RESUMEN

In mammals, early embryonic development exhibits highly unusual spatial positioning of genomic regions at the nuclear lamina, but the mechanisms underpinning this atypical genome organization remain elusive. Here, we generated single-cell profiles of lamina-associated domains (LADs) coupled with transcriptomics, which revealed a striking overlap between preimplantation-specific LAD dissociation and noncanonical broad domains of H3K27me3. Loss of H3K27me3 resulted in a restoration of canonical LAD profiles, suggesting an antagonistic relationship between lamina association and H3K27me3. Tethering of H3K27me3 to the nuclear periphery showed that the resultant relocalization is partially dependent on the underlying DNA sequence. Collectively, our results suggest that the atypical organization of LADs in early developmental stages is the result of a tug-of-war between intrinsic affinity for the nuclear lamina and H3K27me3, constrained by the available space at the nuclear periphery. This study provides detailed insight into the molecular mechanisms regulating nuclear organization during early mammalian development.

RESUMEN

Recent studies have reported the differentiation of pluripotent cells into oocytes in vitro. However, the developmental competence of in vitro-generated oocytes remains low. Here, we perform a comprehensive comparison of mouse germ cell development in vitro over all culture steps versus in vivo with the goal to understand mechanisms underlying poor oocyte quality. We show that the in vitro differentiation of primordial germ cells to growing oocytes and subsequent follicle growth is critical for competence for preimplantation development. Systematic transcriptome analysis of single oocytes that were subjected to different culture steps identifies genes that are normally upregulated during oocyte growth to be susceptible for misregulation during in vitro oogenesis. Many misregulated genes are Polycomb targets. Deregulation of Polycomb repression is therefore a key cause and the earliest defect known in in vitro oocyte differentiation. Conversely, structurally normal in vitro-derived oocytes fail at zygotic genome activation and show abnormal acquisition of 5-hydroxymethylcytosine on maternal chromosomes. Our data identify epigenetic regulation at an early stage of oogenesis limiting developmental competence and suggest opportunities for future improvements.

Asunto(s)

Epigénesis Genética , Oocitos , Femenino , Animales , Ratones , Folículo Ovárico , Oogénesis/genética , Células Germinativas

Asunto(s)

Epigénesis Genética , Padre , ADN/genética , Humanos , Masculino

RESUMEN

Mouse embryonic stem cells (mESCs) are biased toward producing embryonic rather than extraembryonic endoderm fates. Here, we identify the mechanism of this barrier and report that the histone deacetylase Hdac3 and the transcriptional corepressor Dax1 cooperatively limit the lineage repertoire of mESCs by silencing an enhancer of the extraembryonic endoderm-specifying transcription factor Gata6. This restriction is opposed by the pluripotency transcription factors Nr5a2 and Esrrb, which promote cell type conversion. Perturbation of the barrier extends mESC potency and allows formation of 3D spheroids that mimic the spatial segregation of embryonic epiblast and extraembryonic endoderm in early embryos. Overall, this study shows that transcriptional repressors stabilize pluripotency by biasing the equilibrium between embryonic and extraembryonic lineages that is hardwired into the mESC transcriptional network.

Asunto(s)

Receptor Nuclear Huérfano DAX-1 , Histona Desacetilasas , Células Madre Embrionarias de Ratones/citología , Animales , Diferenciación Celular , Células Cultivadas , Receptor Nuclear Huérfano DAX-1/genética , Receptor Nuclear Huérfano DAX-1/metabolismo , Femenino , Factor de Transcripción GATA6/genética , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Masculino , Ratones , ARN Interferente Pequeño/genética , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo

RESUMEN

Ketone bodies are generated in the liver and allow for the maintenance of systemic caloric and energy homeostasis during fasting and caloric restriction. It has previously been demonstrated that neonatal ketogenesis is activated independently of starvation. However, the role of ketogenesis during the perinatal period remains unclear. Here, we show that neonatal ketogenesis plays a protective role in mitochondrial function. We generated a mouse model of insufficient ketogenesis by disrupting the rate-limiting hydroxymethylglutaryl-CoA synthase 2 enzyme gene (Hmgcs2). Hmgcs2 knockout (KO) neonates develop microvesicular steatosis within a few days of birth. Electron microscopic analysis and metabolite profiling indicate a restricted energy production capacity and accumulation of acetyl-CoA in Hmgcs2 KO mice. Furthermore, acetylome analysis of Hmgcs2 KO cells revealed enhanced acetylation of mitochondrial proteins. These findings suggest that neonatal ketogenesis protects the energy-producing capacity of mitochondria by preventing the hyperacetylation of mitochondrial proteins.

Asunto(s)

Metabolismo Energético/fisiología , Cuerpos Cetónicos/biosíntesis , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Ácido 3-Hidroxibutírico/metabolismo , Acetilación , Animales , Animales Recién Nacidos , Hidroximetilglutaril-CoA Sintasa/genética , Hidroximetilglutaril-CoA Sintasa/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Microvasos/fisiología , Consumo de Oxígeno

RESUMEN

Temporal and spatial colinear expression of the Hox genes determines the specification of positional identities during vertebrate development. Post-translational modifications of histones contribute to transcriptional regulation. Lysine demethylase 7A (Kdm7a) demethylates lysine 9 or 27 di-methylation of histone H3 (H3K9me2, H3K27me2) and participates in the transcriptional activation of developmental genes. However, the role of Kdm7a during mouse embryonic development remains to be elucidated. Herein, we show that Kdm7a-/- mouse exhibits an anterior homeotic transformation of the axial skeleton, including an increased number of presacral elements. Importantly, posterior Hox genes (caudally from Hox9) are specifically downregulated in the Kdm7a-/- embryo, which correlates with increased levels of H3K9me2, not H3K27me2. These observations suggest that Kdm7a controls the transcription of posterior Hox genes, likely via its demethylating activity, and thereby regulating the murine anterior-posterior development. Such epigenetic regulatory mechanisms may be harnessed for proper control of coordinate body patterning in vertebrates.

Asunto(s)

Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica/genética , Genes Homeobox/genética , Histona Demetilasas con Dominio de Jumonji , Animales , Embrión de Mamíferos/metabolismo , Femenino , Células HeLa , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Familia de Multigenes/genética

RESUMEN

In mammals, chromatin organization undergoes drastic reorganization during oocyte development. However, the dynamics of three-dimensional chromatin structure in this process is poorly characterized. Using low-input Hi-C (genome-wide chromatin conformation capture), we found that a unique chromatin organization gradually appears during mouse oocyte growth. Oocytes at late stages show self-interacting, cohesin-independent compartmental domains marked by H3K27me3, therefore termed Polycomb-associating domains (PADs). PADs and inter-PAD (iPAD) regions form compartment-like structures with strong inter-domain interactions among nearby PADs. PADs disassemble upon meiotic resumption from diplotene arrest but briefly reappear on the maternal genome after fertilization. Upon maternal depletion of Eed, PADs are largely intact in oocytes, but their reestablishment after fertilization is compromised. By contrast, depletion of Polycomb repressive complex 1 (PRC1) proteins attenuates PADs in oocytes, which is associated with substantial gene de-repression in PADs. These data reveal a critical role of Polycomb in regulating chromatin architecture during mammalian oocyte growth and early development.

Asunto(s)

Cromatina/química , Oocitos/crecimiento & desarrollo , Oogénesis/genética , Proteínas del Grupo Polycomb/fisiología , Animales , Blastocisto/química , Proteínas de Ciclo Celular/fisiología , Proteínas Cromosómicas no Histona/fisiología , Embrión de Mamíferos/química , Silenciador del Gen , Código de Histonas , Ratones , Oocitos/química , Transcripción Genética , Cohesinas

RESUMEN

Accurate lineage reconstruction of mammalian pre-implantation development is essential for inferring the earliest cell fate decisions. Lineage tracing using global fluorescence labeling techniques is complicated by increasing cell density and rapid embryo rotation, which hampers automatic alignment and accurate cell tracking of obtained four-dimensional imaging data sets. Here, we exploit the advantageous properties of primed convertible fluorescent proteins (pr-pcFPs) to simultaneously visualize the global green and the photoconverted red population in order to minimize tracking uncertainties over prolonged time windows. Confined primed conversion of H2B-pr-mEosFP-labeled nuclei combined with light-sheet imaging greatly facilitates segmentation, classification, and tracking of individual nuclei from the 4-cell stage up to the blastocyst. Using green and red labels as fiducial markers, we computationally correct for rotational and translational drift, reduce overall data size, and accomplish high-fidelity lineage tracing even for increased imaging time intervals - addressing major concerns in the field of volumetric embryo imaging.

Asunto(s)

Blastocisto , Luz , Animales , Linaje de la Célula , Desarrollo Embrionario , Fluorescencia , Ratones

RESUMEN

Gnathostome jaws derive from the first pharyngeal arch (PA1), a complex structure constituted by Neural Crest Cells (NCCs), mesodermal, ectodermal and endodermal cells. Here, to determine the regionalized morphogenetic impact of Dlx5/6 expression, we specifically target their inactivation or overexpression to NCCs. NCC-specific Dlx5/6 inactivation (NCC∆Dlx5/6) generates severely hypomorphic lower jaws that present typical maxillary traits. Therefore, differently from Dlx5/6 null-embryos, the upper and the lower jaws of NCC∆Dlx5/6 mice present a different size. Reciprocally, forced Dlx5 expression in maxillary NCCs provokes the appearance of distinct mandibular characters in the upper jaw. We conclude that: (1) Dlx5/6 activation in NCCs invariably determines lower jaw identity; (2) the morphogenetic processes that generate functional matching jaws depend on the harmonization of Dlx5/6 expression in NCCs and in distinct ectodermal territories. The co-evolution of synergistic opposing jaws requires the coordination of distinct regulatory pathways involving the same transcription factors in distant embryonic territories.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/biosíntesis , Mandíbula/embriología , Maxilar/embriología , Cresta Neural/embriología , Animales , Proteínas de Homeodominio/genética , Mandíbula/citología , Maxilar/citología , Ratones , Ratones Mutantes , Cresta Neural/citología

RESUMEN

Age-related fertility decline is hypothesized to occur mainly by the spontaneous exhaustion and deterioration of the ovarian follicle, and the accumulation of ovarian tissue damage resulting from the ovulation cycle may play roles in the process. In this study, we hypothesized that suppressing ovulation would exert protective effects against age-related fertility decline. To test this hypothesis, we established a mouse model in which oral contraceptives (OCs) were administered daily. Female C57BL/6N mice were administered OCs daily from the age of 2 months to 12 months as an ovulation suppression mouse model. Mouse fecundity was investigated by counting oocyte number after ovarian stimulation and by examining live fetuses after mating. We found that compared with control mice administered vehicle alone, 12-month-old mice administered 2-fold dose OCs used for treating humans exhibited a significantly greater average oocyte number after ovarian stimulation (8.5 ± 0.6 vs 5.9 ± 0.6, P < .01). In addition, spontaneous conception with living fetuses after mating was strikingly increased in 12-month-old mice administered OCs relative to controls (6.0 ± 1.2 vs 0.4 ± 0.3, P < .01). In the histological examination of mouse ovarian tissues, we did not detect a significant difference in ovarian follicle number, but reduced amount of brownish foamy fibrous tissues, which might reflect ovarian tissue damage, was detected in aged mice administered OCs. These results suggest the possibility that long-term OC administration might alleviate age-related fertility decline, and the improvement mechanism could be attributed to the prevention of ovarian tissue damage by suppressing ovulation.

Asunto(s)

Envejecimiento/efectos de los fármacos , Anticonceptivos Sintéticos Orales/farmacología , Desogestrel/farmacología , Fertilidad/efectos de los fármacos , Ovario/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Envejecimiento/metabolismo , Animales , Femenino , Fertilidad/fisiología , Peroxidación de Lípido/efectos de los fármacos , Ratones , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Ovario/metabolismo

RESUMEN

Most gnathostomata craniofacial structures derive from pharyngeal arches (PAs), which are colonized by cranial neural crest cells (CNCCs). The anteroposterior and dorsoventral identities of CNCCs are defined by the combinatorial expression of Hox and Dlx genes. The mechanisms associating characteristic Hox/Dlx expression patterns with the topology and morphology of PAs derivatives are only partially known; a better knowledge of these processes might lead to new concepts on the origin of taxon-specific craniofacial morphologies and of certain craniofacial malformations. Here we show that ectopic expression of Hoxa2 in Hox-negative CNCCs results in distinct phenotypes in different CNCC subpopulations. Namely, while ectopic Hoxa2 expression is sufficient for the morphological and molecular transformation of the first PA (PA1) CNCC derivatives into the second PA (PA2)-like structures, this same genetic alteration does not provoke the transformation of derivatives of other CNCC subpopulations, but severely impairs their development. Ectopic Hoxa2 expression results in the transformation of the proximal Meckel's cartilage and of the malleus, two ventral PA1 CNCCs derivatives, into a supernumerary styloid process (SP), a PA2-derived mammalian-specific skeletal structure. These results, together with experiments to inactivate and ectopically activate the Edn1-Dlx5/6 pathway, indicate a dorsoventral PA2 (hyomandibular/ceratohyal) boundary passing through the middle of the SP. The present findings suggest context-dependent function of Hoxa2 in CNCC regional specification and morphogenesis, and provide novel insights into the evolution of taxa-specific patterning of PA-derived structures.

Asunto(s)

Región Branquial/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/metabolismo , Morfogénesis/fisiología , Cresta Neural/metabolismo , Azul Alcián , Animales , Antraquinonas , Región Branquial/metabolismo , Cartilla de ADN/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Hibridación in Situ , Ratones , Ratones Mutantes , Morfogénesis/genética , Cresta Neural/embriología , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

SIRT3 is a member of the sirtuin family and has recently emerged as a vital molecule in controlling the generation of reactive oxygen species (ROS) in oocytes. Appropriate levels of ROS play pivotal roles in human reproductive medicine. The aim of the present study was to investigate SIRT3 expression and analyze the SIRT3-mediated oxidative response in human luteinized granulosa cells (GCs). Human ovarian tissues were subjected to immunohistochemical analysis to localize SIRT3 expression. Hydrogen peroxide and human chorionic gonadotropin were used to analyze the relationship between ROS and SIRT3 by quantitative RT-PCR and Western blotting. Intracellular levels of ROS were investigated by fluorescence after small interfering RNA-mediated knockdown of SIRT3 in human GCs. To uncover the role of SIRT3 in folliculogenesis and luteinization, mRNA levels of related genes and the progesterone concentration were analyzed by quantitative RT-PCR and immunoassays, respectively. We detected the expression of SIRT3 in the GCs of the human ovary. The mRNA levels of SIRT3, catalase, and superoxide dismutase 1 were up-regulated by hydrogen peroxide in both COV434 cells and human GCs and down-regulated by human chorionic gonadotropin. Knockdown of SIRT3 markedly elevated ROS generation in human GCs. In addition, SIRT3 depletion resulted in decreased mRNA expression of aromatase, 17ß-hydroxysteroid dehydrogenase 1, steroidogenic acute regulatory protein, cholesterol side-chain cleavage enzyme, and 3ß-hydroxysteroid dehydrogenase in GCs and thus resulted in decreased progesterone secretion. These results have the important clinical implication that SIRT3 might play a positive role in the folliculogenesis and luteinization processes in GCs, possibly by sensing and regulating the generation of ROS. Activation of SIRT3 function might help to sustain human reproduction by maintaining GCs as well as oocytes.

Asunto(s)

Células de la Granulosa/metabolismo , Luteinización , Estrés Oxidativo , Progesterona/metabolismo , Sirtuina 3/fisiología , Adulto , Antioxidantes/metabolismo , Catalasa/metabolismo , Línea Celular Tumoral , Gonadotropina Coriónica , Femenino , Humanos , Folículo Ovárico/fisiología , Distribución Aleatoria , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1

RESUMEN

Calpains are Ca(2+)-dependent modulator Cys proteases that have a variety of functions in almost all eukaryotes. There are more than 10 well-conserved mammalian calpains, among which eutherian calpain-6 (CAPN6) is unique in that it has amino acid substitutions at the active-site Cys residue (to Lys in humans), strongly suggesting a loss of proteolytic activity. CAPN6 is expressed predominantly in embryonic muscles, placenta, and several cultured cell lines. We previously reported that CAPN6 is involved in regulating microtubule dynamics and actin reorganization in cultured cells. The physiological functions of CAPN6, however, are still unclear. Here, to elucidate CAPN6's in vivo roles, we generated Capn6-deficient mice, in which a lacZ expression cassette was integrated into the Capn6 gene. These Capn6-deficient mouse embryos expressed lacZ predominantly in skeletal muscles, as well as in cartilage and the heart. Histological and biochemical analyses showed that the CAPN6 deficiency promoted the development of embryonic skeletal muscle. In primary cultured skeletal muscle cells that were induced to differentiate into myotubes, Capn6 expression was detected in skeletal myocytes, and Capn6-deficient cultures showed increased differentiation. Furthermore, we found that CAPN6 was expressed in the regenerating skeletal muscles of adult mice after cardiotoxin-induced degeneration. In this experimental system, Capn6-deficient mice exhibited more advanced skeletal-muscle regeneration than heterozygotes or wild-type mice at the same time point. These results collectively showed that a loss of CAPN6 promotes skeletal muscle differentiation during both development and regeneration, suggesting a novel physiological function of CAPN6 as a suppressor of skeletal muscle differentiation.

Asunto(s)

Calpaína/genética , Desarrollo Embrionario/genética , Microtúbulos/metabolismo , Músculo Esquelético/crecimiento & desarrollo , Regeneración/genética , Animales , Calpaína/biosíntesis , Calpaína/deficiencia , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Desarrollo de Músculos/genética

RESUMEN

The avian and mammalian heart originates from two distinct embryonic regions: an early differentiating first heart field and a dorsomedially located second heart field. It remains largely unknown when and how these subdivisions of the heart field divide into regions with different fates. Here, we identify in the mouse a subpopulation of the first (crescent-forming) field marked by endothelin receptor type A (Ednra) gene expression, which contributes to chamber myocardium through a unique type of cell behavior. Ednra-lacZ/EGFP-expressing cells arise in the ventrocaudal inflow region of the early linear heart tube, converge to the midline, move anteriorly along the outer curvature and give rise to chamber myocardium mainly of the left ventricle and both atria. This movement was confirmed by fluorescent dye-labeling and transplantation experiments. The Ednra-lacZ/EGFP-expressing subpopulation is characterized by the presence of Tbx5-expressing cells. Ednra-null embryonic hearts often demonstrate hypoplasia of the ventricular wall, low mitotic activity and decreased Tbx5 expression with reciprocal expansion of Tbx2 expression. Conversely, endothelin 1 stimulates ERK phosphorylation and Tbx5 expression in the early embryonic heart. These results indicate that early Ednra expression defines a subdomain of the first heart field contributing to chamber formation, in which endothelin 1/Ednra signaling is involved. The present finding provides an insight into how subpopulations within the crescent-forming (first) heart field contribute to the coordination of heart morphogenesis through spatiotemporally defined cell movements.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Miocardio/metabolismo , Organogénesis , Receptor de Endotelina A/metabolismo , Animales , Embrión de Mamíferos/metabolismo , Endotelinas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Técnicas de Sustitución del Gen , Ventrículos Cardíacos/embriología , Ratones , Mitosis , Fosforilación , Receptor de Endotelina A/genética , Proteínas de Dominio T Box/metabolismo

RESUMEN

Sirtuins are a phylogenetically conserved NAD+-dependent protein deacetylase/ADP-ribosyltransferase family implicated in diverse biological processes. Several family members localize to mitochondria, the function of which is thought to determine the developmental potential of preimplantation embryos. We have therefore characterized the role of sirtuins in mouse preimplantation development under in vitro culture conditions. All sirtuin members were expressed in eggs, and their expression gradually decreased until the blastocyst stage. Treatment with sirtuin inhibitors resulted in increased intracellular ROS levels and decreased blastocyst formation. These effects were recapitulated by siRNA-induced knockdown of Sirt3, which is involved in mitochondrial energy metabolism, and in Sirt3-/- embryos. The antioxidant N-acetyl-L-cysteine and low-oxygen conditions rescued these adverse effects. When Sirt3-knockdown embryos were transferred to pseudopregnant mice after long-term culture, implantation and fetal growth rates were decreased, indicating that Sirt3-knockdown embryos were sensitive to in vitro conditions and that the effect was long lasting. Further experiments revealed that maternally derived Sirt3 was critical. Sirt3 inactivation increased mitochondrial ROS production, leading to p53 upregulation and changes in downstream gene expression. The inactivation of p53 improved the developmental outcome of Sirt3-knockdown embryos, indicating that the ROS-p53 pathway was responsible for the developmental defects. These results indicate that Sirt3 plays a protective role in preimplantation embryos against stress conditions during in vitro fertilization and culture.

Asunto(s)

Blastocisto/fisiología , Desarrollo Embrionario , Fertilización In Vitro , Estrés Oxidativo , Sirtuina 3/fisiología , Proteína p53 Supresora de Tumor/fisiología , Animales , Femenino , Ratones , Ratones Endogámicos ICR , Mitocondrias/metabolismo , Células 3T3 NIH , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Sirtuina 3/antagonistas & inhibidores , Sirtuina 3/genética

RESUMEN

Dnm3os, a gene that is transcribed into a non-coding RNA (ncRNA), contains three micro RNAs (miRNAs), miR-199a, miR-199a*, and miR-214, whose functions remain unknown in mammals. In this study, we introduced the lacZ gene into the Dnm3os locus to recapitulate its expression pattern and disrupt its function. Dnm3os(+/lacZ) heterozygous embryos showed beta-galactosidase activity, which reflected the authentic expression pattern of Dnm3os RNA. Most of the Dnm3os(lacZ/lacZ) homozygous pups died within one month of birth. After birth, Dnm3os(lacZ/lacZ) mice exhibited several skeletal abnormalities, including craniofacial hypoplasia, defects in dorsal neural arches and spinous processes of the vertebrae, and osteopenia. Importantly, the expression of miR-199a, miR-199a*, and miR-214 was significantly down-regulated in Dnm3os(lacZ/lacZ) embryos, supporting the assumption that Dnm3os serves as a precursor of these three miRNAs. Thus, Dnm3os has emerged as an miRNA-encoding gene that is indispensable for normal skeletal development and body growth in mammals.

Asunto(s)

Desarrollo Óseo , Huesos/embriología , Huesos/metabolismo , ARN no Traducido/genética , Animales , Peso Corporal , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros/genética , Genotipo , Masculino , Ratones , Ratones Transgénicos

RESUMEN

Articulated jaws are highly conserved structures characteristic of gnathostome evolution. Epithelial-mesenchymal interactions within the first pharyngeal arch (PA1) instruct cephalic neural crest cells (CNCCs) to form the different skeletal elements of the jaws. The endothelin-1 (Edn1)/endothelin receptor type-A (Ednra)-->Dlx5/6-->Hand2 signaling pathway is necessary for lower jaw formation. Here, we show that the Edn1 signaling is sufficient for the conversion of the maxillary arch to mandibular identity. Constitutive activation of Ednra induced the transformation of upper jaw, maxillary, structures into lower jaw, mandibular, structures with duplicated Meckel's cartilage and dermatocranial jaws constituted by 4 dentary bones. Misexpression of Hand2 in the Ednra domain caused a similar transformation. Skeletal transformations are accompanied by neuromuscular remodeling. Ednra is expressed by most CNCCs, but its constitutive activation affects predominantly PA1. We conclude that after migration CNCCs are not all equivalent, suggesting that their specification occurs in sequential steps. Also, we show that, within PA1, CNCCs are competent to form both mandibular and maxillary structures and that an Edn1 switch is responsible for the choice of either morphogenetic program.

Asunto(s)

Endotelina-1/metabolismo , Mandíbula/embriología , Maxilar/embriología , Receptor de Endotelina A/metabolismo , Transducción de Señal/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Tipificación del Cuerpo , Endotelina-1/genética , Regulación del Desarrollo de la Expresión Génica , Mandíbula/anatomía & histología , Mandíbula/metabolismo , Maxilar/anatomía & histología , Maxilar/metabolismo , Ratones , Ratones Transgénicos , Receptor de Endotelina A/genética

RESUMEN

The endothelin (Edn) system comprises three ligands (Edn1, Edn2 and Edn3) and their G-protein-coupled type A (Ednra) and type B (Ednrb) receptors. During embryogenesis, the Edn1/Ednra signaling is thought to regulate the dorsoventral axis patterning of pharyngeal arches via Dlx5/Dlx6 upregulation. To further clarify the underlying mechanism, we have established mice in which gene cassettes can be efficiently knocked-in into the Ednra locus using recombinase-mediated cassette exchange (RMCE) based on the Cre-lox system. The first homologous recombination introducing mutant lox-flanked Neo resulted in homeotic transformation of the lower jaw to an upper jaw, as expected. Subsequent RMCE-mediated knock-in of lacZ targeted its expression to the cranial/cardiac neural crest derivatives as well as in mesoderm-derived head mesenchyme. Knock-in of Ednra cDNA resulted in a complete rescue of craniofacial defects of Ednra-null mutants. By contrast, Ednrb cDNA could not rescue them except for the most distal pharyngeal structures. At early stages, the expression of Dlx5, Dlx6 and their downstream genes was downregulated and apoptotic cells distributed distally in the mandible of Ednrb-knock-in embryos. These results, together with similarity in craniofacial defects between Ednrb-knock-in mice and neural-crest-specific Galpha(q)/Galpha(11)-deficient mice, indicate that the dorsoventral axis patterning of pharyngeal arches is regulated by the Ednra-selective, G(q)/G(11)-dependent signaling, while the formation of the distal pharyngeal region is under the control of a G(q)/G(11)-independent signaling, which can be substituted by Ednrb. This RMCE-mediated knock-in system can serve as a useful tool for studies on gene functions in craniofacial development.

Asunto(s)

Región Branquial/embriología , Endotelina-1/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Mutagénesis Insercional , Receptor de Endotelina A/metabolismo , Recombinasas/metabolismo , Transducción de Señal , Animales , Región Branquial/metabolismo , Anomalías Craneofaciales , ADN Complementario , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/enzimología , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Mesodermo/embriología , Mesodermo/enzimología , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Músculo Esquelético/anomalías , Cresta Neural/embriología , Cresta Neural/enzimología , Fenotipo , Receptor de Endotelina A/deficiencia , Receptor de Endotelina A/genética , Receptor de Endotelina B/metabolismo , beta-Galactosidasa/metabolismo

RESUMEN

DNA methylation at cytosine residues in CpG dinucleotides is a component of epigenetic marks crucial to mammalian development. In preimplantation stage embryos, a large part of genomic DNA is extensively demethylated, whereas the methylation patterns are faithfully maintained in certain regions. To date, no enzymes responsible for the maintenance of DNA methylation during preimplantation development have been identified except for the oocyte form of DNA (cytosine-5)-methyltransferase 1 (Dnmt1o) at the 8-cell stage. Herein, we demonstrate that the somatic form of Dnmt1 (Dnmt1s) is present in association with chromatin in MII-stage oocytes as well as in the nucleus throughout preimplantation development. At the early one-cell stage, Dnmt1s is asymmetrically localized in the maternal pronuclei. Thereafter, Dnmt1s is recruited to the paternal genome during pronuclear maturation. During the first two cell cycles after fertilization, Dnmt1s is exported from the nucleus in the G2 phase in a CRM1/exportin-dependent manner. Antibody microinjection and small interfering RNA-mediated knock-down decreases methylated CpG dinucleotides in repetitive intracisternal A-type particle (IAP) sequences and the imprinted gene H19. These results indicate that Dnmt1s is responsible for the maintenance methylation of particular genomic regions whose methylation patterns must be faithfully maintained during preimplantation development.

Asunto(s)

ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Embrión de Mamíferos/metabolismo , Animales , Núcleo Celular/química , Núcleo Celular/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/análisis , ADN (Citosina-5-)-Metiltransferasas/genética , Embrión de Mamíferos/enzimología , Femenino , Isoenzimas/análisis , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Oocitos/enzimología , ARN Interferente Pequeño

RESUMEN

Although several studies have indicated that persons with a high ruminative coping style experience higher depression after the loss of a loved one, the relationship between ruminative coping and the occurrence of clinical depression and anxiety disorders after a loss has not been thoroughly investigated. This study investigated the relationship between response styles (ruminative coping v distractive coping) and the onset of major depression and anxiety disorders in a sample of parents who had experienced sudden child-loss (N = 106). The incidence of major depression after the loss of a child was very high (69%). After controlling for demographic variables and psychiatric history, ruminative coping was significantly associated with the onset of major depression, as defined by DSM-IV, but not with the onset of anxiety disorders. Thus ruminative coping after the loss of a child appears to be a risk factor specifically for major depression.

Asunto(s)

Adaptación Psicológica , Trastornos de Ansiedad/epidemiología , Muerte , Trastorno Depresivo Mayor/epidemiología , Padres/psicología , Adulto , Pueblo Asiatico/psicología , Niño , Preescolar , Femenino , Humanos , Incidencia , Lactante , Recién Nacido , Entrevista Psicológica , Japón/epidemiología , Modelos Logísticos , Masculino , Persona de Mediana Edad , Embarazo , Encuestas y Cuestionarios , Factores de Tiempo