RESUMO
The relationship between the human placenta-the extraembryonic organ made by the fetus, and the decidua-the mucosal layer of the uterus, is essential to nurture and protect the fetus during pregnancy. Extravillous trophoblast cells (EVTs) derived from placental villi infiltrate the decidua, transforming the maternal arteries into high-conductance vessels1. Defects in trophoblast invasion and arterial transformation established during early pregnancy underlie common pregnancy disorders such as pre-eclampsia2. Here we have generated a spatially resolved multiomics single-cell atlas of the entire human maternal-fetal interface including the myometrium, which enables us to resolve the full trajectory of trophoblast differentiation. We have used this cellular map to infer the possible transcription factors mediating EVT invasion and show that they are preserved in in vitro models of EVT differentiation from primary trophoblast organoids3,4 and trophoblast stem cells5. We define the transcriptomes of the final cell states of trophoblast invasion: placental bed giant cells (fused multinucleated EVTs) and endovascular EVTs (which form plugs inside the maternal arteries). We predict the cell-cell communication events contributing to trophoblast invasion and placental bed giant cell formation, and model the dual role of interstitial EVTs and endovascular EVTs in mediating arterial transformation during early pregnancy. Together, our data provide a comprehensive analysis of postimplantation trophoblast differentiation that can be used to inform the design of experimental models of the human placenta in early pregnancy.
Assuntos
Multiômica , Primeiro Trimestre da Gravidez , Trofoblastos , Feminino , Humanos , Gravidez , Movimento Celular , Placenta/irrigação sanguínea , Placenta/citologia , Placenta/fisiologia , Primeiro Trimestre da Gravidez/fisiologia , Trofoblastos/citologia , Trofoblastos/metabolismo , Trofoblastos/fisiologia , Decídua/irrigação sanguínea , Decídua/citologia , Relações Materno-Fetais/fisiologia , Análise de Célula Única , Miométrio/citologia , Miométrio/fisiologia , Diferenciação Celular , Organoides/citologia , Organoides/fisiologia , Células-Tronco/citologia , Transcriptoma , Fatores de Transcrição/metabolismo , Comunicação CelularRESUMO
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/metabolismoRESUMO
OBJECTIVE: To develop a method for the efficient assembly of viral or multimeric proteins into virus-like particles (VLP) or other macro structures. RESULTS: Protein monomers were assembled by eliminating calcium ions through precipitation. The model protein, rotavirus VP6, assembled into stable, long nanotubes with better quality than the assemblies obtained directly from cell culture. Nanotube length was directly proportional to the initial concentration of VP6 monomers, in accordance with the classic nucleation theory of capsid assembly. The quality of the obtained assemblies was confirmed when the nanotubes were functionalized with metals, yielding unique nanobiomaterials. Assembly efficiency was improved in comparison with other previously proposed methods. CONCLUSIONS: The novel method presented here is simpler and faster than other reported methods for the assembly and disassembly of viral proteins, a step needed for most applications.
Assuntos
Antígenos Virais/química , Antígenos Virais/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Rotavirus/metabolismo , Cálcio/química , Precipitação Química , Nanotubos/química , Multimerização ProteicaRESUMO
The placenta is a selective maternal-fetal barrier that provides nourishment and protection from infections. However, certain pathogens can attach to and even cross the placenta, causing pregnancy complications with potential lifelong impacts on the child's health. Here, we profiled at the single-cell level the placental responses to three pathogens associated with intrauterine complications-Plasmodium falciparum, Listeria monocytogenes, and Toxoplasma gondii. We found that upon exposure to the pathogens, all placental lineages trigger inflammatory responses that may compromise placental function. Additionally, we characterized the responses of fetal macrophages known as Hofbauer cells (HBCs) to each pathogen and propose that they are the probable niche for T. gondii. Finally, we revealed how P. falciparum adapts to the placental microenvironment by modulating protein export into the host erythrocyte and nutrient uptake pathways. Altogether, we have defined the cellular networks and signaling pathways mediating acute placental inflammatory responses that could contribute to pregnancy complications.
Assuntos
Placenta , Análise de Célula Única , Humanos , Feminino , Gravidez , Placenta/microbiologia , Placenta/imunologia , Análise de Célula Única/métodos , Plasmodium falciparum , Listeria monocytogenes/patogenicidade , Listeria monocytogenes/fisiologia , Toxoplasma/patogenicidade , Macrófagos/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Toxoplasmose/imunologia , Toxoplasmose/metabolismo , InflamaçãoRESUMO
Inborn errors of T cell development present a pediatric emergency in which timely curative therapy is informed by molecular diagnosis. In 11 affected patients across four consanguineous kindreds, we detected homozygosity for a single deleterious missense variant in the gene NudC domain-containing 3 (NUDCD3). Two infants had severe combined immunodeficiency with the complete absence of T and B cells (T -B- SCID), whereas nine showed classical features of Omenn syndrome (OS). Restricted antigen receptor gene usage by residual T lymphocytes suggested impaired V(D)J recombination. Patient cells showed reduced expression of NUDCD3 protein and diminished ability to support RAG-mediated recombination in vitro, which was associated with pathologic sequestration of RAG1 in the nucleoli. Although impaired V(D)J recombination in a mouse model bearing the homologous variant led to milder immunologic abnormalities, NUDCD3 is absolutely required for healthy T and B cell development in humans.
Assuntos
Imunodeficiência Combinada Severa , Recombinação V(D)J , Humanos , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia , Animais , Camundongos , Recombinação V(D)J/imunologia , Recombinação V(D)J/genética , Masculino , Feminino , Lactente , Linfócitos B/imunologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Linfócitos T/imunologia , Pré-Escolar , Mutação de Sentido IncorretoRESUMO
Totipotency emerges in early embryogenesis, but its molecular underpinnings remain poorly characterized. In the present study, we employed DNA fiber analysis to investigate how pluripotent stem cells are reprogrammed into totipotent-like 2-cell-like cells (2CLCs). We show that totipotent cells of the early mouse embryo have slow DNA replication fork speed and that 2CLCs recapitulate this feature, suggesting that fork speed underlies the transition to a totipotent-like state. 2CLCs emerge concomitant with DNA replication and display changes in replication timing (RT), particularly during the early S-phase. RT changes occur prior to 2CLC emergence, suggesting that RT may predispose to gene expression changes and consequent reprogramming of cell fate. Slowing down replication fork speed experimentally induces 2CLCs. In vivo, slowing fork speed improves the reprogramming efficiency of somatic cell nuclear transfer. Our data suggest that fork speed regulates cellular plasticity and that remodeling of replication features leads to changes in cell fate and reprogramming.
Assuntos
Embrião de Mamíferos , Células-Tronco Pluripotentes , Animais , Diferenciação Celular/genética , Reprogramação Celular/genética , Replicação do DNA/genética , Desenvolvimento Embrionário/genética , CamundongosRESUMO
Totipotent cells hold enormous potential for regenerative medicine. Thus, the development of cellular models recapitulating totipotent-like features is of paramount importance. Cells resembling the totipotent cells of early embryos arise spontaneously in mouse embryonic stem (ES) cell cultures. Such '2-cell-like-cells' (2CLCs) recapitulate 2-cell-stage features and display expanded cell potential. Here, we used 2CLCs to perform a small-molecule screen to identify new pathways regulating the 2-cell-stage program. We identified retinoids as robust inducers of 2CLCs and the retinoic acid (RA)-signaling pathway as a key component of the regulatory circuitry of totipotent cells in embryos. Using single-cell RNA-seq, we reveal the transcriptional dynamics of 2CLC reprogramming and show that ES cells undergo distinct cellular trajectories in response to RA. Importantly, endogenous RA activity in early embryos is essential for zygotic genome activation and developmental progression. Overall, our data shed light on the gene regulatory networks controlling cellular plasticity and the totipotency program.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Totipotentes/citologia , Tretinoína/fisiologia , Acitretina/farmacologia , Animais , Massa Celular Interna do Blastocisto/citologia , Diferenciação Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Feminino , Redes Reguladoras de Genes/genética , Genes Reporter , Isotretinoína/farmacologia , Masculino , Camundongos/embriologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Piperazinas/farmacologia , Pirazóis/farmacologia , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , RNA-Seq , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores do Ácido Retinoico/fisiologia , Transdução de Sinais/efeitos dos fármacos , Células-Tronco Totipotentes/efeitos dos fármacos , Transcrição Gênica , Tretinoína/antagonistas & inibidores , Tretinoína/farmacologia , Receptor gama de Ácido RetinoicoRESUMO
Following fertilization in mammals, the gametes are reprogrammed to create a totipotent zygote, a process that involves de novo establishment of chromatin domains. A major feature occurring during preimplantation development is the dramatic remodelling of constitutive heterochromatin, although the functional relevance of this is unknown. Here, we show that heterochromatin establishment relies on the stepwise expression and regulated activity of SUV39H enzymes. Enforcing precocious acquisition of constitutive heterochromatin results in compromised development and epigenetic reprogramming, which demonstrates that heterochromatin remodelling is essential for natural reprogramming at fertilization. We find that de novo H3K9 trimethylation (H3K9me3) in the paternal pronucleus after fertilization is catalysed by SUV39H2 and that pericentromeric RNAs inhibit SUV39H2 activity and reduce H3K9me3. De novo H3K9me3 is initially non-repressive for gene expression, but instead bookmarks promoters for compaction. Overall, we uncover the functional importance for the restricted transmission of constitutive heterochromatin during reprogramming and a non-repressive role for H3K9me3.