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1.
Gene ; 730: 144318, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-31917231

RESUMO

Although the chicken embryo has been a classical model for developmental studies, the lack of straightforward technologies for chicken transgenesis limited the usefulness of this animal model. Here, we assessed electroporation and lipofection approaches for in ovo transfection of Sleeping Beauty transposon system in stage X-XII chicken embryos. Electroporation of chicken embryos could transfect the trophectodermal cells. Then, a mixture of transposon lipoplexes and high concentrated carboxymethylcellulose (HCC) solution was injected into the subgerminal cavity of day 0 embryos. The lipoplex-HCC mixture substantially increased the number of trophectodermal cells expressing the reporter. Importantly, the fluorescent reporter was detected in cells inside of the embryos as well as circulation cells in the bloodstream during days 3-4 of incubation. This study provided evidence for direct in ovo transfection of early chicken embryos, though the long-term outcome of this approach warrants further studies.


Assuntos
Eletroporação/métodos , Transfecção/métodos , Transposases/genética , Animais , Animais Geneticamente Modificados , Carboximetilcelulose Sódica , Embrião de Galinha , Galinhas/genética , Elementos de DNA Transponíveis/genética , Embrião de Mamíferos/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Transferência de Genes
2.
Nature ; 574(7777): 249-253, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31578523

RESUMO

The integrity of the mammalian epidermis depends on a balance of proliferation and differentiation in the resident population of stem cells1. The kinase RIPK4 and the transcription factor IRF6 are mutated in severe developmental syndromes in humans, and mice lacking these genes display epidermal hyperproliferation and soft-tissue fusions that result in neonatal lethality2-5. Our understanding of how these genes control epidermal differentiation is incomplete. Here we show that the role of RIPK4 in mouse development requires its kinase activity; that RIPK4 and IRF6 expressed in the epidermis regulate the same biological processes; and that the phosphorylation of IRF6 at Ser413 and Ser424 primes IRF6 for activation. Using RNA sequencing (RNA-seq), histone chromatin immunoprecipitation followed by sequencing (ChIP-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) of skin in wild-type and IRF6-deficient mouse embryos, we define the transcriptional programs that are regulated by IRF6 during epidermal differentiation. IRF6 was enriched at bivalent promoters, and IRF6 deficiency caused defective expression of genes that are involved in the metabolism of lipids and the formation of tight junctions. Accordingly, the lipid composition of the stratum corneum of Irf6-/- skin was abnormal, culminating in a severe defect in the function of the epidermal barrier. Collectively, our results explain how RIPK4 and IRF6 function to ensure the integrity of the epidermis and provide mechanistic insights into why developmental syndromes that are characterized by orofacial, skin and genital abnormalities result when this axis goes awry.


Assuntos
Diferenciação Celular , Células Epidérmicas/citologia , Epiderme/fisiologia , Fatores Reguladores de Interferon/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Anormalidades Múltiplas/genética , Animais , Fenda Labial/genética , Fissura Palatina/genética , Cistos/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Células Epidérmicas/metabolismo , Epiderme/embriologia , Anormalidades do Olho/genética , Feminino , Dedos/anormalidades , Regulação da Expressão Gênica , Fatores Reguladores de Interferon/deficiência , Fatores Reguladores de Interferon/genética , Joelho/anormalidades , Articulação do Joelho/anormalidades , Lábio/anormalidades , Metabolismo dos Lipídeos/genética , Deformidades Congênitas das Extremidades Inferiores/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Fosfosserina/metabolismo , Proteínas Serina-Treonina Quinases/genética , Sindactilia/genética , Anormalidades Urogenitais/genética
3.
Nat Commun ; 10(1): 4834, 2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31645568

RESUMO

Tetraploidisation is considered a common event in the evolution of chromosomal instability (CIN) in cancer cells. The current model for how tetraploidy drives CIN in mammalian cells is that a doubling of the number of centrioles that accompany the genome doubling event leads to multipolar spindle formation and chromosome segregation errors. By exploiting the unusual scenario of mouse blastomeres, which lack centrioles until the ~64-cell stage, we show that tetraploidy can drive CIN by an entirely distinct mechanism. Tetraploid blastomeres assemble bipolar spindles dictated by microtubule organising centres, and multipolar spindles are rare. Rather, kinetochore-microtubule turnover is altered, leading to microtubule attachment defects and anaphase chromosome segregation errors. The resulting blastomeres become chromosomally unstable and exhibit a dramatic increase in whole chromosome aneuploidies. Our results thus reveal an unexpected mechanism by which tetraploidy drives CIN, in which the acquisition of chromosomally-unstable microtubule dynamics contributes to chromosome segregation errors following tetraploidisation.


Assuntos
Anáfase , Blastômeros/metabolismo , Instabilidade Cromossômica/genética , Segregação de Cromossomos , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Tetraploidia , Animais , Centríolos , Embrião de Mamíferos/embriologia , Camundongos , Centro Organizador dos Microtúbulos/metabolismo , Mitose , Neoplasias/genética , Fuso Acromático
4.
Nat Commun ; 10(1): 4749, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31628347

RESUMO

Trophectoderm (TE) lineage development is pivotal for proper implantation, placentation, and healthy pregnancy. However, only a few TE-specific transcription factors (TFs) have been systematically characterized, hindering our understanding of the process. To elucidate regulatory mechanisms underlying TE development, here we map super-enhancers (SEs) in trophoblast stem cells (TSCs) as a model. We find both prominent TE-specific master TFs (Cdx2, Gata3, and Tead4), and >150 TFs that had not been previously implicated in TE lineage, that are SE-associated. Mapping targets of 27 SE-predicted TFs reveals a highly intertwined transcriptional regulatory circuitry. Intriguingly, SE-predicted TFs show 4 distinct expression patterns with dynamic alterations of their targets during TSC differentiation. Furthermore, depletion of a subset of TFs results in dysregulation of the markers for specialized cell types in placenta, suggesting a role during TE differentiation. Collectively, we characterize an expanded TE-specific regulatory network, providing a framework for understanding TE lineage development and placentation.


Assuntos
Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Trofoblastos/metabolismo , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Camundongos , Placentação/genética , Gravidez , Fatores de Transcrição/genética , Trofoblastos/citologia
6.
Nat Commun ; 10(1): 3557, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391456

RESUMO

Mammalian embryos change shape dramatically upon implantation. The cellular and molecular mechanism underlying this transition are largely unknown. Here, we show that this transition is directed by cross talk between the embryonic epiblast and the first extra-embryonic tissue, the trophectoderm. Specifically, we show via visualisation of a Cdx2-GFP reporter line and pharmacologically mediated loss and gain of function experiments that the epiblast provides FGF signal that results in differential fate acquisition in the multipotent trophectoderm leading to the formation of a tissue boundary within this tissue. The trophectoderm boundary becomes essential for expansion of the tissue into a multi-layered epithelium. Folding of this multi-layered trophectoderm induces spreading of the second extra-embryonic tissue, the primitive endoderm. Together, these events remodel the pre-implantation embryo into its post-implantation cylindrical shape. Our findings uncover how communication between embryonic and extra-embryonic tissues provides positional cues to drive shape changes in mammalian development during implantation.


Assuntos
Implantação do Embrião/fisiologia , Embrião de Mamíferos/embriologia , Camadas Germinativas/embriologia , Morfogênese/fisiologia , Trofoblastos/fisiologia , Animais , Embrião de Mamíferos/diagnóstico por imagem , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Camadas Germinativas/diagnóstico por imagem , Camadas Germinativas/metabolismo , Ligantes , Masculino , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Trofoblastos/metabolismo
7.
Mol Med Rep ; 20(4): 3326-3336, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432193

RESUMO

The aim of the present study was to determine the association between maternal metabolism and development of the fetal palate, and to suggest a potential non­invasive prenatal diagnostic method for fetal cleft palate (CP). Dexamethasone (DXM) was used to create a CP mouse model. A 9.4­Tesla (T) magnetic resonance spectroscopy (MRS) imager was used to measure an array of metabolites in the maternal serum, placental tissue, amniotic fluid and fetal palates. Multivariate statistical analysis was performed using SIMCA­P 14.1 software. Following DXM treatment, variations were detected in multiple metabolites in the female mice and their fetuses based on 9.4T MRS. It was indicated that in the experimental group during CP formation, leucine, valine, creatine, acetate and citrate levels in the palatal tissue were lower, whereas lactate, alanine, proline/inositol and glutamate­containing metabolite levels were higher, compared with the levels in the control group. In placental tissue and amniotic fluid, succinate and choline levels were lower in the experimental group. The relative concentrations of cholesterol and lipids in palatal tissues from mice treated with DXM were higher compared with the concentrations in tissues from mice in the control group, with the exception of (CH2)n lipids. In the placental tissue, the alteration in cholesterol level exhibited the opposite trend. Lipid levels for the different lipid forms varied and most of them were unsaturated lipids.


Assuntos
Fissura Palatina , Dexametasona/efeitos adversos , Embrião de Mamíferos , Desenvolvimento Embrionário/efeitos dos fármacos , Animais , Fissura Palatina/induzido quimicamente , Fissura Palatina/embriologia , Fissura Palatina/metabolismo , Fissura Palatina/patologia , Dexametasona/farmacologia , Modelos Animais de Doenças , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Feminino , Espectroscopia de Ressonância Magnética , Camundongos
8.
BMC Dev Biol ; 19(1): 13, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31272387

RESUMO

BACKGROUND: The segregation of the hypoblast and the emergence of the pluripotent epiblast mark the final stages of blastocyst formation in mammalian embryos. In bovine embryos the formation of the hypoblast has been partially studied, and evidence shows that MEK signalling plays a limited role in the segregation of this lineage. Here we explored the role of different signalling pathways during lineage segregation in the bovine embryo using immunofluorescence analysis of NANOG and SOX17 as readouts of epiblast and hypoblast, respectively. RESULTS: We show that SOX17 starts to be expressed in 16-32-cell stage embryos, whereas NANOG is first detected from 8-cell stage. SOX17 is first co-expressed with NANOG, but these markers become mutually exclusive by the late blastocyst stage. By assessing the expression kinetics of NANOG/SOX17 we show that inhibition of MEK signalling can eliminate SOX17 expression in bovine blastocysts, without altering NANOG expression. Modulation of WNT, PKC and LIF did not affect NANOG expression in the epiblast when used in combination with the ERK inhibitor. CONCLUSIONS: This study shows that SOX17 can be used as a reliable early marker of hypoblast in the bovine, and based on its expression profile we show that the hypoblast segregates in day 7 blastocysts. Furthermore, SOX17 expression is abolished using 1 µM of PD0325901, without affecting the NANOG population in the epiblast. Modulation of WNT, PKC and LIF are not sufficient to support enhanced NANOG expression in the epiblast when combined with ERK inhibitor, indicating that additional signalling pathways should be examined to determine their potential roles in epiblast expansion.


Assuntos
Blastocisto/citologia , Embrião de Mamíferos/embriologia , Camadas Germinativas/embriologia , Proteína Homeobox Nanog/metabolismo , Fatores de Transcrição SOXF/metabolismo , Animais , Benzamidas/farmacologia , Bovinos , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Camadas Germinativas/citologia , Fator Inibidor de Leucemia/biossíntese , Proteína Homeobox Nanog/genética , Proteína Quinase C/biossíntese , Fatores de Transcrição SOXF/genética , Transdução de Sinais/fisiologia , Proteína Wnt1/biossíntese
9.
Reprod Biol Endocrinol ; 17(1): 54, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31291946

RESUMO

BACKGROUND: Cell-free mitochondrial DNA (cf-mtDNA) in body fluids has attracted much attention for the purpose of monitoring disease because of the clinical advantages. This study investigated whether the cf-mtDNA content in human follicular fluid samples was associated with oocyte and embryo developmental competence. METHODS: We collected 225 individual follicular fluid samples from 92 patients undergoing conventional in vitro fertilization (n = 53) or intracytoplasmic sperm injection (n = 39). cf-mtDNA and cell-free nuclear DNA (cf-nDNA) were measured using real-time quantitative PCR for the ND1 and ß-globin genes. Multivariate logistic regression and linear regression were used to analyze data. RESULTS: The relative cf-mtDNA content (cf-ND1/cf-ß-globin ratio) in follicular fluid was significantly lower in the group showing blastocyst development than in the non-blastocyst group (P = 0.030). Additionally, the relative cf-mtDNA content was significantly and positively correlated with the age of the female patient (P = 0.009), while the relative cf-mtDNA content for older women (≥38 years old) with anti-Müllerian hormone (AMH) ≤1.1 ng/ml was significantly higher than in those with AMH > 1.1 ng/ml (P <0.05). The cf-nDNA content was significantly positively correlated with the antral follicle count (P = 0.012), and significantly negatively correlated with both the number of days of stimulation and the total dose of gonadotropin administration (P = 0.039 and P = 0.015, respectively). Neither cf-mtDNA nor cf-nDNA levels in follicular fluid were associated with oocyte maturation, fertilization, or Day 3 embryo morphological scoring. CONCLUSIONS: The relative cf-mtDNA content in human follicular fluid was negatively correlated with blastulation and positively correlated with the patient age, indicating that it is a promising bio-marker to evaluate oocyte developmental competence.


Assuntos
Biomarcadores/metabolismo , Blastocisto/metabolismo , Ácidos Nucleicos Livres/metabolismo , DNA Mitocondrial/metabolismo , Líquido Folicular/metabolismo , Técnicas de Reprodução Assistida , Adulto , Blastocisto/citologia , Blastocisto/fisiologia , Ácidos Nucleicos Livres/genética , DNA Mitocondrial/genética , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Feminino , Humanos , Pessoa de Meia-Idade , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Gravidez , Adulto Jovem
10.
Zygote ; 27(3): 111-117, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31182179

RESUMO

SummaryIntraspecific and interspecific cloning via somatic cell nuclear transfer (iSCNT) is a biotechnique with great possibilities for wild mammals because it allows the maintenance of biodiversity by recovering species, nuclear reprogramming for the production of pluripotency-induced cells, and studies related to embryonic development. Nevertheless, many areas in cloning, especially those associated with wild mammals, are still in question because of the difficulty in obtaining cytoplasmic donor cells (or cytoplasts). Conversely, donor cell nuclei (or karyoplasts) are widely obtained from the skin of living or post-mortem individuals and often maintained in somatic cell banks. Moreover, the creation of karyoplast-cytoplast complexes by fusion followed by activation and embryo development is one of the most difficult steps that requires further clarification to avoid genetic failures. Although difficult, cloning different species, such as wild carnivores and ungulates, can be successful via iSCNT with embryo development and the birth of offspring. Thus, novel research in the area that contributes to the conservation of biodiversity and knowledge of the physiology of species continues. The present review presents the failures and successes that occurred with the application of the technique in wild mammals, with the goal of helping future work on cloning via iSCNT.


Assuntos
Clonagem de Organismos/métodos , Transferência Embrionária/métodos , Embrião de Mamíferos/embriologia , Técnicas de Transferência Nuclear , Animais , Núcleo Celular/metabolismo , Embrião de Mamíferos/citologia , Feminino , Mamíferos/classificação , Mamíferos/embriologia , Oócitos/citologia , Oócitos/metabolismo , Gravidez
11.
Trop Anim Health Prod ; 51(8): 2641-2644, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31222711

RESUMO

The objective of the study was to evaluate embryo production in middle-aged and mature Bos taurus × Bos indicus cows induced to multiple ovulation (MO) in a tropical environment. Twenty-eight cows were assigned into two groups: (1) middle-aged cows = 4-6 years old (n = 13), and (2) mature cows = 8-12 years old (n = 15). All donors received the same MO protocol with follicle-stimulating hormone in decreasing dose during 4 days and two artificial insemination services. Total numbers of corpora lutea at embryo collection, structures collected, and viable embryos obtained, as well as recovery rate, were higher in middle-aged cows compared with mature cows (P < 0.05). A total number of degenerate embryos and unfertilized oocytes, as well as viability rate, were similar in both groups (P > 0.05). In conclusion, the mature cows responded to the MO treatment, but the average of viable embryos recovered per donor was lower than in middle-aged cows. Therefore, the inclusion of cows ≥ 8 years old as donors in MO programs in tropical environments should be avoided.


Assuntos
Bovinos/fisiologia , Embrião de Mamíferos/embriologia , Hormônio Foliculoestimulante/farmacologia , Inseminação Artificial/veterinária , Indução da Ovulação/veterinária , Ovulação/efeitos dos fármacos , Fatores Etários , Animais , Feminino , Hibridização Genética , México
12.
Exp Anim ; 68(4): 499-509, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31189761

RESUMO

Knockout mouse models are commonly used in developmental biology to investigate the functions of specific genes, and the knowledge obtained in such models has yielded insights into the molecular mechanisms underlying developmental processes. Gastrulation is the most dynamic process in embryogenesis during which differentiation into three germ layers occurs. However, the functions of genes involved in gastrulation are not completely understood. One major reason for this is the technical difficulty of embryo analysis to understand germ layer location. We have generated three reporter mouse strains in which the germ layers are distinguished by different fluorescent reporters. Using CRISPR/Cas9 genome editing in mouse zygotes, the fluorescent reporter genes, EGFP, tdTomato, and TagBFP including 2A peptide sequences were knocked into the appropriate sites before the stop codon of the Sox17 (endoderm marker), Otx2 (ectoderm marker), and T (mesoderm marker) genes, respectively. Founder mice were successfully generated in the Sox17-2A-EGFP, Otx2-2A-tdTomato, and T-2A-TagBFP knockin reporter strains. Further, homozygous knockin mice of all strains appeared morphologically normal and were fertile. On stereomicroscopic analysis, fluorescent signals were detected in a germ layer-specific manner from heterozygous embryos at embryonic day (E) 6.5-8.5 in all strains, and were immunohistochemically demonstrated to match their respective germ layer-specific marker protein at E7.5. Taken together, these observations suggest that the Sox17-2A-EGFP, Otx2-2A-tdTomato, and T-2A-TagBFP knockin reporter mice may be useful for comprehensive analysis of gene function in germ layer formation.


Assuntos
Diferenciação Celular , Embrião de Mamíferos/embriologia , Técnicas de Introdução de Genes/métodos , Genes Reporter , Camadas Germinativas/embriologia , Animais , Proteínas Luminescentes/administração & dosagem , Camundongos , Camundongos Transgênicos
13.
Nature ; 571(7763): 112-116, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31189957

RESUMO

Size control is fundamental in tissue development and homeostasis1,2. Although the role of cell proliferation in these processes has been widely studied, the mechanisms that control embryo size-and how these mechanisms affect cell fate-remain unknown. Here we use the mouse blastocyst as a model to unravel a key role of fluid-filled lumen in the control of embryo size and specification of cell fate. We find that there is a twofold increase in lumenal pressure during blastocyst development, which translates into a concomitant increase in cell cortical tension and tissue stiffness of the trophectoderm that lines the lumen. Increased cortical tension leads to vinculin mechanosensing and maturation of functional tight junctions, which establishes a positive feedback loop to accommodate lumen growth. When the cortical tension reaches a critical threshold, cell-cell adhesion cannot be sustained during mitotic entry, which leads to trophectoderm rupture and blastocyst collapse. A simple theory of hydraulically gated oscillations recapitulates the observed dynamics of size oscillations, and predicts the scaling of embryo size with tissue volume. This theory further predicts that disrupted tight junctions or increased tissue stiffness lead to a smaller embryo size, which we verified by biophysical, embryological, pharmacological and genetic perturbations. Changes in lumenal pressure and size can influence the cell division pattern of the trophectoderm, and thereby affect cell allocation and fate. Our study reveals how lumenal pressure and tissue mechanics control embryo size at the tissue scale, which is coupled to cell position and fate at the cellular scale.


Assuntos
Diferenciação Celular , Linhagem da Célula , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário , Mecanotransdução Celular/fisiologia , Animais , Blastocisto/citologia , Adesão Celular , Divisão Celular , Forma Celular , Embrião de Mamíferos/anatomia & histologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Junções Íntimas , Vinculina/metabolismo
14.
Andrologia ; 51(8): e13340, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31197867

RESUMO

Our objective was to evaluate the effect of IMSI on embryo kinetics and clinical outcomes in patients with different aetiologies of male infertility. A total of 150 couples with different aetiologies of male infertility were randomly divided into ICSI and IMSI treatment groups (n = 75). ICSI was done accordingly. For IMSI group, the sperm selection was done using MSOME criteria and then injected. The zygotes were cultured in time-lapse monitoring system (TLM) for 3 days. A total of 650 embryos were developed and assessed using TLM in two groups. Data showed the rate of fragmentation had significant correlation with different aetiologies (p = 0.01), and the timing of s1, t4, s2 and t5 occurred significantly later in oligoasthenoteratozoospermia (OAT) patients compared with others (p < 0.05). In IMSI group, there were no differences in the TLM parameters among different aetiologies (p > 0.05). The rates of chemical pregnancy and implantation (37.8% and 38.2% respectively) were insignificantly higher in OAT patients compare to others (p > 0.05). Also, the clinical pregnancy and live birth rates (32% and 32% respectively) were insignificantly higher in teratozoospermia (T) cases. Sperm selection with MSOME parameters and IMSI can improve the embryo morphokinetics and clinical outcomes in couples with male factor infertility, especially for OAT and T patients.


Assuntos
Embrião de Mamíferos/diagnóstico por imagem , Desenvolvimento Embrionário , Infertilidade Masculina/terapia , Injeções de Esperma Intracitoplásmicas/métodos , Adulto , Coeficiente de Natalidade , Técnicas de Cultura Embrionária , Embrião de Mamíferos/embriologia , Feminino , Humanos , Masculino , Microinjeções , Indução da Ovulação/métodos , Gravidez , Taxa de Gravidez , Análise do Sêmen/métodos , Imagem com Lapso de Tempo , Resultado do Tratamento
15.
Nature ; 569(7758): 729-733, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31118510

RESUMO

In mammals, the emergence of totipotency after fertilization involves extensive rearrangements of the spatial positioning of the genome1,2. However, the contribution of spatial genome organization to the regulation of developmental programs is unclear3. Here we generate high-resolution maps of genomic interactions with the nuclear lamina (a filamentous meshwork that lines the inner nuclear membrane) in mouse pre-implantation embryos. We reveal that nuclear organization is not inherited from the maternal germline but is instead established de novo shortly after fertilization. The two parental genomes establish lamina-associated domains (LADs)4 with different features that converge after the 8-cell stage. We find that the mechanism of LAD establishment is unrelated to DNA replication. Instead, we show that paternal LAD formation in zygotes is prevented by ectopic expression of Kdm5b, which suggests that LAD establishment may be dependent on remodelling of H3K4 methylation. Our data suggest a step-wise assembly model whereby early LAD formation precedes consolidation of topologically associating domains.


Assuntos
Posicionamento Cromossômico , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Genoma/fisiologia , Lâmina Nuclear/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário , Feminino , Fertilização , Histona Desmetilases com o Domínio Jumonji/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/citologia , Oócitos/metabolismo , Zigoto/citologia , Zigoto/metabolismo
16.
Dev Growth Differ ; 61(5): 327-336, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31111476

RESUMO

Development of an embryo is driven by a series of molecular instructions that control the differentiation of tissue precursor cells and shape the tissues into major body parts. LIM homeobox 1 (LHX1) is a transcription factor that plays a major role in the development of the embryonic head of the mouse. Loss of LHX1 function disrupts the morphogenetic movement of head tissue precursors and impacts on the function of molecular factors in modulating the activity of the WNT signaling pathway. LHX1 acts with a transcription factor complex to regulate the transcription of target genes in multiple phases of development and in a range of embryonic tissues of the mouse and Xenopus. Determining the interacting factors and transcriptional targets of LHX1 will be key to unraveling the ensemble of factors involved in head development and building a head gene regulatory network.


Assuntos
Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Redes Reguladoras de Genes , Cabeça/embriologia , Proteínas com Homeodomínio LIM/metabolismo , Animais , Redes Reguladoras de Genes/genética , Humanos , Proteínas com Homeodomínio LIM/deficiência , Proteínas com Homeodomínio LIM/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
17.
Dev Biol ; 452(1): 1-7, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31042497

RESUMO

Cardiomyocytes undergo dramatic changes during the fetal to neonatal transition stage to adapt to the new environment. The molecular and genetic mechanisms regulating these changes remain elusive. In this study, we showed Sema6D as a novel signaling molecule regulating perinatal cardiomyocyte proliferation and maturation. SEMA6D is a member of the Semaphorin family of signaling molecules. To reveal its function during cardiogenesis, we specifically inactivated Sema6D in embryonic cardiomyocytes using a conditional gene deletion approach. All mutant animals showed hypoplastic myocardial walls in neonatal hearts due to reduced cell proliferation. We further revealed that expression of MYCN and its downstream cell cycle regulators is impaired in late fetal hearts in which Sema6D is deleted, suggesting that SEMA6D acts through MYCN to regulate cardiomyocyte proliferation. In early postnatal mutant hearts, expression of adult forms of sarcomeric proteins is increased, while expression of embryonic forms is decreased. These data collectively suggest that SEMA6D is required to maintain late fetal/early neonatal cardiomyocytes at a proliferative and less mature status. Deletion of Sema6D in cardiomyocytes led to reduced proliferation and accelerated maturation. We further examined the consequence of these defects through echocardiographic analysis. Embryonic heart deletion of Sema6D significantly impaired the cardiac contraction of male adult hearts, while having a minor effect on female mutant hearts, suggesting that the effect of Sema6D-deletion in adult hearts is sex dependent.


Assuntos
Proliferação de Células , Embrião de Mamíferos/embriologia , Coração/embriologia , Miócitos Cardíacos/metabolismo , Organogênese , Semaforinas/metabolismo , Animais , Ecocardiografia , Embrião de Mamíferos/citologia , Deleção de Genes , Coração/diagnóstico por imagem , Masculino , Camundongos , Camundongos Transgênicos , Contração Miocárdica , Miócitos Cardíacos/citologia , Semaforinas/genética , Desenvolvimento Sexual
18.
Nature ; 570(7759): 77-82, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31086336

RESUMO

Ontogeny describes the emergence of complex multicellular organisms from single totipotent cells. This field is particularly challenging in mammals, owing to the indeterminate relationship between self-renewal and differentiation, variation in progenitor field sizes, and internal gestation in these animals. Here we present a flexible, high-information, multi-channel molecular recorder with a single-cell readout and apply it as an evolving lineage tracer to assemble mouse cell-fate maps from fertilization through gastrulation. By combining lineage information with single-cell RNA sequencing profiles, we recapitulate canonical developmental relationships between different tissue types and reveal the nearly complete transcriptional convergence of endodermal cells of extra-embryonic and embryonic origins. Finally, we apply our cell-fate maps to estimate the number of embryonic progenitor cells and their degree of asymmetric partitioning during specification. Our approach enables massively parallel, high-resolution recording of lineage and other information in mammalian systems, which will facilitate the construction of a quantitative framework for understanding developmental processes.


Assuntos
Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/embriologia , Endoderma/metabolismo , Feminino , Fertilização , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , Especificidade de Órgãos/genética , Fenótipo , Análise de Sequência de RNA , Análise de Célula Única
19.
Int J Dev Biol ; 63(3-4-5): 143-155, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31058293

RESUMO

Monozygotic (MZ) polyembryony is a strategy to increase the output of a single zygote, thereby producing more offspring from a limited number of oocytes. However, MZ twins and multiples (multiplets) of mammals occur rarely in nature, while their generation has been more successful experimentally. In this work, we review some of the methodological, biological and field aspects of experimental MZ polyembryony in mammals. First attempts of mechanical bisection of 2-cell stage rodent embryos provided a proof-of-principle for the survival and independent development of both blastomeres. Subsequently, experiments in other species, particularly sheep and bovine, allowed 2 methods of embryo multiplication to become routine: the separation or biopsy of blastomeres from cleavage-stage embryos and the bisection of morulae and blastocysts. We discuss how the preferable stage of bisection and the success rate can be species-specific. The scope that profited most from experimental MZ polyembryony is the production of additional copies of elite livestock individuals, the reduction of interindividual variation in test groups and the possibility of investigating discordant phenotypic traits in the same genomic background, for instance, comparing an affected twin with its healthy co-twin. By contrast, the original motivation for experimental polyembryony - efficiently generating more offspring out of the same zygote - has not been fulfilled yet. Although embryo splitting leads to an increase in quantity, there is a loss of embryo quality, thus, there is no real gain from artificially generated embryos (yet) in the field of medically assisted reproduction. In conclusion, mammalian zygotes have the regulative capacity to be polyembryonic, but this is not obligate.


Assuntos
Blastômeros/citologia , Gêmeos Monozigóticos , Animais , Blastocisto/citologia , Cruzamento , Bovinos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário , Feminino , Ovinos/embriologia , Zigoto/citologia
20.
Morphologie ; 103(341 Pt 2): 72-79, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31092318

RESUMO

BACKGROUND AND AIM: Difficulties are encountered in embryology learning such as imagining embryo modifications in three-dimensions and time. We provided an experimentation to evaluate if short videos during magisterial lecture could increase the quality and the efficiency of embryology teaching. METHODS: The study was conducted amongst students in first year of medical studies in France. It is an intense and highly competitive year at the end of which students can engage in medical or paramedical specialties depending on their rank. In a first step, pre-implantation embryo development and microscopic videos of in vitro Fertilization were presented during a course of medical ethics. Three months later, students gave their opinion on this presentation in a satisfaction survey using a Likert scale. In a second step (the two following years), similar videos were integrated in the regular embryology lectures and the results of the subsequent embryology test were analyzed. RESULTS: In the first step, students declared that movies could increase their interest in embryology and significantly help to the comprehension and memorization of embryologic processes. In the second step, we found that students answered better to the video-related questions of the test even if globally in the first year, results were weaker compared to previous years. DISCUSSION: The effects of movies in pedagogy are discussed, especially the accelerated rhythm imposed by this medium. Adverse consequences could be balanced by traditional drawing. CONCLUSIONS: The association of complementary pedagogic methods like movies and drawing could allow an optimization of embryo teaching.


Assuntos
Embriologia/educação , Ensino , Gravação em Vídeo , Currículo , Educação em Odontologia/métodos , Educação de Graduação em Medicina/métodos , Educação em Farmácia/métodos , Avaliação Educacional/estatística & dados numéricos , Embrião de Mamíferos/diagnóstico por imagem , Embrião de Mamíferos/embriologia , França , Humanos , Aprendizagem , Avaliação de Programas e Projetos de Saúde , Estudantes de Medicina/psicologia , Estudantes de Medicina/estatística & dados numéricos
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