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1.
Sci Transl Med ; 13(587)2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33790026

RESUMO

The lateral ventricle (LV) is flanked by the subventricular zone (SVZ), a neural stem cell (NSC) niche rich in extrinsic growth factors regulating NSC maintenance, proliferation, and neuronal differentiation. Dysregulation of the SVZ niche causes LV expansion, a condition known as hydrocephalus; however, the underlying pathological mechanisms are unclear. We show that deficiency of the proteoglycan Tsukushi (TSK) in ependymal cells at the LV surface and in the cerebrospinal fluid results in hydrocephalus with neurodevelopmental disorder-like symptoms in mice. These symptoms are accompanied by altered differentiation and survival of the NSC lineage, disrupted ependymal structure, and dysregulated Wnt signaling. Multiple TSK variants found in patients with hydrocephalus exhibit reduced physiological activity in mice in vivo and in vitro. Administration of wild-type TSK protein or Wnt antagonists, but not of hydrocephalus-related TSK variants, in the LV of TSK knockout mice prevented hydrocephalus and preserved SVZ neurogenesis. These observations suggest that TSK plays a crucial role as a niche molecule modulating the fate of SVZ NSCs and point to TSK as a candidate for the diagnosis and therapy of hydrocephalus.

2.
Sci Rep ; 11(1): 5857, 2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712686

RESUMO

Epithelial cells organize an ordered array of non-centrosomal microtubules, the minus ends of which are regulated by CAMSAP3. The role of these microtubules in epithelial functions, however, is poorly understood. Here, we show that the kidneys of mice in which Camsap3 is mutated develop cysts at the proximal convoluted tubules (PCTs). PCTs were severely dilated in the mutant kidneys, and they also exhibited enhanced cell proliferation. In these PCTs, epithelial cells became flattened along with perturbation of microtubule arrays as well as of certain subcellular structures such as interdigitating basal processes. Furthermore, YAP and PIEZO1, which are known as mechanosensitive regulators for cell shaping and proliferation, were activated in these mutant PCT cells. These observations suggest that CAMSAP3-mediated microtubule networks are important for maintaining the proper mechanical properties of PCT cells, and its loss triggers cell deformation and proliferation via activation of mechanosensors, resulting in the dilation of PCTs.

3.
Nat Metab ; 2020 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-33288951

RESUMO

Osteoclasts are the exclusive bone-resorbing cells, playing a central role in bone metabolism, as well as the bone damage that occurs under pathological conditions1,2. In postnatal life, haematopoietic stem-cell-derived precursors give rise to osteoclasts in response to stimulation with macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand, both of which are produced by osteoclastogenesis-supporting cells such as osteoblasts and osteocytes1-3. However, the precise mechanisms underlying cell fate specification during osteoclast differentiation remain unclear. Here, we report the transcriptional profiling of 7,228 murine cells undergoing in vitro osteoclastogenesis, describing the stepwise events that take place during the osteoclast fate decision process. Based on our single-cell transcriptomic dataset, we find that osteoclast precursor cells transiently express CD11c, and deletion of receptor activator of nuclear factor-κB specifically in CD11c-expressing cells inhibited osteoclast formation in vivo and in vitro. Furthermore, we identify Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (Cited2) as the molecular switch triggering terminal differentiation of osteoclasts, and deletion of Cited2 in osteoclast precursors in vivo resulted in a failure to commit to osteoclast fate. Together, the results of this study provide a detailed molecular road map of the osteoclast differentiation process, refining and expanding our understanding of the molecular mechanisms underlying osteoclastogenesis.

4.
Proc Natl Acad Sci U S A ; 117(36): 22193-22203, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32839317

RESUMO

The establishment of axon/dendrite polarity is fundamental for neurons to integrate into functional circuits, and this process is critically dependent on microtubules (MTs). In the early stages of the establishment process, MTs in axons change dramatically with the morphological building of neurons; however, how the MT network changes are triggered is unclear. Here we show that CAMSAP1 plays a decisive role in the neuronal axon identification process by regulating the number of MTs. Neurons lacking CAMSAP1 form a multiple axon phenotype in vitro, while the multipolar-bipolar transition and radial migration are blocked in vivo. We demonstrate that the polarity regulator MARK2 kinase phosphorylates CAMSAP1 and affects its ability to bind to MTs, which in turn changes the protection of MT minus-ends and also triggers asymmetric distribution of MTs. Our results indicate that the polarized MT network in neurons is a decisive factor in establishing axon/dendritic polarity and is initially triggered by polarized signals.


Assuntos
Polaridade Celular/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/fisiologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Imunoprecipitação , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Neurônios , Paclitaxel , Ligação Proteica
5.
Cell Rep ; 31(7): 107653, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32433962

RESUMO

In CRISPR-Cas9-assisted knockin (KI) in zygotes, a remaining challenge is routinely achieving high-efficiency KI of large (kilobase-sized) DNA elements. Here, we focus on the timing of pronuclear injection and establish a reliable homologous recombination (HR)-based method to generate large KIs in zygotes compared with two other types of KI strategies involving distinct DNA repair pathways. At the ROSA26 locus, pronuclear injection with CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and Cas9 protein at the S phase by using the HR-based method yields the most efficient and accurate KIs (up to 70%). This approach is also generally effective for generating large KI alleles at other gene loci. We further apply our method to efficiently obtain biallelic ROSA26 KIs by sequential injection into both pronuclei. Our results suggest that delivery of genome editing components and donor DNA into S-phase zygotes is critical for efficient KI of large DNA elements.

6.
Development ; 147(9)2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398354

RESUMO

Osteoblasts arise from bone-surrounding connective tissue containing tenocytes and fibroblasts. Lineages of these cell populations and mechanisms of their differentiation are not well understood. Screening enhancer-trap lines of zebrafish allowed us to identify Ebf3 as a transcription factor marking tenocytes and connective tissue cells in skeletal muscle of embryos. Knockout of Ebf3 in mice had no effect on chondrogenesis but led to sternum ossification defects as a result of defective generation of Runx2+ pre-osteoblasts. Conditional and temporal Ebf3 knockout mice revealed requirements of Ebf3 in the lateral plate mesenchyme cells (LPMs), especially in tendon/muscle connective tissue cells, and a stage-specific Ebf3 requirement at embryonic day 9.5-10.5. Upregulated expression of connective tissue markers, such as Egr1/2 and Osr1, increased number of Islet1+ mesenchyme cells, and downregulation of gene expression of the Runx2 regulator Shox2 in Ebf3-deleted thoracic LPMs suggest crucial roles of Ebf3 in the onset of lateral plate mesoderm differentiation towards osteoblasts forming sternum tissues.

7.
Cell Rep ; 31(5): 107592, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32375034

RESUMO

The six-layered neocortex is a shared characteristic of all mammals, but not of non-mammalian species, and its formation requires an inside-out pattern of neuronal migration. The extant reptilian dorsal cortex is thought to represent an ancestral form of the neocortex, although how the reptilian three-layered cortex is formed is poorly understood. Here, we show unique patterns of lamination and neuronal migration in the developing reptilian cortex. While the multipolar-to-bipolar transition of migrating neurons is essential for mammalian cortical development, the reptilian cortex lacks bipolar-shaped migrating neurons, resulting in an outside-in pattern of cortical development. Furthermore, dynamic regulation of Wnt signal strengths contributes to neuronal morphological changes, which is conserved across species. Our data preclude the idea that the six-layered mammalian neocortex emerged by simple addition to the reptilian dorsal cortex but suggest that the acquisition of a novel neuronal morphology based on conserved developmental programs contributed to neocortical evolution.

9.
Blood ; 136(6): 684-697, 2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32325488

RESUMO

The linear ubiquitin chain assembly complex (LUBAC) is a key regulator of NF-κB signaling. Activating single-nucleotide polymorphisms of HOIP, the catalytic subunit of LUBAC, are enriched in patients with activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL), and expression of HOIP, which parallels LUBAC activity, is elevated in ABC-DLBCL samples. Thus, to clarify the precise roles of LUBAC in lymphomagenesis, we generated a mouse model with augmented expression of HOIP in B cells. Interestingly, augmented HOIP expression facilitated DLBCL-like B-cell lymphomagenesis driven by MYD88-activating mutation. The developed lymphoma cells partly shared somatic gene mutations with human DLBCLs, with increased frequency of a typical AID mutation pattern. In vitro analysis revealed that HOIP overexpression protected B cells from DNA damage-induced cell death through NF-κB activation, and analysis of the human DLBCL database showed that expression of HOIP positively correlated with gene signatures representing regulation of apoptosis signaling, as well as NF-κB signaling. These results indicate that HOIP facilitates lymphomagenesis by preventing cell death and augmenting NF-κB signaling, leading to accumulation of AID-mediated mutations. Furthermore, a natural compound that specifically inhibits LUBAC was shown to suppress the tumor growth in a mouse transplantation model. Collectively, our data indicate that LUBAC is crucially involved in B-cell lymphomagenesis through protection against DNA damage-induced cell death and is a suitable therapeutic target for B-cell lymphomas.

10.
Sci Rep ; 10(1): 2518, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054870

RESUMO

The field of genome editing was founded on the establishment of methods, such as the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR/Cas) system, used to target DNA double-strand breaks (DSBs). However, the efficiency of genome editing also largely depends on the endogenous cellular repair machinery. Here, we report that the specific modulation of targeting vectors to provide 3' overhangs at both ends increased the efficiency of homology-directed repair (HDR) in embryonic stem cells. We applied the modulated targeting vectors to produce homologous recombinant mice directly by pronuclear injection, but the frequency of HDR was low. Furthermore, we combined our method with the CRISPR/Cas9 system, resulting in a significant increase in HDR frequency. Thus, our HDR-based method, enhanced homologous recombination for genome targeting (eHOT), is a new and powerful method for genome engineering.


Assuntos
Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , Edição de Genes , Marcação de Genes , Recombinação Homóloga , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Edição de Genes/métodos , Marcação de Genes/métodos , Vetores Genéticos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Reparo de DNA por Recombinação
11.
Dev Dyn ; 249(6): 698-710, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32012381

RESUMO

BACKGROUND: During development, Cajal-Retzius (CR) cells are the first generated and essential pioneering neurons that control neuronal migration and arealization in the mammalian cortex. CR cells are derived from specific regions within the telencephalon, that is, the pallial septum in the rostromedial cortex, the pallial-subpallial boundary, and the cortical hem (CH) in the caudomedial cortex. However, the molecular mechanism underlying the generation of CR cell subtypes in distinct regions of origin is poorly understood. RESULTS: We found that double-sex and mab-3 related transcription factor (Dmrt) genes, that is, Dmrta1 and Dmrt3, were expressed in the progenitor domains that produce CR cells. The number of CH-derived CR cells was severely decreased in Dmrt3 mutants, especially in Dmrta1 and Dmrt3 double mutants. The reduced production of the CR cells was consistent with the developmental impairment of the CH structures in the medial telencephalon from which the CR cells are produced. CONCLUSION: Dmrta1 and Dmrt3 cooperatively regulate patterning of the CH structure and production of the CR cells from the CH during cortical development.

12.
Nat Ecol Evol ; 4(2): 261-269, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31907383

RESUMO

Unidirectional fluid flow generated by motile cilia at the left-right organizer (LRO) breaks left-right (L-R) symmetry during early embryogenesis in mouse, frog and zebrafish. The chick embryo, however, does not require motile cilia for L-R symmetry breaking. The diversity of mechanisms for L-R symmetry breaking among vertebrates and the trigger for such symmetry breaking in non-mammalian amniotes have remained unknown. Here we examined how L-R asymmetry is established in two reptiles, Madagascar ground gecko and Chinese softshell turtle. Both of these reptiles appear to lack motile cilia at the LRO. The expression of the Nodal gene at the LRO in the reptilian embryos was found to be asymmetric, in contrast to that in vertebrates such as mouse that are dependent on cilia for L-R patterning. Two paralogues of the Nodal gene derived from an ancient gene duplication are retained and expressed differentially in cilia-dependent and cilia-independent vertebrates. The expression of these two Nodal paralogues is similarly controlled in the lateral plate mesoderm but regulated differently at the LRO. Our in-depth analysis of reptilian embryos thus suggests that mammals and non-mammalian amniotes deploy distinct strategies dependent on different Nodal paralogues for rendering Nodal activity asymmetric at the LRO.


Assuntos
Padronização Corporal , Cílios , Animais , Embrião de Galinha , Madagáscar , Camundongos , Répteis , Peixe-Zebra
13.
Mol Brain ; 12(1): 94, 2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31718706

RESUMO

Grb2-associated regulator of Erk/MAPK (GAREM), is an adaptor protein related to the several cell growth factor receptor-signaling. The GAREM family has two subtypes, GAREM1 and GAREM2, both encoded in the human and mouse genome. Recent genome-wide research identified GAREM2 as a candidate of neurodegenerative diseases. Here, we use knockout (KO) mice to show the role of GAREM2, that is highly expressed in the brain. According to the comprehensive behavioral battery, they exhibited less anxiety both in elevated plus maze and open field tests, mildly increased social approaching behavior in the reciprocal social interaction test, and longer latency to immobility in the tail suspension test as compared to wild-type (WT). Additionally, the extension of neurites in the primary cultured neurons was suppressed in ones derived from GAREM2 KO mice. Furthermore, we also identified Intersectin, as a binding partner of GAREM2 in this study. Intersectin is also a multi-domain adaptor protein that regulates endocytosis and cell signaling, which can potentially alter the subcellular localization of GAREM2. The important molecules, such as the neurotrophin receptor and Erk family, that are involved in the signaling pathway of the neural cell growth in the mouse brain, have been reported to participate in emotional behavior. As GAREM plays a role in the cellular growth factor receptor signaling pathway, GAREM2 may have a common role related to the transduction of Erk signaling in the higher brain functions.


Assuntos
Comportamento Animal , Encéfalo/metabolismo , Proteína Adaptadora GRB2/deficiência , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Ansiedade/patologia , Linhagem Celular , Comportamento Exploratório , Feminino , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Humanos , Aprendizagem em Labirinto , Camundongos Knockout , Crescimento Neuronal , Neurônios/metabolismo , Tempo de Reação , Comportamento Social
14.
Genes Cells ; 24(12): 762-767, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31701596

RESUMO

The 15th Transgenic Technology (TT) Meeting of the International Society for Transgenic Technologies (ISTT) was held for the first time in Japan in April of 2019 (TT2019). Delegates from around the world, including researchers, technicians and trainees, spent an exciting 4 days, engaging in scientific and technical discussions, information sharing and networking. In the era of genome editing, CRISPR technologies prevailed as popular subjects of discussion throughout the meeting on topics ranging from their applications in the mouse and growing number of other research and industrial animal species, technical challenges in the production of genome-edited animals, to ethical considerations in biomedical research. In particular, impressive progress was reported on the use of CRISPR technologies in nonconventional animal models and large mammalian species in biomedical and industrial settings, indicating areas of expanding frontiers in animal transgenesis. Amid growing excitement with genome editing technologies, reports on conventional genetic engineering approaches and reproductive biology techniques demonstrated that these techniques remain essential to meet the demanding needs of generating complex genome modifications, complementing genome editing approaches. TT2019 was a great success, concluding with a widely shared appreciation of the current field and hints for future directions of animal transgenesis.


Assuntos
Animais Geneticamente Modificados/genética , Congressos como Assunto , Edição de Genes/métodos , Animais , Japão
15.
Sci Rep ; 9(1): 15911, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31685837

RESUMO

Membrane morphology is an important structural determinant as it reflects cellular functions. The pentaspan membrane protein Prominin-1 (Prom1/CD133) is known to be localised to protrusions and plays a pivotal role in migration and the determination of cellular morphology; however, the underlying mechanism of its action have been elusive. Here, we performed molecular characterisation of Prom1, focussing primarily on its effects on cell morphology. Overexpression of Prom1 in RPE-1 cells triggers multiple, long, cholesterol-enriched fibres, independently of actin and microtubule polymerisation. A five amino acid stretch located at the carboxyl cytosolic region is essential for fibre formation. The small GTPase Rho and its downstream Rho-associated coiled-coil-containing protein kinase (ROCK) are also essential for this process, and active Rho colocalises with Prom1 at the site of initialisation of fibre formation. In mouse embryonic fibroblast (MEF) cells we show that Prom1 is required for chloride ion efflux induced by calcium ion uptake, and demonstrate that fibre formation is closely associated with chloride efflux activity. Collectively, these findings suggest that Prom1 affects cell morphology and contributes to chloride conductance.


Assuntos
Antígeno AC133/metabolismo , Cálcio/metabolismo , Extensões da Superfície Celular/metabolismo , Cloretos/metabolismo , Quinases Associadas a rho/metabolismo , Antígeno AC133/química , Antígeno AC133/genética , Citoesqueleto de Actina/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Colesterol/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtúbulos/metabolismo , Transdução de Sinais
16.
Proc Natl Acad Sci U S A ; 116(47): 23653-23661, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31694883

RESUMO

The activation of innate immune receptors by pathogen-associated molecular patterns (PAMPs) is central to host defense against infections. On the other hand, these receptors are also activated by immunogenic damage-associated molecular patterns (DAMPs), typically released from dying cells, and the activation can evoke chronic inflammatory or autoimmune disorders. One of the best known receptors involved in the immune pathogenesis is Toll-like receptor 7 (TLR7), which recognizes RNA with single-stranded structure. However, the causative DAMP RNA(s) in the pathogenesis has yet to be identified. Here, we first developed a chemical compound, termed KN69, that suppresses autoimmunity in several established mouse models. A subsequent search for KN69-binding partners led to the identification of U11 small nuclear RNA (U11snRNA) as a candidate DAMP RNA involved in TLR7-induced autoimmunity. We then showed that U11snRNA robustly activated the TLR7 pathway in vitro and induced arthritis disease in vivo. We also found a correlation between high serum level of U11snRNA and autoimmune diseases in human subjects and established mouse models. Finally, by revealing the structural basis for U11snRNA's ability to activate TLR7, we developed more potent TLR7 agonists and TLR7 antagonists, which may offer new therapeutic approaches for autoimmunity or other immune-driven diseases. Thus, our study has revealed a hitherto unknown immune function of U11snRNA, providing insight into TLR7-mediated autoimmunity and its potential for further therapeutic applications.


Assuntos
Glicoproteínas de Membrana/agonistas , RNA Nuclear Pequeno/imunologia , Receptor 7 Toll-Like/agonistas , Adulto , Alarminas/química , Animais , Artrite Reumatoide/sangue , Artrite Reumatoide/imunologia , Doenças Autoimunes/sangue , Doenças Autoimunes/imunologia , Sequência de Bases , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Imunossupressores/síntese química , Imunossupressores/farmacologia , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Pessoa de Meia-Idade , RNA/imunologia , RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/imunologia , Análise de Sequência de RNA , Receptor 7 Toll-Like/deficiência , Adulto Jovem
17.
eNeuro ; 6(6)2019.
Artigo em Inglês | MEDLINE | ID: mdl-31672846

RESUMO

A fundamental strategy in sensory coding is parallel processing, whereby unique, distinct features of sensation are computed and projected to the central target in the form of submodal maps. It remains unclear, however, whether such parallel processing strategy is employed in the main olfactory system, which codes the complex hierarchical odor and behavioral scenes. A potential scheme is that distinct subsets of projection neurons in the olfactory bulb (OB) form parallel projections to the targets. Taking advantage of the observation that the distinct projection neurons develop at different times, we developed a Cre-loxP-based method that allows for birthdate-specific labeling of cell bodies and their axon projections in mice. This birthdate tag analysis revealed that the mitral cells (MCs) born in an early developmental stage and the external tufted cells (TCs) born a few days later form segregated parallel projections. Specifically, the latter subset converges the axons onto only two small specific targets, one of which, located at the anterolateral edge of the olfactory tubercle (OT), excludes widespread MC projections. This target is made up of neurons that express dopamine D1 but not D2 receptor and corresponds to the most anterolateral isolation of the CAP compartments (aiCAP) that were defined previously. This finding of segregated projections suggests that olfactory sensing does indeed involve parallel processing of functionally distinct submodalities. Importantly, the birthdate tag method used here may pave the way for deciphering the functional meaning of these individual projection pathways in the future.


Assuntos
Neurônios/citologia , Bulbo Olfatório/citologia , Condutos Olfatórios/citologia , Animais , Camundongos , Camundongos Transgênicos , Neurônios/fisiologia , Bulbo Olfatório/fisiologia , Condutos Olfatórios/fisiologia , Olfato/fisiologia
18.
Cell Rep ; 29(3): 603-616.e5, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31618630

RESUMO

In higher vertebrates, cephalic neural crest cells (NCCs) form craniofacial skeleton by differentiating into chondrocytes and osteoblasts. A subpopulation of cephalic NCCs, cardiac NCCs (CNCCs), migrates to the heart. However, CNCCs mostly do not yield skeletogenic derivatives, and the molecular mechanisms of this fate restriction remain elusive. We identify a disintegrin and metalloprotease 19 (Adam19) as a position-specific fate regulator of NCCs. Adam19-depleted mice abnormally form NCC-derived cartilage in their hearts through the upregulation of Sox9 levels in CNCCs. Moreover, NCC-lineage-specific Sox9-overexpressing mice recapitulate CNCC chondrogenesis. In vitro experiments show that Adam19 mediates the cleavage of bone morphogenic protein (BMP) type I receptor Alk2 (Acvr1), whereas pharmacogenetic approaches reveal that Adam19 inhibits CNCC chondrogenesis by suppressing the BMP-Sox9 cascade, presumably through processing Alk2. These findings suggest a metalloprotease-dependent mechanism attenuating cellular responsiveness to BMP ligands, which is essential for both the positional restriction of NCC skeletogenesis and normal heart development.


Assuntos
Proteínas ADAM/metabolismo , Crista Neural/metabolismo , Transdução de Sinais , Proteínas ADAM/deficiência , Proteínas ADAM/genética , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Animais , Proteína Morfogenética Óssea 6/metabolismo , Cartilagem/crescimento & desenvolvimento , Cartilagem/metabolismo , Cartilagem/patologia , Diferenciação Celular , Condrogênese , Embrião de Mamíferos/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Miocárdio/citologia , Miocárdio/metabolismo , Crista Neural/citologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Regulação para Cima
19.
Stem Cell Reports ; 13(3): 485-498, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31402336

RESUMO

Soon after fertilization, the few totipotent cells of mammalian embryos diverge to form a structure called the blastocyst (BC). Although numerous cell types, including germ cells and extended-pluripotency stem cells, have been developed from pluripotent stem cells (PSCs) in vitro, generating functional BCs only from PSCs remains elusive. Here, we describe induced self-organizing 3D BC-like cysts (iBLCs) generated from mouse PSC culture. Resembling natural BCs, iBLCs have a blastocoel-like cavity and were formed with outer cells expressing trophectoderm lineage markers and with inner cells expressing pluripotency markers. iBLCs transplanted to pseudopregnant mice uteruses implanted, induced decidualization, and exhibited growth and development before resorption, demonstrating that iBLCs are implantation competent. iBLC precursor intermediates required the transcription factor Prdm14 and concomitantly activated the totipotency-related cleavage-stage MERVL reporter and 2C genes. Thus, our system may contribute to the understanding of molecular mechanisms underpinning totipotency, embryogenesis, and implantation.


Assuntos
Blastocisto/metabolismo , Células-Tronco Pluripotentes/citologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Blastocisto/citologia , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Linhagem da Célula , Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário , Feminino , Genes Reporter , Camundongos , Camundongos Endogâmicos ICR , Células-Tronco Pluripotentes/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Útero/patologia
20.
Development ; 146(15)2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31371378

RESUMO

The spatiotemporal identity of neural progenitors and the regional control of neurogenesis are essential for the development of cerebral cortical architecture. Here, we report that mammalian DM domain factors (Dmrt) determine the identity of cerebral cortical progenitors. Among the Dmrt family genes expressed in the developing dorsal telencephalon, Dmrt3 and Dmrta2 show a medialhigh/laterallow expression gradient. Their simultaneous loss confers a ventral identity to dorsal progenitors, resulting in the ectopic expression of Gsx2 and massive production of GABAergic olfactory bulb interneurons in the dorsal telencephalon. Furthermore, double-mutant progenitors in the medial region exhibit upregulated Pax6 and more lateral characteristics. These ventral and lateral shifts in progenitor identity depend on Dmrt gene dosage. We also found that Dmrt factors bind to Gsx2 and Pax6 enhancers to suppress their expression. Our findings thus reveal that the graded expression of Dmrt factors provide positional information for progenitors by differentially repressing downstream genes in the developing cerebral cortex.


Assuntos
Córtex Cerebral/embriologia , Células-Tronco Neurais/citologia , Neurogênese/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Córtex Cerebral/citologia , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição PAX6/biossíntese , Fator de Transcrição PAX6/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética
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