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1.
Adv Mater ; 35(36): e2301673, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37269532

RESUMO

In living tissues, cells express their functions following complex signals from their surrounding microenvironment. Capturing both hierarchical architectures at the micro- and macroscale, and anisotropic cell patterning remains a major challenge in bioprinting, and a bottleneck toward creating physiologically-relevant models. Addressing this limitation, a novel technique is introduced, termed Embedded Extrusion-Volumetric Printing (EmVP), converging extrusion-bioprinting and layer-less, ultra-fast volumetric bioprinting, allowing spatially pattern multiple inks/cell types. Light-responsive microgels are developed for the first time as bioresins (µResins) for light-based volumetric bioprinting, providing a microporous environment permissive for cell homing and self-organization. Tuning the mechanical and optical properties of gelatin-based microparticles enables their use as support bath for suspended extrusion printing, in which features containing high cell densities can be easily introduced. µResins can be sculpted within seconds with tomographic light projections into centimeter-scale, granular hydrogel-based, convoluted constructs. Interstitial microvoids enhanced differentiation of multiple stem/progenitor cells (vascular, mesenchymal, neural), otherwise not possible with conventional bulk hydrogels. As proof-of-concept, EmVP is applied to create complex synthetic biology-inspired intercellular communication models, where adipocyte differentiation is regulated by optogenetic-engineered pancreatic cells. Overall, EmVP offers new avenues for producing regenerative grafts with biological functionality, and for developing engineered living systems and (metabolic) disease models.


Assuntos
Bioimpressão , Microgéis , Engenharia Tecidual/métodos , Hidrogéis , Bioimpressão/métodos , Impressão Tridimensional , Alicerces Teciduais
3.
Proc Natl Acad Sci U S A ; 119(28): e2118182119, 2022 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-35787055

RESUMO

X chromosome inactivation (XCI) is the process of silencing one of the X chromosomes in cells of the female mammal which ensures dosage compensation between the sexes. Although theoretically random in somatic tissues, the choice of which X chromosome is chosen to be inactivated can be biased in mice by genetic element(s) associated with the so-called X-controlling element (Xce). Although the Xce was first described and genetically localized nearly 40 y ago, its mode of action remains elusive. In the approach presented here, we identify a single long noncoding RNA (lncRNA) within the Xce locus, Lppnx, which may be the driving factor in the choice of which X chromosome will be inactivated in the developing female mouse embryo. Comparing weak and strong Xce alleles we show that Lppnx modulates the expression of Xist lncRNA, one of the key factors in XCI, by controlling the occupancy of pluripotency factors at Intron1 of Xist. This effect is counteracted by enhanced binding of Rex1 in DxPas34, another key element in XCI regulating the activity of Tsix lncRNA, the main antagonist of Xist, in the strong but not in the weak Xce allele. These results suggest that the different susceptibility for XCI observed in weak and strong Xce alleles results from differential transcription factor binding of Xist Intron 1 and DxPas34, and that Lppnx represents a decisive factor in explaining the action of the Xce.


Assuntos
RNA Longo não Codificante , Inativação do Cromossomo X , Alelos , Animais , Mecanismo Genético de Compensação de Dose , Feminino , Mamíferos/genética , Camundongos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Cromossomo X/genética
4.
Commun Biol ; 4(1): 478, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846535

RESUMO

Mutations in the gene encoding Lamin B receptor (LBR), a nuclear-membrane protein with sterol reductase activity, have been linked to rare human disorders. Phenotypes range from a benign blood disorder, such as Pelger-Huet anomaly (PHA), affecting the morphology and chromatin organization of white blood cells, to embryonic lethality as for Greenberg dysplasia (GRBGD). Existing PHA mouse models do not fully recapitulate the human phenotypes, hindering efforts to understand the molecular etiology of this disorder. Here we show, using CRISPR/Cas-9 gene editing technology, that a 236bp N-terminal deletion in the mouse Lbr gene, generating a protein missing the N-terminal domains of LBR, presents a superior model of human PHA. Further, we address recent reports of a link between Lbr and defects in X chromosome inactivation (XCI) and show that our mouse mutant displays minor X chromosome inactivation defects that do not lead to any overt phenotypes in vivo. We suggest that our N-terminal deletion model provides a valuable pre-clinical tool to the research community and will aid in further understanding the etiology of PHA and the diverse functions of LBR.


Assuntos
Anomalia de Pelger-Huët/genética , Receptores Citoplasmáticos e Nucleares/genética , Inativação do Cromossomo X/genética , Animais , Camundongos , Camundongos Knockout , Fenótipo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptor de Lamina B
5.
Commun Biol ; 4(1): 485, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33859315

RESUMO

Female mammals achieve dosage compensation by inactivating one of their two X chromosomes during development, a process entirely dependent on Xist, an X-linked long non-coding RNA (lncRNA). At the onset of X chromosome inactivation (XCI), Xist is up-regulated and spreads along the future inactive X chromosome. Contextually, it recruits repressive histone and DNA modifiers that transcriptionally silence the X chromosome. Xist regulation is tightly coupled to differentiation and its expression is under the control of both pluripotency and epigenetic factors. Recent evidence has suggested that chromatin remodelers accumulate at the X Inactivation Center (XIC) and here we demonstrate a new role for Chd8 in Xist regulation in differentiating ES cells, linked to its control and prevention of spurious transcription factor interactions occurring within Xist regulatory regions. Our findings have a broader relevance, in the context of complex, developmentally-regulated gene expression.


Assuntos
Proteínas de Ligação a DNA/genética , Inativação do Cromossomo X , Cromossomo X/genética , Animais , Proteínas de Ligação a DNA/metabolismo , Mecanismo Genético de Compensação de Dose , Feminino , Camundongos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo
6.
Science ; 336(6088): 1549-54, 2012 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-22723415

RESUMO

Telomerase activity controls telomere length and plays a pivotal role in stem cells, aging, and cancer. Here, we report a molecular link between Wnt/ß-catenin signaling and the expression of the telomerase subunit Tert. ß-Catenin-deficient mouse embryonic stem (ES) cells have short telomeres; conversely, ES cell expressing an activated form of ß-catenin (ß-cat(ΔEx3/+)) have long telomeres. We show that ß-catenin regulates Tert expression through the interaction with Klf4, a core component of the pluripotency transcriptional network. ß-Catenin binds to the Tert promoter in a mouse intestinal tumor model and in human carcinoma cells. We uncover a previously unknown link between the stem cell and oncogenic potential whereby ß-catenin regulates Tert expression, and thereby telomere length, which could be critical in human regenerative therapy and cancer.


Assuntos
Células-Tronco Adultas/metabolismo , Células-Tronco Embrionárias/metabolismo , Neoplasias/metabolismo , Telomerase/genética , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Neoplasias/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Telomerase/metabolismo , Telômero/metabolismo , Telômero/ultraestrutura , Homeostase do Telômero , Sítio de Iniciação de Transcrição , Proteínas Wnt/metabolismo , beta Catenina/genética
7.
Breast Cancer Res ; 13(5): R104, 2011 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-22030022

RESUMO

INTRODUCTION: E-cadherin (E-cad; cadherin 1) and N-cadherin (N-cad; cadherin 2) are the most prominent members of the cadherin family of cell adhesion molecules. Although they share many structural and functional features, they are expressed in an almost mutually exclusive manner in vivo. METHODS: To explore functional differences between the two cadherins in vivo, we recently generated a knock-in line in which N-cad is expressed from the E-cad locus. In combination with a conditional gene inactivation approach, we expressed N-cad in the absence of E-cad (referred to as Ncadk.i.) in alveolar epithelial cells of the mammary gland starting in late pregnancy. RESULTS: We found that the sole presence of N-cad induces constitutively active fibroblast growth factor (Fgf) signaling and a precocious involution resulting in massive apoptosis of alveolar cells. To block apoptosis, we conditionally deleted one allele of p53 in Ncadk.i. mice and observed a temporal rescue of alveolar morphology and function. However, an accumulation of fibrotic tissue and cysts with increasing age and lactation cycles was observed. This phenotype closely resembled fibrocystic mastopathy (FM), a common disorder in humans, which is thought to precede breast cancer. Concordantly, 55% of Ncadk.i. mice harboring a heterozygous p53 deletion developed malignant and invasive tumors. CONCLUSIONS: Our results demonstrate a possible role for N-cad in the formation of fibrosis and cysts in the mammary gland. Moreover, we show that these lesions precede the development of malignant tumors. Thus, we provide a new mouse model to investigate the molecular mechanisms of fibrocystic mastopathy and the transition from benign to malignant tumors.


Assuntos
Cisto Mamário/genética , Caderinas/genética , Glândulas Mamárias Animais/patologia , Fatores Etários , Animais , Apoptose/genética , Cisto Mamário/patologia , Caderinas/metabolismo , Movimento Celular/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Doença da Mama Fibrocística/genética , Doença da Mama Fibrocística/patologia , Fibrose/genética , Inativação Gênica , Genes p53 , Lactação/genética , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Transgênicos , Gravidez
8.
Development ; 137(14): 2297-305, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20534673

RESUMO

We conditionally substituted E-cadherin (E-cad; cadherin 1) with N-cadherin (N-cad; cadherin 2) during intestine development by generating mice in which an Ncad cDNA was knocked into the Ecad locus. Mutant mice were born, demonstrating that N-cad can structurally replace E-cad and establish proper organ architecture. After birth, mutant mice gradually developed a mutant phenotype in both the small and large intestine and died at ~2-3 weeks of age, probably due to malnutrition during the transition to solid food. Molecular analysis revealed an extended domain of cells from the crypt into the villus region, with nuclear localization of beta-catenin (beta-cat; Ctnnb1) and enhanced expression of several beta-cat target genes. In addition, the BMP signaling pathway was suppressed in the intestinal epithelium of the villi, suggesting that N-cad might interfere with BMP signaling in the intestinal epithelial cell layer. Interestingly, mutant mice developed severe dysplasia and clusters of cells with neoplastic features scattered along the crypt-villus axis in the small and large intestine. Our experimental model indicates that, in the absence of E-cad, the sole expression of N-cad in an epithelial environment is sufficient to induce neoplastic transformations.


Assuntos
Caderinas/genética , Caderinas/metabolismo , Mucosa Intestinal/metabolismo , Animais , DNA Complementar/metabolismo , Células Epiteliais/metabolismo , Camundongos , Camundongos Transgênicos , Pólipos/genética , Pólipos/metabolismo , Transdução de Sinais/genética , beta Catenina/genética , beta Catenina/metabolismo
9.
Dev Dyn ; 239(1): 191-9, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19705445

RESUMO

beta-Catenin plays two major roles during the development of multicellular organisms. It is the downstream effector of the canonical Wnt signaling cascade, which is involved in many developmental processes and in tumor formation. Additionally, it is linked to classic cadherins and is required for the correct assembly and function of adherens junctions. beta-Catenin loss of function mutants show early gastrulation lethality. To address the role of beta-catenin in postgastrulation stages and to overcome the early embryonic lethality, we performed conditional gene targeting, using Cdx1::Cre, a newly established mouse line. By this approach, beta-catenin was depleted in the entire posterior embryo after the gastrulation process at embryonic day 8.0, when the three germ layers were established. We observed defects in signaling and adhesion which are temporarily separated. At an early event, known targets of Wnt/beta-catenin are down-regulated in the paraxial mesoderm. Moreover, Fgf8 and Wnt3a, the key players of the segmentation process, are down-regulated in the neural ectoderm (NE). Wnt3a expression was rescued in mutant embryos by exogenous Fgf and inhibition of Fgf signaling in wild-type embryos resulted in Wnt3a down-regulation. Based on these results, we assume the existence of an autoregulatory feedback loop in the NE where Fgf8 regulates Wnt3a, which in turn, by means of beta-catenin, maintains Fgf8 expression. In later stages, the lack of beta-catenin caused a progressive posterior disintegration. We found that beta-catenin is required for the correct localization of N-cadherin at the membrane of neural ectodermal cells and that its absence causes a disintegration of the neural tube.


Assuntos
Adesão Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Tubo Neural/embriologia , Transdução de Sinais/fisiologia , beta Catenina/metabolismo , Animais , Fator 8 de Crescimento de Fibroblasto/metabolismo , Marcação de Genes , Imuno-Histoquímica , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Mesoderma/metabolismo , Camundongos , Tubo Neural/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt3 , Proteína Wnt3A , beta Catenina/genética
10.
Genesis ; 47(3): 204-9, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19241391

RESUMO

Transgenic mice with a defined cell- or tissues-specific expression of Cre-recombinase are essential tools to study gene function. Here we report the generation and analysis of a transgenic mouse line (Cdx1::Cre) with restricted Cre-expression from Cdx1 regulatory elements. The expression of Cre-recombinase mimicked the endogenous expression pattern of Cdx1 at midgastrulation (from E7.5 to early headfold stage) inducing recombination in the three germlayers of the primitive streak region throughout the posterior embryo and caudal to the heart. This enables gene modifications to investigate patterning of the caudal embryo during and after gastrulation. Interestingly, we identified Cdx1 expression in the trophectoderm (TE) of blastocyst stage embryos. Concordantly, we detected extensive Cre-mediated recombination in the polar TE and, although to lesser extent, in the mural TE. In E7.5 postimplantation embryos, almost all cells of the extraembryonic ectoderm (ExE), which are derived from the polar TE, are recombined although the ExE itself is negative for Cdx1 and Cre at this stage. These results indicate that Cdx1::Cre mice are also a valuable tool to study gene function in tissues essential for placental development.


Assuntos
Blastocisto/metabolismo , Ectoderma/embriologia , Gastrulação/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/biossíntese , Integrases/biossíntese , Alelos , Animais , Blastocisto/citologia , Ectoderma/citologia , Implantação do Embrião/fisiologia , Proteínas de Homeodomínio/genética , Integrases/genética , Camundongos , Camundongos Transgênicos
11.
Development ; 131(23): 5817-24, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15525667

RESUMO

Many components of the Wnt/beta-catenin signaling pathway are expressed during mouse pre-implantation embryo development, suggesting that this pathway may control cell proliferation and differentiation at this time. We find no evidence for a functional activity of this pathway in cleavage-stage embryos using the Wnt-reporter line, BAT-gal. To further probe the activity of this pathway, we activated beta-catenin signaling by mating a zona pellucida3-cre (Zp3-cre) transgenic mouse line with a mouse line containing an exon3-floxed beta-catenin allele. The result is expression of a stabilized form of beta-catenin, resistant to degradation by the GSK3beta-mediated proteasome pathway, expressed in the developing oocyte and in each cell of the resulting embryos. Nuclear localization and signaling function of beta-catenin were not observed in cleavage-stage embryos derived from these oocytes. These results indicate that in pre-implantation embryos, molecular mechanisms independent of the GSK3beta-mediated ubiquitination and proteasome degradation pathway inhibit the nuclear function of beta-catenin. Although the mutant blastocysts initially developed normally, they then exhibited a specific phenotype in the embryonic ectoderm layer of early post-implantation embryos. We show a nuclear function of beta-catenin in the mutant epiblast that leads to activation of Wnt/beta-catenin target genes. As a consequence, cells of the embryonic ectoderm change their fate, resulting in a premature epithelial-mesenchymal transition.


Assuntos
Blastocisto/metabolismo , Proteínas do Citoesqueleto/química , Epitélio/metabolismo , Mesoderma/metabolismo , Transativadores/química , Zigoto/metabolismo , Alelos , Animais , Padronização Corporal , Diferenciação Celular , Núcleo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Ectoderma/metabolismo , Éxons , Genes Reporter , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Imuno-Histoquímica , Hibridização In Situ , Óperon Lac , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Oócitos/metabolismo , Fenótipo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , Fatores de Tempo , Transativadores/metabolismo , Transgenes , Ubiquitina/metabolismo , beta Catenina , beta-Galactosidase/metabolismo
12.
Mech Dev ; 115(1-2): 53-62, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12049767

RESUMO

The Ca(++)-dependent cell adhesion molecule E-cadherin is expressed throughout mouse development and in adult tissues. Classical gene targeting has demonstrated that E-cadherin-deficient embryos die at the blastocyst stage. To study the involvement of E-cadherin in organogenesis, a conditional gene inactivation scheme was undertaken using the bacteriophage P1 recombinase Cre/loxP system. Mice with homozygous loxP sites in both alleles of the E-cadherin (Cdh1) gene were generated and these mice were crossed with transgenic mice with the Cre recombinase under the control of the hormone-inducible MMTV promoter. This resulted in deletion of the E-cadherin gene in the differentiating alveolar epithelial cells of the mammary gland. The mutant mammary gland developed normally up to 16-18 days of pregnancy but exhibited a dramatic phenotype around parturition. The production of milk proteins was so drastically reduced that adult mutant mothers could not suckle their offspring. Thus, the lack of E-cadherin affected the terminal differentiation program of the lactating mammary gland. In concordance with this finding, the prolactin-dependent activation of the transcription factor Stat5a was initiated but not maintained in the mutant gland. Instead, without E-cadherin massive cell death was observed at parturition and the mutant mammary gland at this stage resembled that of the involuted gland normally seen after weaning. These results demonstrate an essential role for E-cadherin in the function of differentiated alveolar epithelial cells. No tumors were detected in mutant glands lacking E-cadherin.


Assuntos
Mama/fisiologia , Caderinas/fisiologia , Lactação/fisiologia , Animais , Apoptose , Mama/citologia , Caderinas/genética , Diferenciação Celular , Células Epiteliais/citologia , Feminino , Masculino , Camundongos , Camundongos Transgênicos
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