RESUMO
The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development.
Assuntos
Movimento Celular , Rastreamento de Células , Células/citologia , Biologia do Desenvolvimento/métodos , Embrião de Mamíferos/citologia , Feto/citologia , Disseminação de Informação , Organogênese , Adulto , Animais , Atlas como Assunto , Técnicas de Cultura de Células , Sobrevivência Celular , Visualização de Dados , Feminino , Humanos , Imageamento Tridimensional , Masculino , Modelos Animais , Organogênese/genética , Organoides/citologia , Células-Tronco/citologiaRESUMO
We review here some of the historical highlights in exploratory studies of the vertebrate embryonic structure known as the neural crest. The study of the molecular properties of the cells that it produces, their migratory capacities and plasticity, and the still-growing list of tissues that depend on their presence for form and function, continue to enrich our understanding of congenital malformations, paediatric cancers and evolutionary biology. Developmental biology has been key to our understanding of the neural crest, starting with the early days of experimental embryology and through to today, when increasingly powerful technologies contribute to further insight into this fascinating vertebrate cell population.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Crista Neural/fisiologia , Animais , Evolução Biológica , Diferenciação Celular , Linhagem da Célula , Movimento Celular , Embrião de Galinha , Coturnix , Biologia do Desenvolvimento , Predisposição Genética para Doença , Humanos , Neoplasias/metabolismoRESUMO
Forty percent of somatotroph tumors harbor recurrent activating GNAS mutations, historically called the gsp oncogene. In gsp-negative somatotroph tumors, GNAS expression itself is highly variable; those with GNAS overexpression most resemble phenotypically those carrying the gsp oncogene. GNAS is monoallelically expressed in the normal pituitary due to methylation-based imprinting. We hypothesize that changes in GNAS imprinting of gsp-negative tumors affect GNAS expression levels and tumorigenesis. We characterized the GNAS locus in two independent somatotroph tumor cohorts: one of 23 tumors previously published (PMID: 31883967) and classified by pan-genomic analysis, and a second with 82 tumors. Multi-omics analysis of the first cohort identified a significant difference between gsp-negative and gsp-positive tumors in the methylation index at the known differentially methylated region (DMR) of the GNAS A/B transcript promoter, which was confirmed in the larger series of 82 tumors. GNAS allelic expression was analyzed using a polymorphic Fok1 cleavage site in 32 heterozygous gsp-negative tumors. GNAS expression was significantly reduced in the 14 tumors with relaxed GNAS imprinting and biallelic expression, compared to 18 tumors with monoallelic expression. Tumors with relaxed GNAS imprinting showed significantly lower SSTR2 and AIP expression levels. Altered A/B DMR methylation was found exclusively in gsp-negative somatotroph tumors. 43% of gsp-negative tumors showed GNAS imprinting relaxation, which correlated with lower GNAS, SSTR2 and AIP expression, indicating lower sensitivity to somatostatin analogues and potentially aggressive behavior.
Assuntos
Cromograninas/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Neoplasias Hipofisárias/genética , Somatotrofos/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Cromograninas/metabolismo , Metilação de DNA , Epigênese Genética , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Regulação Neoplásica da Expressão Gênica , Impressão Genômica , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/patologia , Somatotrofos/patologia , Adulto JovemRESUMO
Mutations in effectors of the hedgehog signaling pathway are responsible for a wide variety of ocular developmental anomalies. These range from massive malformations of the brain and ocular primordia, not always compatible with postnatal life, to subtle but damaging functional effects on specific eye components. This review will concentrate on the effects and effectors of the major vertebrate hedgehog ligand for eye and brain formation, Sonic hedgehog (SHH), in tissues that constitute the eye directly and also in those tissues that exert indirect influence on eye formation. After a brief overview of human eye development, the many roles of the SHH signaling pathway during both early and later morphogenetic processes in the brain and then eye and periocular primordia will be evoked. Some of the unique molecular biology of this pathway in vertebrates, particularly ciliary signal transduction, will also be broached within this developmental cellular context.
Assuntos
Olho/metabolismo , Proteínas Hedgehog/genética , Transdução de Sinais/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , HumanosRESUMO
Ocular developmental anomalies (ODA) such as anophthalmia/microphthalmia (AM) or anterior segment dysgenesis (ASD) have an estimated combined prevalence of 3.7 in 10,000 births. Mutations in SOX2 are the most frequent contributors to severe ODA, yet account for a minority of the genetic drivers. To identify novel ODA loci, we conducted targeted high-throughput sequencing of 407 candidate genes in an initial cohort of 22 sporadic ODA patients. Patched 1 (PTCH1), an inhibitor of sonic hedgehog (SHH) signaling, harbored an enrichment of rare heterozygous variants in comparison to either controls, or to the other candidate genes (four missense and one frameshift); targeted resequencing of PTCH1 in a second cohort of 48 ODA patients identified two additional rare nonsynonymous changes. Using multiple transient models and a CRISPR/Cas9-generated mutant, we show physiologically relevant phenotypes altering SHH signaling and eye development upon abrogation of ptch1 in zebrafish for which in vivo complementation assays using these models showed that all six patient missense mutations affect SHH signaling. Finally, through transcriptomic and ChIP analyses, we show that SOX2 binds to an intronic domain of the PTCH1 locus to regulate PTCH1 expression, findings that were validated both in vitro and in vivo. Together, these results demonstrate that PTCH1 mutations contribute to as much as 10% of ODA, identify the SHH signaling pathway as a novel effector of SOX2 activity during human ocular development, and indicate that ODA is likely the result of overactive SHH signaling in humans harboring mutations in either PTCH1 or SOX2.
Assuntos
Anormalidades do Olho/genética , Anormalidades do Olho/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Receptor Patched-1/genética , Fatores de Transcrição SOXB1/metabolismo , Alelos , Animais , Estudos de Casos e Controles , Modelos Animais de Doenças , Loci Gênicos , Heterozigoto , Humanos , Mutação , Receptor Patched-1/metabolismo , Fenótipo , Análise de Sequência de DNA , Peixe-ZebraRESUMO
Noonan syndrome and related disorders are a group of clinically and genetically heterogeneous conditions caused by mutations in genes of the RAS/MAPK pathway. Noonan syndrome causes multiple congenital anomalies, which are frequently accompanied by hypertrophic cardiomyopathy (HCM). We report here a Tunisian patient with a severe phenotype of Noonan syndrome including neonatal HCM, facial dysmorphism, severe failure to thrive, cutaneous abnormalities, pectus excavatum and severe stunted growth, who died in her eighth month of life. Using whole exome sequencing, we identified a de novo mutation in exon 7 of the RAF1 gene: c.776C > A (p.Ser259Tyr). This mutation affects a highly conserved serine residue, a main mediator of Raf-1 inhibition via phosphorylation. To our knowledge the c.776C > A mutation has been previously reported in only one case with prenatally diagnosed Noonan syndrome. Our study further supports the striking correlation of RAF1 mutations with HCM and highlights the clinical severity of Noonan syndrome associated with a RAF1 p.Ser259Tyr mutation.
Assuntos
Cardiomiopatia Hipertrófica/fisiopatologia , Síndrome de Noonan/fisiopatologia , Proteínas Proto-Oncogênicas c-raf/genética , Cardiomiopatia Hipertrófica/genética , Feminino , Humanos , Lactente , Mutação , Síndrome de Noonan/genética , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , TunísiaRESUMO
Members of the large family of Hox transcription factors are encoded by genes whose tightly regulated expression in development and in space within different embryonic tissues confer positional identity from the neck to the tips of the limbs. Many structures of the face, head, and heart develop from cell populations expressing few or no Hox genes. Hoxb1 is the member of its chromosomal cluster expressed in the most rostral domain during vertebrate development, but never by the multipotent neural crest cell population anterior to the cerebellum. We have developed a novel floxed transgenic mouse line, CAG-Hoxb1,-EGFP (CAG-Hoxb1), which upon recombination by Cre recombinase conditionally induces robust Hoxb1 and eGFP overexpression. When induced within the neural crest lineage, pups die at birth. A variable phenotype develops from E11.5 on, associating frontonasal hypoplasia/aplasia, micrognathia/agnathia, major ocular and forebrain anomalies, and cardiovascular malformations. Neural crest derivatives in the body appear unaffected. Transcription of effectors of developmental signaling pathways (Bmp, Shh, Vegfa) and transcription factors (Pax3, Sox9) is altered in mutants. These outcomes emphasize that repression of Hoxb1, along with other paralog group 1 and 2 Hox genes, is strictly necessary in anterior cephalic NC for craniofacial, visual, auditory, and cardiovascular development.
Assuntos
Anormalidades Craniofaciais/genética , Proteínas de Homeodomínio/fisiologia , Animais , Linhagem da Célula/fisiologia , Movimento Celular , Anormalidades Craniofaciais/embriologia , Expressão Ectópica do Gene/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Homeobox/genética , Cabeça/embriologia , Coração/embriologia , Cardiopatias Congênitas/embriologia , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Transgênicos , Crista Neural/metabolismo , Transdução de SinaisRESUMO
Neuroblastoma is a childhood cancer in which many children still have poor outcomes, emphasising the need to better understand its pathogenesis. Despite recent genome-wide mutation analyses, many primary neuroblastomas do not contain recognizable driver mutations, implicating alternate molecular pathologies such as epigenetic alterations. To discover genes that become epigenetically deregulated during neuroblastoma tumorigenesis, we took the novel approach of comparing neuroblastomas to neural crest precursor cells, using genome-wide DNA methylation analysis. We identified 93 genes that were significantly differentially methylated of which 26 (28%) were hypermethylated and 67 (72%) were hypomethylated. Concentrating on hypermethylated genes to identify candidate tumor suppressor loci, we found the cell engulfment and adhesion factor gene MEGF10 to be epigenetically repressed by DNA hypermethylation or by H3K27/K9 methylation in neuroblastoma cell lines. MEGF10 showed significantly down-regulated expression in neuroblastoma tumor samples; furthermore patients with the lowest-expressing tumors had reduced relapse-free survival. Our functional studies showed that knock-down of MEGF10 expression in neuroblastoma cell lines promoted cell growth, consistent with MEGF10 acting as a clinically relevant, epigenetically deregulated neuroblastoma tumor suppressor gene. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.
Assuntos
Metilação de DNA , Genes Supressores de Tumor , Proteínas de Membrana/genética , Neuroblastoma/genética , Linhagem Celular Tumoral , Criança , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Código das Histonas , HumanosRESUMO
BACKGROUND: Retinoic acid (RA), the bioactive derivative of vitamin A, is essential for vertebrate heart development. Both excess and reduced RA signaling lead to cardiovascular malformations affecting the outflow tract (OFT). To address the cellular mechanisms underlying the effects of RA signaling during OFT morphogenesis, we used transient maternal RA supplementation to rescue the early lethality resulting from inactivation of the murine retinaldehyde dehydrogenase 2 (Raldh2) gene. RESULTS: By embryonic day 13.5, all rescued Raldh2(-/-) hearts exhibit severe, reproducible OFT septation defects, although wild-type and Raldh2(+/-) littermates have normal hearts. Cardiac neural crest cells (cNCC) were present in OFT cushions of Raldh2(-/-) mutant embryos but ectopically located in the periphery of the endocardial cushions, rather than immediately underlying the endocardium. Excess mesenchyme was generated by Raldh2(-/-) mutant endocardium, which displaced cNCC derivatives from their subendocardial, medial position. CONCLUSIONS: RA signaling affects not only cNCC numbers but also their position relative to endocardial mesenchyme during the septation process. Our study shows that inappropriate coordination between the different cell types of the OFT perturbs its morphogenesis and leads to a severe congenital heart defect, persistent truncus arteriosus.
Assuntos
Aldeído Oxirredutases/metabolismo , Linhagem da Célula/fisiologia , Coração/embriologia , Organogênese/fisiologia , Transdução de Sinais/fisiologia , Tretinoína/metabolismo , Aldeído Oxirredutases/genética , Animais , Camundongos , Camundongos KnockoutRESUMO
Neural progenitor proliferation and migration influence brain size during neurogenesis. We report an autosomal recessive microcephaly syndrome cosegregating with a homozygous balanced translocation between chromosomes 3p and 10q, and we show that a position effect at the breakpoint on chromosome 3 silences the eomesodermin transcript (EOMES), also known as T-box-brain2 (TBR2). Together with the expression pattern of EOMES in the developing human brain, our data suggest that EOMES is involved in neuronal division and/or migration. Thus, mutations in genes encoding not only mitotic and apoptotic proteins but also transcription factors may be responsible for malformative microcephaly syndromes.
Assuntos
Agenesia do Corpo Caloso , Inativação Gênica , Homozigoto , Microcefalia/genética , Proteínas com Domínio T/genética , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Cromossomos Humanos Par 10 , Cromossomos Humanos Par 3 , Análise Mutacional de DNA , Humanos , Masculino , Linhagem , Translocação GenéticaRESUMO
A new consensus-based classification of congenital melanocytic nevi (CMN) has recently been proposed. It includes categories for projected adult size (PAS) and location, satellite nevi counts, and morphologic characteristics (color heterogeneity, rugosity, nodularity, and hypertrichosis). The objective of the current study was to test the applicability of the new categorization scheme and to correlate classification outcome with the patient's history of melanoma and neurocutaneous melanocytosis (NCM). Children and adults with CMN attending a patient conference in Dallas, Texas, in 2012 were invited to participate in the study. Anamnestical data were collected using a standardized questionnaire. Two dermatologists performed clinical examinations. Of 45 patients enrolled, 33 had a giant CMN (G1 [>40 cm PAS], n = 13; G2 [>60 cm PAS], n = 20), 12 had an NCM (5 symptomatic, 7 asymptomatic), and 1 had a history of melanoma. CMN size was positively correlated with NCM (p < 0.05). The classification system allowed an easy and detailed phenotypic characterization of each individual CMN. CMN size and morphology were difficult to assess in patients after surgical removal, and the number of satellite nevi at birth or during infancy was not always known. Our report provides practical aids for the application of the newly proposed CMN classification. Prospective evaluation of accurately classified patients in CMN registries will reveal the predictive value of the scheme. The small study sample limits meaningful conclusions regarding the correlation between CMN parameters and the risk of NCM and melanoma.
Assuntos
Nevo Pigmentado , Guias de Prática Clínica como Assunto , Neoplasias Cutâneas , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Adulto Jovem , Melanoma/diagnóstico , Melanoma/epidemiologia , Melanoma/patologia , Melanose/diagnóstico , Melanose/epidemiologia , Melanose/patologia , Síndromes Neurocutâneas/diagnóstico , Síndromes Neurocutâneas/epidemiologia , Síndromes Neurocutâneas/patologia , Nevo Pigmentado/classificação , Nevo Pigmentado/diagnóstico , Nevo Pigmentado/epidemiologia , Nevo Pigmentado/patologia , Exame Físico , Sistema de Registros , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/epidemiologia , Neoplasias Cutâneas/patologia , Inquéritos e QuestionáriosRESUMO
BACKGROUND & AIMS: Short-bowel syndrome usually results from surgical resection of the small intestine for diseases such as intestinal atresias, volvulus, and necrotizing enterocolitis. Patients with congenital short-bowel syndrome (CSBS) are born with a substantial shortening of the small intestine, to a mean length of 50 cm, compared with a normal length at birth of 190-280 cm. They also are born with intestinal malrotation. Because CSBS occurs in many consanguineous families, it is considered to be an autosomal-recessive disorder. We aimed to identify and characterize the genetic factor causing CSBS. METHODS: We performed homozygosity mapping using 610,000 K single-nucleotide polymorphism arrays to analyze the genomes of 5 patients with CSBS. After identifying a gene causing the disease, we determined its expression pattern in human embryos. We also overexpressed forms of the gene product that were and were not associated with CSBS in Chinese Hamster Ovary and T84 cells and generated a zebrafish model of the disease. RESULTS: We identified loss-of-function mutations in Coxsackie- and adenovirus receptor-like membrane protein (CLMP) in CSBS patients. CLMP is a tight-junction-associated protein that is expressed in the intestine of human embryos throughout development. Mutations in CLMP prevented its normal localization to the cell membrane. Knock-down experiments in zebrafish resulted in general developmental defects, including shortening of the intestine and the absence of goblet cells. Because goblet cells are characteristic for the midintestine in zebrafish, which resembles the small intestine in human beings, the zebrafish model mimics CSBS. CONCLUSIONS: Loss-of-function mutations in CLMP cause CSBS in human beings, likely by interfering with tight-junction formation, which disrupts intestinal development. Furthermore, we developed a zebrafish model of CSBS.
Assuntos
Intestino Delgado/anormalidades , Mutação de Sentido Incorreto , Receptores Virais/genética , Síndrome do Intestino Curto/genética , Adolescente , Adulto , Animais , Células CHO , Criança , Pré-Escolar , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Cricetinae , Cricetulus , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença , Heterozigoto , Homozigoto , Humanos , Lactente , Recém-Nascido , Intestino Delgado/metabolismo , Masculino , Morfogênese , Fenótipo , Polimorfismo de Nucleotídeo Único , Receptores Virais/metabolismo , Síndrome do Intestino Curto/embriologia , Síndrome do Intestino Curto/metabolismo , Síndrome do Intestino Curto/patologia , Transfecção , Adulto Jovem , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
BACKGROUND: Otocephaly or dysgnathia complex is characterised by mandibular hypoplasia/agenesis, ear anomalies, microstomia, and microglossia; the molecular basis of this developmental defect is largely unknown in humans. METHODS AND RESULTS: This study reports a large family in which two cousins with micro/anophthalmia each gave birth to at least one child with otocephaly, suggesting a genetic relationship between anophthalmia and otocephaly. OTX2, a known microphthalmia locus, was screened in this family and a frameshifting mutation was found. The study subsequently identified in one unrelated otocephalic patient a sporadic OTX2 mutation. Because OTX2 mutations may not be sufficient to cause otocephaly, the study assayed the potential of otx2 to modify craniofacial phenotypes in the context of known otocephaly gene suppression in vivo. It was found that otx2 can interact genetically with pgap1, prrx1, and msx1 to exacerbate mandibular and midline defects during zebrafish development. However, sequencing of these loci in the OTX2-positive families did not unearth likely pathogenic lesions, suggesting further genetic heterogeneity and complexity. CONCLUSION: Identification of OTX2 involvement in otocephaly/dysgnathia in humans, even if loss of function mutations at this locus does not sufficiently explain the complex anatomical defects of these patients, suggests the requirement for a second genetic hit. Consistent with this notion, trans suppression of otx2 and other developmentally related genes recapitulate aspects of the otocephaly phenotype in zebrafish. This study highlights the combined utility of genetics and functional approaches to dissect both the regulatory pathways that govern craniofacial development and the genetics of this disease group.
Assuntos
Holoprosencefalia/genética , Anormalidades Maxilomandibulares/genética , Fatores de Transcrição Otx/genética , Animais , Sequência de Bases , Modelos Animais de Doenças , Embrião não Mamífero/anormalidades , Embrião não Mamífero/patologia , Feminino , Holoprosencefalia/patologia , Humanos , Anormalidades Maxilomandibulares/patologia , Dados de Sequência Molecular , Linhagem , Análise de Sequência de DNA , Peixe-ZebraRESUMO
Congenital central hypoventilation syndrome (CCHS or Ondine's curse; OMIM 209880) is a life-threatening disorder involving an impaired ventilatory response to hypercarbia and hypoxemia. This core phenotype is associated with lower-penetrance anomalies of the autonomic nervous system (ANS) including Hirschsprung disease and tumors of neural-crest derivatives such as ganglioneuromas and neuroblastomas. In mice, the development of ANS reflex circuits is dependent on the paired-like homeobox gene Phox2b. Thus, we regarded its human ortholog, PHOX2B, as a candidate gene in CCHS. We found heterozygous de novo mutations in PHOX2B in 18 of 29 individuals with CCHS. Most mutations consisted of 5-9 alanine expansions within a 20-residue polyalanine tract probably resulting from non-homologous recombination. We show that PHOX2B is expressed in both the central and the peripheral ANS during human embryonic development. Our data support an essential role of PHOX2B in the normal patterning of the autonomous ventilation system and, more generally, of the ANS in humans.
Assuntos
Mutação da Fase de Leitura , Proteínas de Homeodomínio/genética , Peptídeos , Apneia do Sono Tipo Central/genética , Fatores de Transcrição/genética , Alanina/genética , Tronco Encefálico/metabolismo , Heterozigoto , Humanos , Mutação , Sistema Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fenótipo , Recombinação GenéticaRESUMO
Metastatic melanoma develops once transformed melanocytic cells begin to de-differentiate into migratory and invasive melanoma cells with neural crest cell (NCC)-like and epithelial-to-mesenchymal transition (EMT)-like features. However, it is still unclear how transformed melanocytes assume a metastatic melanoma cell state. Here, we define DNA methylation changes that accompany metastatic progression in melanoma patients and discover Nuclear Receptor Subfamily 2 Group F, Member 2 - isoform 2 (NR2F2-Iso2) as an epigenetically regulated metastasis driver. NR2F2-Iso2 is transcribed from an alternative transcriptional start site (TSS) and it is truncated at the N-terminal end which encodes the NR2F2 DNA-binding domain. We find that NR2F2-Iso2 expression is turned off by DNA methylation when NCCs differentiate into melanocytes. Conversely, this process is reversed during metastatic melanoma progression, when NR2F2-Iso2 becomes increasingly hypomethylated and re-expressed. Our functional and molecular studies suggest that NR2F2-Iso2 drives metastatic melanoma progression by modulating the activity of full-length NR2F2 (Isoform 1) over EMT- and NCC-associated target genes. Our findings indicate that DNA methylation changes play a crucial role during metastatic melanoma progression, and their control of NR2F2 activity allows transformed melanocytes to acquire NCC-like and EMT-like features. This epigenetically regulated transcriptional plasticity facilitates cell state transitions and metastatic spread.
Assuntos
Melanoma , Neoplasias Cutâneas , Humanos , Linhagem Celular Tumoral , Melanoma/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Transição Epitelial-Mesenquimal/genética , Epigênese Genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator II de Transcrição COUP/metabolismoRESUMO
FTO is a nuclear protein belonging to the AlkB-related non-haem iron- and 2-oxoglutarate-dependent dioxygenase family. Although polymorphisms within the first intron of the FTO gene have been associated with obesity, the physiological role of FTO remains unknown. Here we show that a R316Q mutation, inactivating FTO enzymatic activity, is responsible for an autosomal-recessive lethal syndrome. Cultured skin fibroblasts from affected subjects showed impaired proliferation and accelerated senescence. These findings indicate that FTO is essential for normal development of the central nervous and cardiovascular systems in human and establish that a mutation in a human member of the AlkB-related dioxygenase family results in a severe polymalformation syndrome.
Assuntos
Anormalidades Múltiplas/genética , Predisposição Genética para Doença , Transtornos do Crescimento/genética , Mutação , Proteínas/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Linhagem , Alinhamento de SequênciaRESUMO
BACKGROUND: Neural tube defects (NTDs) are common human birth defects with a complex etiology. To develop a comprehensive knowledge of the genes expressed during normal neurulation, we established transcriptomes from human neural tube fragments during and after neurulation using long Serial Analysis of Gene Expression (long-SAGE). METHODS: Rostral and caudal neural tubes were dissected from normal human embryos aged between 26 and 32 days of gestation. Tissues from the same region and Carnegie stage were pooled (n ≥ 4) and total RNA extracted to construct four long-SAGE libraries. Tags were mapped using the UniGene Homo sapiens 17 bp tag-to-gene best mapping set. Differentially expressed genes were identified by chi-square or Fisher's exact test, and validation was performed for a subset of those transcripts using in situ hybridization. In silico analyses were performed with BinGO and EXPANDER. RESULTS: We observed most genes to be similarly regulated in rostral and caudal regions, but expression profiles differed during and after closure. In silico analysis found similar enrichments in both regions for biologic process terms, transcription factor binding and miRNA target motifs. Twelve genes potentially expressing alternate isoforms by region or developmental stage, and the microRNAs miR-339-5p, miR-141/200a, miR-23ab, and miR-129/129-5p are among several potential candidates identified here for future research. CONCLUSIONS: Time appears to influence gene expression in the developing central nervous system more than location. These data provide a novel complement to traditional strategies of identifying genes associated with human NTDs and offer unique insight into the genes associated with normal human neurulation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Tubo Neural/metabolismo , Neurulação/genética , RNA Mensageiro/genética , Aborto Legal , Mapeamento Cromossômico , Simulação por Computador , Embrião de Mamíferos , Feminino , Perfilação da Expressão Gênica , Biblioteca Genômica , Humanos , Hibridização In Situ , Modelos Genéticos , Tubo Neural/citologia , Defeitos do Tubo Neural/genética , Defeitos do Tubo Neural/metabolismo , Defeitos do Tubo Neural/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Fatores de Tempo , Transcrição GênicaRESUMO
Hirschsprung disease (HSCR) is a common, multigenic neurocristopathy characterized by incomplete innervation along a variable length of the gut. The pivotal gene in isolated HSCR cases, either sporadic or familial, is RET. HSCR also presents in various syndromes, including Shah-Waardenburg syndrome (WS), Down (DS), and Bardet-Biedl (BBS). Here, we report 3 families with BBS and HSCR with concomitant mutations in BBS genes and regulatory RET elements, whose functionality is tested in physiologically relevant assays. Our data suggest that BBS mutations can potentiate HSCR predisposing RET alleles, which by themselves are insufficient to cause disease. We also demonstrate that these genes interact genetically in vivo to modulate gut innervation, and that this interaction likely occurs through complementary, yet independent, pathways that converge on the same biological process.
Assuntos
Epistasia Genética , Doença de Hirschsprung/genética , Mutação , Proteínas/genética , Proteínas Proto-Oncogênicas c-ret/genética , Estômago/inervação , Alelos , Citoplasma/metabolismo , Elementos Facilitadores Genéticos , Saúde da Família , Feminino , Genótipo , Humanos , Masculino , Proteínas Associadas aos Microtúbulos , LinhagemRESUMO
The MAPK/ERK pathway regulates a variety of physiological cellular functions, including cell proliferation and survival. It is abnormally activated in many types of human cancers in response to driver mutations in regulators of this pathway that trigger tumor initiation. The early steps of oncogenic progression downstream of ERK overactivation are poorly understood due to a lack of appropriate models. We show here that ERK1/2 overactivation in the trunk neural tube of the chicken embryo through expression of a constitutively active form of the upstream kinase MEK1 (MEK1ca), rapidly provokes a profound change in the transcriptional signature of developing spinal cord cells. These changes are concordant with a previously established role of the tyrosine kinase receptor ligand FGF8 acting via the ERK1/2 effectors to maintain an undifferentiated state. Furthermore, we show that MEK1ca-transfected spinal cord cells lose neuronal identity, retain caudal markers, and ectopically express potential effector oncogenes, such as AQP1. MEK1ca expression in the developing spinal cord from the chicken embryo is thus a tractable in vivo model to identify the mechanisms fostering neoplasia and malignancy in ERK-induced tumorigenesis of neural origins.
Assuntos
Transformação Celular Neoplásica/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Transdução de Sinais , Medula Espinal/metabolismo , Animais , Galinhas , Modelos Animais de Doenças , Humanos , Medula Espinal/patologiaRESUMO
Recurrent missense mutations of the PIK3CA oncogene are among the most frequent drivers of human cancers. These often lead to constitutive activation of its product p110α, a phosphatidylinositol 3-kinase (PI3K) catalytic subunit. In addition to causing a broad range of cancers, the H1047R mutation is also found in affected tissues of a distinct set of congenital tumors and malformations. Collectively termed PIK3CA-related disorders (PRDs), these lead to overgrowth of brain, adipose, connective and musculoskeletal tissues and/or blood and lymphatic vessel components. Vascular malformations are frequently observed in PRD, due to cell-autonomous activation of PI3K signaling within endothelial cells. These, like most muscle, connective tissue and bone, are derived from the embryonic mesoderm. However, important organ systems affected in PRDs are neuroectodermal derivatives. To further examine their development, we drove the most common post-zygotic activating mutation of Pik3ca in neural crest and related embryonic lineages. Outcomes included macrocephaly, cleft secondary palate and more subtle skull anomalies. Surprisingly, Pik3ca-mutant subpopulations of neural crest origin were also associated with widespread cephalic vascular anomalies. Mesectodermal neural crest is a major source of non-endothelial connective tissue in the head, but not the body. To examine the response of vascular connective tissues of the body to constitutive Pik3ca activity during development, we expressed the mutation by way of an Egr2 (Krox20) Cre driver. Lineage tracing led us to observe new lineages that had normally once expressed Krox20 and that may be co-opted in pathogenesis, including vascular pericytes and perimysial fibroblasts. Finally, Schwann cell precursors having transcribed either Krox20 or Sox10 and induced to express constitutively active PI3K were associated with vascular and other tumors. These murine phenotypes may aid discovery of new candidate human PRDs affecting craniofacial and vascular smooth muscle development as well as the reciprocal paracrine signaling mechanisms leading to tissue overgrowth.