metabolismo , Elementos Facilitadores Genéticos/genética , Imunofluorescência/métodos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hibridização in Situ Fluorescente/métodos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Prosencéfalo/embriologia , Ligação Proteica , RNA Longo não Codificante/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Primary neurulation is the process by which the neural tube, the central nervous system precursor, is formed from the neural plate. Incomplete neural tube closure occurs frequently, yet underlying causes remain poorly understood. Developmental studies in amniotes and amphibians have identified hingepoint and neural fold formation as key morphogenetic events and hallmarks of primary neurulation, the disruption of which causes neural tube defects. In contrast, the mode of neurulation in teleosts has remained highly debated. Teleosts are thought to have evolved a unique mode of neurulation, whereby the neural plate infolds in absence of hingepoints and neural folds, at least in the hindbrain/trunk where it has been studied. Using high-resolution imaging and time-lapse microscopy, we show here the presence of these morphological landmarks in the zebrafish anterior neural plate. These results reveal similarities between neurulation in teleosts and other vertebrates and hence the suitability of zebrafish to understand human neurulation.
Assuntos
Células Epiteliais/fisiologia , Placa Neural/embriologia , Tubo Neural/embriologia , Neurulação , Prosencéfalo/embriologia , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Movimento Celular , Forma Celular , Células Epiteliais/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Morfogênese , Placa Neural/metabolismo , Tubo Neural/metabolismo , Defeitos do Tubo Neural/embriologia , Prosencéfalo/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Imagem com Lapso de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
The developing brain is under the risk of exposure to a multitude of environmental stressors. While perinatal exposure to excessive levels of environmental stress is responsible for a wide spectrum of neurological and psychiatric conditions, the developing brain is equipped with intrinsic cell protection, the mechanisms of which remain unknown. Here we show, using neonatal mouse as a model system, that primary cilia, hair-like protrusions from the neuronal cell body, play an essential role in protecting immature neurons from the negative impacts of exposure to environmental stress. More specifically, we found that primary cilia prevent the degeneration of dendritic arbors upon exposure to alcohol and ketamine, two major cell stressors, by activating cilia-localized insulin-like growth factor 1 receptor and downstream Akt signaling. We also found that activation of this pathway inhibits Caspase-3 activation and caspase-mediated cleavage/fragmentation of cytoskeletal proteins in stress-exposed neurons. These results indicate that primary cilia play an integral role in mitigating adverse impacts of environmental stressors such as drugs on perinatal brain development.