RESUMO
Presomitic mesoderm (PSM) cells are the precursors of the somites, which flank both sides of the neural tube and give rise to the musculo-skeletal system shaping the vertebrate body. WNT and FGF signaling control the formation of both the PSM and the somites and show a graded distribution with highest levels in the posterior PSM. We have used reporters for the mesoderm/PSM control genes T, Tbx6, and Msgn1 to investigate the differentiation of mouse ESCs from the naïve state via EpiSCs to PSM cells. Here we show that the activation of WNT signaling by CHIR99021 (CH) in combination with FGF ligand induces embryo-like PSM at high efficiency. By varying the FGF ligand concentration, the state of PSM cells formed can be altered. High FGF concentration supports posterior PSM formation, whereas low FGF generates anterior/differentiating PSM, in line with in vivo data. Furthermore, the level of Msgn1 expression depends on the FGF ligand concentration. We also show that Activin/Nodal signaling inhibits CH-mediated PSM induction in EpiSCs, without affecting T-expression. Inversely, Activin/Nodal inhibition enhances PSM induction by WNT/high FGF signaling. The ability to generate PSM cells of either posterior or anterior PSM identity with high efficiency in vitro will promote the investigation of the gene regulatory networks controlling the formation of nascent PSM cells and their switch to differentiating/somitic paraxial mesoderm. Stem Cells 2016;34:1790-1800.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Mesoderma/embriologia , Somitos/embriologia , Proteínas Wnt/metabolismo , Ativinas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Ligantes , Mesoderma/citologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Somitos/citologiaRESUMO
BACKGROUND: Mutations in the Lamin A/C (LMNA) gene are responsible for about 6% of all familial dilated cardiomyopathy (DCM) cases which tend to present at a young age and follow a fulminant course. METHODS: We report a 47-year-old DCM patient with severely impaired left ventricular ejection fraction and NYHA functional class IV despite optimal heart failure treatment. Whole-exome sequencing revealed an LMNA E161K missense mutation as the pathogenetic cause for DCM in this patient. We generated a patient-specific LMNA-knock in (LMNA-KI) in vitro model using mES cells. RESULTS: Beta adrenergic stimulation of cardiomyocytes derived from LMNA-KI mES cells resulted in augmented mTOR signaling and increased dysregulation of action potentials, which could be effectively prevented by the mTOR-inhibitor rapamycin. A cardiac biopsy confirmed strong activation of the mTOR-signaling pathway in the patient. An off-label treatment with oral rapamycin was initiated and resulted in an improvement in left ventricular ejection fraction (27.8% to 44.5%), NT-BNP (8120 ng/L to 2210 ng/L) and NYHA functional class. CONCLUSION: We have successfully generated the first in vitro model to recapitulate a patient-specific LMNA E161K mutation which leads to a severe form of DCM. The model may serve as a template for individualized and specific treatment of heart failure.
RESUMO
Esophageal atresia with or without tracheoesophageal fistula (EA/TEF) is the most common congenital malformation of the upper digestive tract. This study represents the first genome-wide association study (GWAS) to identify risk loci for EA/TEF. We used a European case-control sample comprising 764 EA/TEF patients and 5,778 controls and observed genome-wide significant associations at three loci. On chromosome 10q21 within the gene CTNNA3 (p = 2.11 × 10-8; odds ratio [OR] = 3.94; 95% confidence interval [CI], 3.10-5.00), on chromosome 16q24 next to the FOX gene cluster (p = 2.25 × 10-10; OR = 1.47; 95% CI, 1.38-1.55) and on chromosome 17q12 next to the gene HNF1B (p = 3.35 × 10-16; OR = 1.75; 95% CI, 1.64-1.87). We next carried out an esophageal/tracheal transcriptome profiling in rat embryos at four selected embryonic time points. Based on these data and on already published data, the implicated genes at all three GWAS loci are promising candidates for EA/TEF development. We also analyzed the genetic EA/TEF architecture beyond the single marker level, which revealed an estimated single-nucleotide polymorphism (SNP)-based heritability of around 37% ± 14% standard deviation. In addition, we examined the polygenicity of EA/TEF and found that EA/TEF is less polygenic than other complex genetic diseases. In conclusion, the results of our study contribute to a better understanding on the underlying genetic architecture of ET/TEF with the identification of three risk loci and candidate genes.
RESUMO
The WW-and-C2-domain-containing (WWC) protein family is involved in the regulation of cell differentiation, cell proliferation, and organ growth control. As upstream components of the Hippo signaling pathway, WWC proteins activate the Large tumor suppressor (LATS) kinase that in turn phosphorylates Yes-associated protein (YAP) and its paralog Transcriptional coactivator-with-PDZ-binding motif (TAZ) preventing their nuclear import and transcriptional activity. Inhibition of WWC expression leads to downregulation of the Hippo pathway, increased expression of YAP/TAZ target genes and enhanced organ growth. In mice, a ubiquitous Wwc1 knockout (KO) induces a mild neurological phenotype with no impact on embryogenesis or organ growth. In contrast, we could show here that ubiquitous deletion of Wwc2 in mice leads to early embryonic lethality. Wwc2 KO embryos display growth retardation, a disturbed placenta development, impaired vascularization, and finally embryonic death. A whole-transcriptome analysis of embryos lacking Wwc2 revealed a massive deregulation of gene expression with impact on cell fate determination, cell metabolism, and angiogenesis. Consequently, a perinatal, endothelial-specific Wwc2 KO in mice led to disturbed vessel formation and vascular hypersprouting in the retina. In summary, our data elucidate a novel role for Wwc2 as a key regulator in early embryonic development and sprouting angiogenesis in mice.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Desenvolvimento Embrionário/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Diferenciação Celular/fisiologia , Feminino , Via de Sinalização Hippo , Masculino , Camundongos , Camundongos Knockout , Neovascularização Fisiológica/fisiologia , Transdução de SinaisRESUMO
BACKGROUND: Syndromic brain malformations comprise a large group of anomalies with a birth prevalence of about 1 in 1,000 live births. Their etiological factors remain largely unknown. To identify causative mutations, we used whole-exome sequencing (WES) in aborted fetuses and children with syndromic brain malformations in which chromosomal microarray analysis was previously unremarkable. METHODS: WES analysis was applied in eight case-parent trios, six aborted fetuses, and two children. RESULTS: WES identified a novel de novo mutation (p.Gly268Arg) in ACTB (Baraitser-Winter syndrome-1), a homozygous stop mutation (p.R2442*) in ASPM (primary microcephaly type 5), and a novel hemizygous X-chromosomal mutation (p.I250V) in SLC9A6 (X-linked syndromic mentaly retardation, Christianson type). Furthermore, WES identified a de novo mutation (p.Arg1093Gln) in BAZ1A. This mutation was previously reported in only one allele in 121.362 alleles tested (dbSNP build 147). BAZ1A has been associated with neurodevelopmental impairment and dysregulation of several pathways including vitamin D metabolism. Here, serum vitamin-D (25-(OH)D) levels were insufficient and gene expression comparison between the child and her parents identified 27 differentially expressed genes. Of note, 10 out of these 27 genes are associated to cytoskeleton, integrin and synaptic related pathways, pinpointing to the relevance of BAZ1A in neural development. In situ hybridization in mouse embryos between E10.5 and E13.5 detected Baz1a expression in the central and peripheral nervous system. CONCLUSION: In syndromic brain malformations, WES is likely to identify causative mutations when chromosomal microarray analysis is unremarkable. Our findings suggest BAZ1A as a possible new candidate gene.
Assuntos
Actinas/genética , Encéfalo/anormalidades , Sequenciamento do Exoma , Mutação , Trocadores de Sódio-Hidrogênio/genética , Fatores de Transcrição/genética , Animais , Criança , Proteínas Cromossômicas não Histona , Feminino , Humanos , Masculino , CamundongosRESUMO
The mouse t haplotype, a variant 20â cM genomic region on Chromosome 17, harbors 16 embryonic control genes identified by recessive lethal mutations isolated from wild mouse populations. Due to technical constraints so far only one of these, the tw5 lethal, has been cloned and molecularly characterized. Here we report the molecular isolation of the tw18 lethal. Embryos carrying the tw18 lethal die from major gastrulation defects commencing with primitive streak formation at E6.5. We have used transcriptome and marker gene analyses to describe the molecular etiology of the tw18 phenotype. We show that both WNT and Nodal signal transduction are impaired in the mutant epiblast, causing embryonic patterning defects and failure of primitive streak and mesoderm formation. By using a candidate gene approach, gene knockout by homologous recombination and genetic rescue, we have identified the gene causing the tw18 phenotype as Ppp2r1a, encoding the PP2A scaffolding subunit PR65alpha. Our work highlights the importance of phosphatase 2A in embryonic patterning, primitive streak formation, gastrulation, and mesoderm formation downstream of WNT and Nodal signaling.
RESUMO
Colorectal cancer is driven by cooperating oncogenic mutations. In this study, we use organotypic cultures derived from transgenic mice inducibly expressing oncogenic ß-catenin and/or PIK3CAH1047R to follow sequential changes in cancer-related signaling networks, intestinal cell metabolism, and physiology in a three-dimensional environment mimicking tissue architecture. Activation of ß-catenin alone results in the formation of highly clonogenic cells that are nonmotile and prone to undergo apoptosis. In contrast, coexpression of stabilized ß-catenin and PIK3CAH1047R gives rise to intestinal cells that are apoptosis-resistant, proliferative, stem cell-like, and motile. Systematic inhibitor treatments of organoids followed by quantitative phenotyping and phosphoprotein analyses uncover key changes in the signaling network topology of intestinal cells after induction of stabilized ß-catenin and PIK3CAH1047R We find that survival and motility of organoid cells are associated with 4EBP1 and AKT phosphorylation, respectively. Our work defines phenotypes, signaling network states, and vulnerabilities of transgenic intestinal organoids as a novel approach to understanding oncogene activities and guiding the development of targeted therapies.
Assuntos
Transformação Celular Neoplásica/metabolismo , Neoplasias Intestinais/enzimologia , Intestino Delgado/enzimologia , Células-Tronco Neoplásicas/enzimologia , Organoides/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose , Adesão Celular , Proteínas de Ciclo Celular , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Cultivadas , Classe I de Fosfatidilinositol 3-Quinases , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Humanos , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , Intestino Delgado/patologia , Camundongos Transgênicos , Mutação , Células-Tronco Neoplásicas/patologia , Organoides/patologia , Fenótipo , Fosfatidilinositol 3-Quinases/genética , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Fatores de Tempo , Transcriptoma , Transfecção , beta Catenina/genéticaRESUMO
During somitogenesis differential gene expression can be observed for so-called cyclic genes, which display expression changes with a periodicity of 120min in the mouse. In screens to identify novel cyclic genes in murine embryos, Fam181b was predicted to be an oscillating gene in the presomitic mesoderm (psm). This gene, and its closely related paralog Fam181a, belong to the thus far uncharacterized Fam181 gene family. Here we describe the expression of Fam181b and Fam181a during murine embryonic development. In addition, we confirm oscillation of Fam181b in the psm in-phase with targets of, and regulated by, Notch signaling. Fam181b expression in the psm, as well as in the lateral plate mesoderm, was found to be affected by genetic background. We show that Fam181a and b exhibit partially overlapping mRNA expression patterns, and encode for proteins containing highly-conserved motifs, which predominantly localize to the nucleus. A Fam181b loss-of-function model was generated and found to result in no obvious phenotype.
Assuntos
Desenvolvimento Embrionário , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Somitos/crescimento & desenvolvimento , Animais , Relógios Biológicos , Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Camundongos , Família Multigênica , Proteínas do Tecido Nervoso/química , Filogenia , Receptores Notch/metabolismo , Transdução de SinaisRESUMO
Mesoderm formation in the mouse embryo initiates around E6.5 at the primitive streak and continues until the end of axis extension at E12.5. It requires the process of epithelial-to-mesenchymal transition (EMT), wherein cells detach from the epithelium, adopt mesenchymal cell morphology, and gain competence to migrate. It was shown previously that, prior to mesoderm formation, the transcription factor SRF (Serum Response Factor) is essential for the formation of the primitive streak. To elucidate the role of murine Srf in mesoderm formation during axis extension we conditionally inactivated Srf in nascent mesoderm using the T(s)::Cre driver mouse. Defects in mutant embryos became apparent at E8.75 in the heart and in the allantois. From E9.0 onwards body axis elongation was arrested. Using genome-wide expression analysis, combined with SRF occupancy data from ChIP-seq analysis, we identified a set of direct SRF target genes acting in posterior nascent mesoderm which are enriched for transcripts associated with migratory function. We further show that cell migration is impaired in Srf mutant embryos. Thus, the primary role for SRF in the nascent mesoderm during elongation of the embryonic body axis is the activation of a migratory program, which is a prerequisite for axis extension.