RESUMO
Nucleosome positioning can alter the accessibility of DNA-binding proteins to their cognate DNA elements, and thus its precise control is essential for cell identity and function. Mammalian preimplantation embryos undergo temporal changes in gene expression and cell potency, suggesting the involvement of dynamic epigenetic control during this developmental phase. However, the dynamics of nucleosome organization during early development are poorly understood. In this study, using a low-input MNase-seq method, we show that nucleosome positioning is globally obscure in zygotes but becomes well defined during subsequent development. Down-regulation of the chromatin assembly in embryonic stem cells can partially reverse nucleosome organization into a zygote-like pattern, suggesting a possible link between the chromatin assembly pathway and fuzzy nucleosomes in zygotes. We also reveal that YY1, a zinc finger-containing transcription factor expressed upon zygotic genome activation, regulates the de novo formation of well-positioned nucleosome arrays at the regulatory elements through identifying YY1-binding sites in eight-cell embryos. The YY1-binding regions acquire H3K27ac enrichment around the eight-cell and morula stages, and YY1 depletion impairs the morula-to-blastocyst transition. Thus, our study delineates the remodeling of nucleosome organization and its underlying mechanism during early mouse development.
Assuntos
Nucleossomos , Fatores de Transcrição , Animais , Camundongos , Cromatina , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Mamíferos/genética , Nucleossomos/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Early organogenesis represents a key step in animal development, during which pluripotent cells diversify to initiate organ formation. Here, we sampled 300,000 single-cell transcriptomes from mouse embryos between E8.5 and E9.5 in 6-h intervals and combined this new dataset with our previous atlas (E6.5-E8.5) to produce a densely sampled timecourse of >400,000 cells from early gastrulation to organogenesis. Computational lineage reconstruction identified complex waves of blood and endothelial development, including a new programme for somite-derived endothelium. We also dissected the E7.5 primitive streak into four adjacent regions, performed scRNA-seq and predicted cell fates computationally. Finally, we defined developmental state/fate relationships by combining orthotopic grafting, microscopic analysis and scRNA-seq to transcriptionally determine cell fates of grafted primitive streak regions after 24â h of in vitro embryo culture. Experimentally determined fate outcomes were in good agreement with computationally predicted fates, demonstrating how classical grafting experiments can be revisited to establish high-resolution cell state/fate relationships. Such interdisciplinary approaches will benefit future studies in developmental biology and guide the in vitro production of cells for organ regeneration and repair.
Assuntos
Gastrulação , Organogênese , Camundongos , Animais , Diferenciação Celular , Organogênese/genética , Linha Primitiva , Endotélio , Embrião de Mamíferos , MamíferosRESUMO
Signaling pathways regulate the patterns of Hox gene expression that underlie their functions in the specification of axial identity. Little is known about the properties of cis-regulatory elements and underlying transcriptional mechanisms that integrate graded signaling inputs to coordinately control Hox expression. Here, we optimized a single molecule fluorescent in situ hybridization (smFISH) technique with probes spanning introns to evaluate how three shared retinoic acid response element (RARE)-dependent enhancers in the Hoxb cluster regulate patterns of nascent transcription in vivo at the level of single cells in wild-type and mutant embryos. We predominately detect nascent transcription of only a single Hoxb gene in each cell, with no evidence for simultaneous co-transcriptional coupling of all or specific subsets of genes. Single and/or compound RARE mutations indicate that each enhancer differentially impacts global and local patterns of nascent transcription, suggesting that selectivity and competitive interactions between these enhancers is important to robustly maintain the proper levels and patterns of nascent Hoxb transcription. This implies that rapid and dynamic regulatory interactions potentiate transcription of genes through combined inputs from these enhancers in coordinating the retinoic acid response.
Assuntos
Proteínas de Homeodomínio , Tretinoína , Camundongos , Animais , Tretinoína/metabolismo , Proteínas de Homeodomínio/metabolismo , Camundongos Transgênicos , Tubo Neural/metabolismo , Hibridização in Situ Fluorescente , Elementos Facilitadores GenéticosRESUMO
Tfap2b, a pivotal transcription factor, plays critical roles within neural crest cells and their derived lineage. To unravel the intricate lineage dynamics and contribution of these Tfap2b+ cells during craniofacial development, we established a Tfap2b-CreERT2 knock-in transgenic mouse line using the CRISPR-Cas9-mediated homologous direct repair. By breeding with tdTomato reporter mice and initiating Cre activity through tamoxifen induction at distinct developmental time points, we show the Tfap2b lineage within the key neural crest-derived domains, such as the facial mesenchyme, midbrain, cerebellum, spinal cord, and limbs. Notably, the migratory neurons stemming from the dorsal root ganglia are visible subsequent to Cre activity initiated at E8.5. Intriguingly, Tfap2b+ cells, serving as the progenitors for limb development, show activity predominantly commencing at E10.5. Across the mouse craniofacial landscape, Tfap2b exhibits a widespread presence throughout the facial organs. Here we validate its role as a marker of progenitors in tooth development and have confirmed that this process initiates from E12.5. Our study not only validates the Tfap2b-CreERT2 transgenic line, but also provides a powerful tool for lineage tracing and genetic targeting of Tfap2b-expressing cells and their progenitor in a temporally and spatially regulated manner during the intricate process of development and organogenesis.
Assuntos
Sistemas CRISPR-Cas , Tamoxifeno , Camundongos , Animais , Tamoxifeno/farmacologia , Camundongos Transgênicos , Proteína Vermelha Fluorescente , Integrases/genética , Integrases/metabolismoRESUMO
The primordia of the post-otic mouse embryo forms largely from a bipotential cell population containing neuromesodermal progenitors (NMP) which reside in the tail bud and contribute to the elaboration of the major body axis after gastrulation. The mechanisms by which the NMP population is both maintained and subsequently directed down mesodermal and neural lineages is incompletely understood. The vertebrate transcription factor Cdx2, is essential for axial elongation and has been implicated in maintaining the NMP niche and in specification of NMP derivatives. To better understand the role of the Cdx family in axial elongation, we employed a conditional mutant allele which evokes total loss of Cdx function, and enriched for tail bud progenitors through the use of a Pax2-GFP transgenic reporter. Using this approach, we identified 349 Cdx-dependent genes by RNA sequencing (RNA-seq). From these, Gene Ontology and chromatin immunoprecipitation analysis further revealed a number of putative direct Cdx candidate target genes implicated in axial elongation, including Sp8, Isl1, Evx1, Zic3 and Nr2f1. Additional analysis of available single-cell RNA-seq data from mouse tail buds revealed the co-expression of Sp8, Isl1, Evx1 and Zic3 with Cdx2 in putative NMP cells, while Nr2f1 was excluded from this population. These findings identify a number of novel Cdx targets and provide further insight into the critical roles for Cdx in elaborating the post-otic embryo.
Assuntos
Padronização Corporal/genética , Fator de Transcrição CDX2/metabolismo , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Proteínas de Homeodomínio/metabolismo , Transdução de Sinais/genética , Animais , Fator de Transcrição CDX2/genética , Fator I de Transcrição COUP/genética , Imunoprecipitação da Cromatina/métodos , Proteínas de Ligação a DNA/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM/genética , Masculino , Mesoderma/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , RNA-Seq/métodos , Células-Tronco/metabolismo , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: H3K9me3 and DNA methylation co-marked CpG-rich regions (CHMs) are functionally important in mouse pre-implantation embryos, but their characteristics in other biological processes are still largely unknown. RESULTS: In this study, we performed a comprehensive analysis to characterize CHMs during 6 mouse developmental processes, identifying over 2,600 CHMs exhibiting stable co-mark of H3K9me3 and DNA methylation patterns at CpG-rich regions. We revealed the distinctive features of CHMs, including elevated H3K9me3 signals and a significant presence in euchromatin and the potential role in silencing younger long terminal repeats (LTRs), especially in some ERVK subfamilies. The results highlight the distinct nature of universal CHMs compared to CpG-rich nonCHMs in terms of location, LTR enrichment, and DNA sequence features, enhancing our understanding of CpG-rich regions' regulatory roles. CONCLUSIONS: This study characterizes the features of CHMs in multiple developmental processes and broadens our understanding of the regulatory roles of CpG-rich regions.
Assuntos
Metilação de DNA , Histonas , Animais , Camundongos , Histonas/metabolismo , DNA , Sequências Repetidas Terminais , Ilhas de CpGRESUMO
Many loci maintain parent-of-origin DNA methylation only briefly after fertilization during mammalian development: Whether this form of transient genomic imprinting can impact the early embryonic transcriptome or even have life-long consequences on genome regulation and possibly phenotypes is currently unknown. Here, we report a maternal germline differentially methylated region (DMR) at the mouse Gpr1/Zdbf2 (DBF-type zinc finger-containing protein 2) locus, which controls the paternal-specific expression of long isoforms of Zdbf2 (Liz) in the early embryo. This DMR loses parental specificity by gain of DNA methylation at implantation in the embryo but is maintained in extraembryonic tissues. As a consequence of this transient, tissue-specific maternal imprinting, Liz expression is restricted to the pluripotent embryo, extraembryonic tissues, and pluripotent male germ cells. We found that Liz potentially functions as both Zdbf2-coding RNA and cis-regulatory RNA. Importantly, Liz-mediated events allow a switch from maternal to paternal imprinted DNA methylation and from Liz to canonical Zdbf2 promoter use during embryonic differentiation, which are stably maintained through somatic life and conserved in humans. The Gpr1/Zdbf2 locus lacks classical imprinting histone modifications, but analysis of mutant embryonic stem cells reveals fine-tuned regulation of Zdbf2 dosage through DNA and H3K27 methylation interplay. Together, our work underlines the developmental and evolutionary need to ensure proper Liz/Zdbf2 dosage as a driving force for dynamic genomic imprinting at the Gpr1/Zdbf2 locus.
Assuntos
Metilação de DNA , Impressão Genômica/genética , Mamíferos/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Animais , Células-Tronco Embrionárias/metabolismo , Evolução Molecular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Humanos , Masculino , Mamíferos/embriologia , Mamíferos/metabolismo , Camundongos , Regiões Promotoras Genéticas , Espermatogênese/genéticaRESUMO
X inactivation in mammals is regulated by epigenetic modifications. Functional deficiency of SmcHD1 has been shown to cause de-repression of X-inactivated genes in post-implantation female mouse embryos, suggesting a role of SmcHD1 in the maintenance of X inactivation. Here, we show that de-repression of X-inactivated genes accompanied a local reduction in the enrichment of H3K27me3 in mouse embryonic fibroblasts deficient for SmcHD1. Furthermore, many of these genes overlapped with those having a significantly lower enrichment of H3K27me3 at the blastocyst stage in wild type. Intriguingly, however, depletion of SmcHD1 did not compromise the X-inactivated state in immortalized female mouse embryonic fibroblasts, in which X inactivation had been established and maintained. Taking all these findings together, we suggest that SmcHD1 facilitates the incorporation of H3K27me3 and perhaps other epigenetic modifications at gene loci that are silenced even with the lower enrichment of H3K27me3 at the early stage of X inactivation. The epigenetic state at these loci would, however, remain as it is at the blastocyst stage in the absence of SmcHD1 after implantation, which would eventually compromise the maintenance of the X-inactivated state at later stages.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Epigênese Genética/genética , Genes Ligados ao Cromossomo X/genética , Inativação do Cromossomo X/genética , Animais , Células Cultivadas , Proteínas Cromossômicas não Histona/genética , Embrião de Mamíferos/embriologia , Feminino , Fibroblastos/citologia , Histonas/genética , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
The orientation of mouse hair follicles is controlled by the planar cell polarity (PCP) pathway. Mutations in PCP genes result in two categories of hair mis-orientation phenotype: randomly oriented and vertically oriented to the skin surface. Here, we demonstrate that the randomly oriented hair phenotype observed in frizzled 6 (Fzd6) mutants results from a partial loss of the polarity, due to the functional redundancy of another closely related frizzled gene, Fzd3 Double knockout of Fzd3 and Fzd6 globally, or only in the skin, led to vertically oriented hair follicles and a total loss of anterior-posterior polarity. Furthermore, we provide evidence that, contrary to the prevailing model, asymmetrical localization of the Fzd6 protein is not observed in skin epithelial cells. Through transcriptome analyses and in vitro studies, we show collagen triple helix repeat containing 1 (Cthrc1) to be a potential downstream effector of Fzd6, but not of Fzd3. Cthrc1 binds directly to the extracellular domains of Fzd3 and Fzd6 to enhance the Wnt/PCP signaling. These results suggest that Fzd3 and Fzd6 play a redundant role in controlling the polarity of developing skin, but through non-identical mechanisms.
Assuntos
Polaridade Celular , Receptores Frizzled/metabolismo , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Animais , Animais Recém-Nascidos , Células Epiteliais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Retroalimentação Fisiológica , Células HEK293 , Humanos , Integrases/metabolismo , Camundongos Knockout , Modelos Biológicos , Mosaicismo , Ligação Proteica , Transdução de Sinais , Pele/citologiaRESUMO
The GATA family of transcription factors is of crucial importance during embryonic development, playing complex and widespread roles in cell fate decisions and tissue morphogenesis. GATA proteins are essential for the development of tissues derived from all three germ layers, including the skin, brain, gonads, liver, hematopoietic, cardiovascular and urogenital systems. The crucial activity of GATA factors is underscored by the fact that inactivating mutations in most GATA members lead to embryonic lethality in mouse models and are often associated with developmental diseases in humans. In this Primer, we discuss the unique and redundant functions of GATA proteins in tissue morphogenesis, with an emphasis on their regulation of lineage specification and early organogenesis.
Assuntos
Doença , Fatores de Transcrição GATA/metabolismo , Crescimento e Desenvolvimento , Animais , Diferenciação Celular , Humanos , Organogênese , Transcrição GênicaRESUMO
To determine the developmental stage of embryonic mice, we apply a geometric morphometric approach to the changing shape of the mouse limb bud as it grows from embryonic day 10 to embryonic day 15 post-conception. As the ontogenetic sequence results in the de novo emergence of shape features not present in the early stages, we have created a standard ontogenetic trajectory for limb bud development - a quantitative characterization of shape change during limb morphogenesis. This trajectory of form as a function of time also gives us the reverse function: the ability to infer developmental stage from form, with a typical uncertainty of 2â h. We introduce eMOSS (embryonic mouse ontogenetic staging system) as a fast, reliable, convenient and freely available online tool for staging embryos from two-dimensional images of their limb buds, and illustrate its use in phenotyping early limb abnormalities.
Assuntos
Embrião de Mamíferos/embriologia , Idade Gestacional , Membro Posterior/embriologia , Botões de Extremidades/embriologia , Morfogênese/fisiologia , Algoritmos , Animais , Regulação da Expressão Gênica no Desenvolvimento , CamundongosRESUMO
In addition to its function as an inhibitor of histone acetyltransferases, Nir (Noc2l) binds to p53 and TAp63 to regulate their activity. Here, we show that epidermis-specific ablation of Nir impairs epidermal stratification and barrier function, resulting in perinatal lethality. Nir-deficient epidermis lacks appendages and remains single layered during embryogenesis. Cell proliferation is inhibited, whereas apoptosis and p53 acetylation are increased, indicating that Nir is controlling cell proliferation by limiting p53 acetylation. Transcriptome analysis revealed that Nir regulates the expression of essential factors in epidermis development, such as keratins, integrins and laminins. Furthermore, Nir binds to and controls the expression of p63 and limits H3K18ac at the p63 promoter. Corroborating the stratification defects, asymmetric cell divisions were virtually absent in Nir-deficient mice, suggesting that Nir is required for correct mitotic spindle orientation. In summary, our data define Nir as a key regulator of skin development.
Assuntos
Epiderme/metabolismo , Histona Acetiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Animais , Apoptose/genética , Divisão Celular Assimétrica/genética , Técnicas de Cultura de Células , Divisão Celular , Proliferação de Células/genética , Imunoprecipitação da Cromatina , Epiderme/crescimento & desenvolvimento , Imunofluorescência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mouse Developmental and Metabolic Phenotype Repository (MDMPR) is an open access, real-time database which dedicates to share mouse resources and phenotype data. MDMPR is supported by the National Key Research and Development Project "Establishment of Mouse Developmental and Metabolic Phenotype Repository" within the Key Project of "Developmental Programming and Its Metabolic Regulation" from the Ministry of Science and Technology of the People's Republic of China's program. In the next 5 years, MDMPR will create 500 mutant mouse models related to development and metabolism, perform standard phenotyping analysis, and establish a phenotype database. MDMPR is a combination of resources and data repository, has several sub-systems, including the ES cell database, the project management system, the breeding management system, the sperm bank management system and the phenotyping database. These systems digitalize all data and ensure their authenticity in real-time. Besides the gradual increase of data during the project, MDMPR will also integrate other resources, such as human KO ES cell database, STRING database, database of Core Transcriptional Regulatory Circuitries and Enhancer-Indel database. MDMPR is anticipated to contribute to various areas of developmental and metabolic research to investigators through more convenient accesses to the resources and data in one-stop manner, thereby accelerating the research processes and ultimately serving the medical causes of human health.
Assuntos
Fenótipo , Animais , Bases de Dados Factuais , Modelos Animais de Doenças , CamundongosRESUMO
Elastic fibres provide tissues with elasticity and flexibility. In the healthy human cornea, elastic fibres are limited to the posterior region of the peripheral stroma, but their specific functional role remains elusive. Here, we examine the physical and structural characteristics of the cornea during development in the mgΔloxPneo dominant-negative mouse model for Marfan syndrome, in which the physiological extracellular matrix of its elastic-fibre rich tissues is disrupted by the presence of a dysfunctional fibrillin-1 glycoprotein. Optical coherence tomography demonstrated a reduced corneal thickness in the mutant compared to wild type mice from embryonic day 16.5 until adulthood. X-ray scattering and electron microscopy revealed a disruption to both the elastic fibre and collagen fibril ultrastructure in the knockout mice, as well as abnormally low levels of the proteoglycan decorin. It is suggested that these alterations might be a result of increased transforming growth factor beta signalling. To conclude, this study has demonstrated corneal structure and ultrastructure to be altered when fibrillin-1 is disrupted and has provided insights into the role of fibrillin-1 in developing a functional cornea.
Assuntos
Córnea/anormalidades , Matriz Extracelular/metabolismo , Síndrome de Marfan/patologia , Animais , Córnea/metabolismo , Córnea/ultraestrutura , Modelos Animais de Doenças , Elasticidade , Feminino , Fibrilina-1/metabolismo , Síndrome de Marfan/metabolismo , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Tomografia de Coerência Óptica/métodosRESUMO
A sequencing-based profiling method (RiboMeth-seq) for ribose methylations was used to study methylation patterns in mouse adult tissues and during development. In contrast to previous reports based on studies of human cancer cell lines, almost all methylation sites were close to fully methylated in adult tissues. A subset of sites was differentially modified in developing tissues compared to their adult counterparts and showed clear developmental dynamics. This provides the first evidence for ribosome heterogeneity at the level of rRNA modifications during mouse development. In a prominent example, the expression levels of SNORD78 during development appeared to be regulated by alternative splicing of the Gas5 host-gene and to correlate with the methylation level of its target site at LSU-G4593. The results are discussed in the context of the specialized ribosome hypothesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Ribose/metabolismo , Processamento Alternativo , Animais , Biologia Computacional/métodos , Desenvolvimento Embrionário/genética , Perfilação da Expressão Gênica , Íntrons , Metilação , Camundongos , Especificidade de Órgãos/genéticaRESUMO
The BMP pathway regulates developmental processes including angiogenesis, yet its signaling outputs are complex and context-dependent. Recently, we showed that SMAD6, an intracellular BMP inhibitor expressed in endothelial cells, decreases vessel sprouting and branching both in vitro and in zebrafish. Genetic deletion of SMAD6 in mice results in poorly characterized cardiovascular defects and lethality. Here, we analyzed the effects of SMAD6 loss on vascular function during murine development. SMAD6 was expressed in a subset of blood vessels throughout development, primarily in arteries, while expression outside of the vasculature was largely confined to developing cardiac valves with no obvious embryonic phenotype. Mice deficient in SMAD6 died during late gestation and early stages of postnatal development, and this lethality was associated with vessel hemorrhage. Mice that survived past birth had increased branching and sprouting of developing postnatal retinal vessels and disorganized tight and adherens junctions. In vitro, knockdown of SMAD6 led to abnormal endothelial cell adherens junctions and increased VE-cadherin endocytosis, indicative of activated endothelium. Thus, SMAD6 is essential for proper blood vessel function during murine development, where it appears to stabilize endothelial junctions to prevent hemorrhage and aberrant angiogenesis.
Assuntos
Vasos Sanguíneos/fisiologia , Proteína Smad6/genética , Proteína Smad6/fisiologia , Junções Aderentes/metabolismo , Animais , Artérias/metabolismo , Vasos Sanguíneos/metabolismo , Células Endoteliais/fisiologia , Endotélio Vascular/metabolismo , Hemorragia/sangue , Junções Intercelulares/fisiologia , Camundongos , Neovascularização Patológica/genética , Neovascularização Fisiológica/genética , Vasos Retinianos , Transdução de SinaisRESUMO
Smoothened (Smo) is the essential transducer of Sonic hedgehog (Shh) signaling, which regulates cell fate and proliferation during embryogenesis. We identified a novel mouse mutant, cabbie (cbb), and found that its cause is a missense mutation in Smo. We showed the Smocbb mutation is insensitive to the Shh agonist SAG, perhaps due to the disruption of SAG binding. We characterized Smocbb for defects in craniofacial and skeletal development, as well as neural tube patterning, and revealed Smocbb affected processes that require the highest levels of Shh activity. Smo is normally enriched in cilia upon Shh stimulation; however, we detected inefficient enrichment of Smo in Smocbb mutants whether we stimulated with Shh or SAG. Taken together, our data suggest that the highest levels of vertebrate Hedgehog signaling activity require efficient Smo ciliary enrichment.
Assuntos
Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Receptor Smoothened/genética , Animais , Padronização Corporal/genética , Técnicas de Cultura de Células , Camundongos , Mutação , Organogênese/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Vertebrados/metabolismoRESUMO
Pluripotent stem cells can be classified into two distinct states, naïve and primed, which show different degrees of potency. One difficulty in stem cell research is the inability to distinguish these states in live cells. Studies on female mice have shown that reactivation of inactive X chromosomes occurs in the naïve state, while one of the X chromosomes is inactivated in the primed state. Therefore, we aimed to distinguish the two states by monitoring X chromosome reactivation. Thus far, X chromosome reactivation has been analysed using fixed cells; here, we inserted different fluorescent reporter gene cassettes (mCherry and eGFP) into each X chromosome. Using these knock-in 'Momiji' mice, we detected X chromosome reactivation accurately in live embryos, and confirmed that the pluripotent states of embryos were stable ex vivo, as represented by embryonic and epiblast stem cells in terms of X chromosome reactivation. Thus, Momiji mice provide a simple and accurate method for identifying stem cell status based on X chromosome reactivation.
Assuntos
Embrião de Mamíferos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Inativação do Cromossomo X/fisiologia , Cromossomo X/metabolismo , Animais , Feminino , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Camundongos , Camundongos Mutantes , Fosfoglicerato Quinase/genética , Fosfoglicerato Quinase/metabolismo , Células-Tronco Pluripotentes/citologia , Cromossomo X/genética , Inativação do Cromossomo X/genéticaRESUMO
Adenosine to inosine RNA editing in protein-coding messenger RNAs (mRNAs) potentially leads to changes in the amino acid composition of the encoded proteins. The mRNAs encoding the ubiquitously expressed actin-crosslinking proteins Filamin A and Filamin B undergo RNA editing leading to a highly conserved glutamine to arginine exchange at the identical position in either protein. Here, by targeted amplicon sequencing we analysed the RNA editing of Filamin B across several mouse tissues during post-natal development. We find highest filamin B editing levels in skeletal muscles, cartilage and bones, tissues where Filamin B function seems most important. Through the analysis of Filamin B editing in mice deficient in either ADAR1 or 2, we identified ADAR2 as the enzyme responsible for Filamin B RNA editing. We show that in neuronal tissues Filamin B editing drops in spliced transcripts indicating regulated maturation of edited transcripts. We show further that the variability of Filamin B editing across several organs correlates with its mRNA expression.
Assuntos
Osso e Ossos/metabolismo , Cartilagem/metabolismo , Filaminas/genética , Músculo Esquelético/metabolismo , Edição de RNA , RNA Mensageiro/metabolismo , Adenosina/genética , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Humanos , Inosina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
In mammals, hair follicles cover most of the body surface and exhibit precise and stereotyped orientations relative to the body axes. Follicle orientation is controlled by the planar cell polarity (PCP; or, more generally, tissue polarity) system, as determined by the follicle mis-orientation phenotypes observed in mice with PCP gene mutations. The present study uses conditional knockout alleles of the PCP genes Frizzled6 (Fz6), Vangl1, and Vangl2, together with a series of Cre drivers to interrogate the spatio-temporal domains of PCP gene action in the developing mouse epidermis required for follicle orientation. Fz6 is required starting between embryonic day (E)11.5 and E12.5. Eliminating Fz6 in either the anterior or the posterior halves of the embryo or in either the feet or the torso leads to follicle mis-orientation phenotypes that are limited to the territories associated with Fz6 loss, implying either that PCP signaling is required for communicating polarity information on a local but not a global scale, or that there are multiple independent sources of global polarity information. Eliminating Fz6 in most hair follicle cells or in the inter-follicular epidermis at E15.5 suggests that PCP signaling in developing follicles is not required to maintain their orientation. The asymmetric arrangement of Merkel cells around the base of each guard hair follicle dependents on Fz6 expression in the epidermis but not in differentiating Merkel cells. These experiments constrain current models of PCP signaling and the flow of polarity information in mammalian skin.