RESUMO
Only 30% of embryos from in vitro fertilized oocytes successfully implant and develop to term, leading to repeated transfer cycles. To reduce time-to-pregnancy and stress for patients, there is a need for a diagnostic tool to better select embryos and oocytes based on their physiology. The current standard employs brightfield imaging, which provides limited physiological information. Here, we introduce METAPHOR: Metabolic Evaluation through Phasor-based Hyperspectral Imaging and Organelle Recognition. This non-invasive, label-free imaging method combines two-photon illumination and AI to deliver the metabolic profile of embryos and oocytes based on intrinsic autofluorescence signals. We used it to classify i) mouse blastocysts cultured under standard conditions or with depletion of selected metabolites (glucose, pyruvate, lactate); and ii) oocytes from young and old mouse females, or in vitro-aged oocytes. The imaging process was safe for blastocysts and oocytes. The METAPHOR classification of control vs. metabolites-depleted embryos reached an area under the ROC curve (AUC) of 93.7%, compared to 51% achieved for human grading using brightfield imaging. The binary classification of young vs. old/in vitro-aged oocytes and their blastulation prediction using METAPHOR reached an AUC of 96.2% and 82.2%, respectively. Finally, organelle recognition and segmentation based on the flavin adenine dinucleotide signal revealed that quantification of mitochondria size and distribution can be used as a biomarker to classify oocytes and embryos. The performance and safety of the method highlight the accuracy of noninvasive metabolic imaging as a complementary approach to evaluate oocytes and embryos based on their physiology.
Assuntos
Blastocisto , Oócitos , Animais , Blastocisto/metabolismo , Camundongos , Oócitos/metabolismo , Feminino , Organelas/metabolismo , Imagem Óptica/métodosRESUMO
Human meiosis in oocytes entails an intricate regulation of the transcriptome to support late oocyte growth and early embryo development, both crucial to reproductive success. Currently, little is known about the co- and post-transcriptional mRNA processing mechanisms regulating the last meiotic phases, which contribute to transcriptome complexity and influence translation rates. We analyzed gene expression changes, splicing and pre-mRNA processing in an RNA sequencing set of 40 human oocytes at different meiotic maturation stages, matured both in vivo and in vitro. We found abundant untranslated region (UTR) processing, mostly at the 3' end, of meiosis-related genes between the germinal vesicle (GV) and metaphase II (MII) stages, supported by the differential expression of spliceosome and pre-mRNA processing related genes. Importantly, we found very few differences among GV oocytes across several durations of IVM, as long as they did not reach MII, suggesting an association of RNA processing and successful meiosis transit. Changes in protein isoforms are minor, although specific and consistent for genes involved in chromosome organization and spindle assembly. In conclusion, we reveal a dynamic transcript remodeling during human female meiosis, and show how pre-mRNA processing, specifically 3'UTR shortening, drives a selective translational regulation of transcripts necessary to reach final meiotic maturation.
Assuntos
Técnicas de Maturação in Vitro de Oócitos , Precursores de RNA , Humanos , Feminino , Precursores de RNA/genética , Precursores de RNA/metabolismo , Oócitos/metabolismo , Meiose/genética , Oogênese/genéticaRESUMO
The mechanism of conversion of the human sperm basal body to a centrosome after fertilization, and its role in supporting human early embryogenesis, has not been directly addressed so far. Using proteomics and immunofluorescence studies, we show here that the human zygote inherits a basal body enriched with centrosomal proteins from the sperm, establishing the first functional centrosome of the new organism. Injection of human sperm tails containing the basal body into human oocytes followed by parthenogenetic activation, showed that the centrosome contributes to the robustness of the early cell divisions, increasing the probability of parthenotes reaching the compaction stage. In the absence of the sperm-derived centrosome, pericentriolar material (PCM) components stored in the oocyte can form de novo structures after genome activation, suggesting a tight PCM expression control in zygotes. Our results reveal that the sperm basal body is a complex organelle which converts to a centrosome after fertilization, ensuring the early steps of embryogenesis and successful compaction. However, more experiments are needed to elucidate the exact molecular mechanisms of centrosome inheritance in humans.
Assuntos
Corpos Basais/metabolismo , Blastocisto/metabolismo , Centrossomo/metabolismo , Injeções de Esperma Intracitoplásmicas , Interações Espermatozoide-Óvulo , Espermatozoides/metabolismo , Adolescente , Adulto , Desenvolvimento Embrionário , Feminino , Células HeLa , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Gravidez , Adulto JovemRESUMO
The transition from a transcriptionally active state (GV) to a transcriptionally inactive state (mature MII oocytes) is required for the acquisition of oocyte developmental competence. We hypothesize that the expression of specific genes at the in vivo matured (MII) stage could be modulated by posttranscriptional mechanisms, particularly regulation of alternative splicing (AS). In this study, we examined the transcriptional activity of GV oocytes after ovarian stimulation followed by oocyte pick-up and the landscape of alternatively spliced isoforms in human MII oocytes. Individual oocytes were processed and analyzed for transcriptional activity (GV), gene expression (GV and MII), and AS signatures (GV and MII) on HTA 2.0 microarrays. Samples were grouped according to maturation stage, and then subgrouped according to women's age and antral follicular count (AFC); array results were validated by quantitative polymerase chain reaction. Differentially expressed genes between GV and MII oocytes clustered mainly in biological processes related to mitochondrial metabolism. Interestingly, 16 genes that were related to the regulation of transcription and mitochondrial translation showed differences in alternatively spliced isoform profiles despite not being differentially expressed between groups. Altogether, our results contribute to our understanding of the role of AS in oocyte developmental competence acquisition.
Assuntos
Oócitos , Oogênese , Feminino , Humanos , Mitocôndrias/fisiologia , Oócitos/metabolismo , Oogênese/genética , Indução da Ovulação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
STUDY QUESTION: Are phospholipase C zeta 1 (PLCZ1) mutations associated with fertilization failure (FF) after ICSI? SUMMARY ANSWER: New mutations in the PLCZ1 sequence are associated with FFs after ICSI. WHAT IS KNOWN ALREADY: FF occurs in 1-3% of ICSI cycles, mainly due to oocyte activation failure (OAF). The sperm PLCζ/PLCZ1 protein hydrolyzes phosphatidylinositol (4, 5)-bisphosphate in the oocyte, leading to intracellular calcium release and oocyte activation. To date, few PLCZ1 point mutations causing decreased protein levels or activity have been linked to FF. However, functional alterations of PLCζ/PLCZ1 in response to both described and novel mutations have not been investigated. STUDY DESIGN, SIZE, DURATION: We performed a study including 37 patients presenting total or partial FF (fertilization rate (FR), ≤25%) after ICSI occurring between 2014 and 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were divided into two groups based on oocyte evaluation 19 h post ICSI: FF due to a defect in oocyte activation (OAF, n = 22) and FF due to other causes ('no-OAF', n = 15). Samples from 13 men with good fertilization (FR, >50%) were used as controls. PLCζ/PLCZ1 protein localization and levels in sperm were evaluated by immunofluorescence and western blot, respectively. Sanger sequencing on genomic DNA was used to identify PLCZ1 mutations in exonic regions. The effect of the mutations on protein functionality was predicted in silico using the MODICT algorithm. Functional assays were performed by cRNA injection of wild-type and mutated forms of PLCZ1 into human in vitro matured metaphase II oocytes, and fertilization outcomes (second polar body extrusion, pronucleus appearance) scored 19 h after injection. MAIN RESULTS AND THE ROLE OF CHANCE: In the OAF group, 12 (54.6%) patients carried at least one mutation in the PLCZ1 coding sequence, one patient out of 15 (6.7%) in the no-OAF group (P < 0.05) and none of the 13 controls (P < 0.05). A total of six different mutations were identified. Five of them were single-nucleotide missense mutations: p.I120M, located at the end of the EF-hand domain; p.R197H, p.L224P and p.H233L, located at the X catalytic domain; and p.S500 L, located at the C2 domain. The sixth mutation, a frameshift variant (p.V326K fs*25), generates a truncated protein at the X-Y linker region. In silico analysis with MODICT predicted all the mutations except p.I120M to be potentially deleterious for PLCζ/PLCZ1 activity. After PLCZ1 cRNA injection, a significant decrease in the percentage of activated oocytes was observed for three mutations (p.R197H, p.H233L and p.V326K fs*25), indicating a deleterious effect on enzymatic activity. PLCZ1 protein localization and expression levels in sperm were similar across groups. FRs were restored (to >60%) in patients carrying PLCZ1 mutations (n = 10) after assisted oocyte activation (AOA), with seven patients achieving pregnancy and live birth. LIMITATIONS, REASONS FOR CAUTION: Caution should be exerted when comparing the cRNA injection results with fertilization outcomes after ICSI, especially in patients presenting mutations in heterozygosis. WIDER IMPLICATIONS OF THE FINDINGS: PLCZ1 mutations were found in high frequency in patients presenting OAF. Functional analysis of three mutations in human oocytes confirms alteration of PLCζ/PLCZ1 activity and their likely involvement in impaired oocyte activation. Our results suggest that PLCZ1 gene sequencing could be useful as a tool for the diagnosis and counseling of couples presenting FF after ICSI due to OAF. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by intramural funding of Clínica EUGIN, by the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia (GENCAT 2015 DI 049 to M. T.-M. and GENCAT 2015 DI 048 to D. C.-B.) and by the Torres Quevedo Program from the Spanish Ministry of Economy and Competitiveness to A. F.-V. No competing interest declared.
Assuntos
Mutação , Fosfoinositídeo Fosfolipase C/genética , Injeções de Esperma Intracitoplásmicas , Espermatozoides/metabolismo , Adulto , Forma Celular/genética , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Oócitos/citologia , Gravidez , Análise do Sêmen , Motilidade dos Espermatozoides/genética , Espermatozoides/citologia , Falha de TratamentoRESUMO
Most facial bones, including frontal bones, are derived from neural crest cells through intramembranous ossification. Fibroblast growth factor receptor 1 (Fgfr1) plays a pivotal role in craniofacial bone development, and loss of Fgfr1 leads to cleft palate and facial cleft defects in newborn mice. However, the potential role of the Fgfr1 gene in neural crest cell-mediated craniofacial development remains unclear. To investigate the role of Fgfr1 in neural crest cells, we analyzed Wnt1-Cre;Fgfr1flox/flox mice. Our results show that specific knockout of Fgfr1 in neural crest cells induced heterotopic chondrogenesis and osteogenesis at the interface of the anterior portions of frontal bones. We observed that heterotopic bone formation continued through postnatal day 28, whereas heterotopic chondrogenesis lasted only through the embryonic period. In summary, our results indicate that loss of Fgfr1 in neural crest cells leads to heterotopic chondrogenesis and osteogenesis.
Assuntos
Condrogênese , Osso Frontal/crescimento & desenvolvimento , Crista Neural/crescimento & desenvolvimento , Osteogênese , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Osso Frontal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Knockout , Crista Neural/citologia , Crista Neural/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genéticaRESUMO
Successful fertilization in mammals depends on the sperm's ability to initiate intracellular Ca2+ oscillations in the egg, a phenomenon that is elicited by Sperm-oocyte activating factors (SOAFs), whose quantitative and/or qualitative defect might result in fertilization failure. One such proposed factor is Post-acrosomal WW domain-binding protein (PAWP/WBP2NL), although its ability to activate human oocytes has been questioned and its implication in human fertilization failure remains unknown. Here, we sought to determine if PAWP/WBP2NL expression and distribution in sperm cells associate with low/complete fertilization failure in males participating in intracytoplasmic sperm injection (ICSI) cycles. This prospective study was conducted on eight couples referred for elective ICSI with either the woman's own (n = 4) or a donor eggs (n = 4). Eight sperm donor samples used in ICSI, which resulted in normal fertilization rates, were used as the control group. For each male patient and donor sperm, PAWP/WBP2NL sequence, protein expression, and cellular distribution were analyzed by PCR amplification-sequencing, Western blot, and immunofluorescence, respectively. PAWP/WBP2NL was present in all samples, and no significant differences were detected between patients with fertilization failure and donors in sequence variants or mean protein expression, or in the proportion of PAWP/WBP2NL-positive sperm. In conclusion, no clear association between PAWP/WBP2NL protein expression in sperm and fertilization outcome in ICSI were observed from this cohort.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Fertilização in vitro , Infertilidade , Proteínas de Plasma Seminal/genética , Proteínas de Plasma Seminal/metabolismo , Injeções de Esperma Intracitoplásmicas , Espermatozoides/metabolismo , Adulto , Sequência de Bases , Estudos de Casos e Controles , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Expressão Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Infertilidade/genética , Infertilidade/metabolismo , Infertilidade/patologia , Infertilidade/terapia , Masculino , Pessoa de Meia-Idade , Distribuição Tecidual , Doadores de Tecidos , Falha de Tratamento , Adulto JovemRESUMO
The maintenance of pluripotency in mouse embryonic stem cells (mESCs) relies on the activity of a transcriptional network that is fuelled by the activity of three transcription factors (Nanog, Oct4 and Sox2) and balanced by the repressive activity of Tcf3. Extracellular signals modulate the activity of the network and regulate the differentiation capacity of the cells. Wnt/ß-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of ß-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency. A recent report shows that ß-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain. Here, we show that during self-renewal there is negligible transcriptional activity of ß-catenin and that this is due to its tight association with membranes, where we find it in a complex with Oct4 and E-cadherin. Differentiation triggers a burst of Wnt/ß-catenin transcriptional activity that coincides with the disassembly of the complex. Our results establish that ß-catenin, but not its transcriptional activity, is central to pluripotency acting through a ß-catenin/Oct4 complex.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/fisiologia , beta Catenina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Camundongos , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/fisiologia , Fator 3 de Transcrição de Octâmero/fisiologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , RNA Interferente Pequeno/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , beta Catenina/fisiologiaRESUMO
PURPOSE: Sperm-borne PLCζ protein induces Ca(2+) oscillations in the oocyte and is believed to play a major role during oocyte activation. However, its implication in fertilization failure following ICSI is still debated. We analyzed PLCζ gene sequence, protein expression level, and localization in both patients with previous failed fertilization by ICSI and sperm donors with proven fertility in order to assess the association of PLCζ with both sperm characteristics and ability to fertilize. METHODS: Semen from 15 patients and 13 sperm donors with proven fertility was included in the study. Analysis of the PLCζ gene sequence, protein expression through Western blot, and protein localization by immunofluorescence were performed. RESULTS: Two patients with total fertilization failure presented mutations in heterozygosis in the PLCζ gene. Comparison with donor sample sequences displayed comparable SNP allele frequency. Distribution pattern of PLCζ did not vary significantly between donor and patient samples. Levels of PLCζ protein in sperm cells showed an interindividual variability both in patient and donor samples. Several SNPs previously reported in infertile patients were also present in fertile men. CONCLUSION: Failed fertilization occurs even when levels and distribution of PLCζ protein are within normal range. PLCζ seems to be a necessary but not sufficient factor in determining the molecular pathway involved in oocyte activation.
Assuntos
Fosfoinositídeo Fosfolipase C/fisiologia , Interações Espermatozoide-Óvulo , Espermatozoides/metabolismo , Biomarcadores/metabolismo , Fertilização , Humanos , Infertilidade Masculina/metabolismo , Masculino , Fosfoinositídeo Fosfolipase C/química , Fosfoinositídeo Fosfolipase C/metabolismo , Injeções de Esperma Intracitoplásmicas , Espermatozoides/fisiologiaRESUMO
The canonical Wnt/ß-catenin signaling pathway is known to play crucial roles in organogenesis by regulating both proliferation and differentiation. In the inner ear, this pathway has been shown to regulate the size of the otic placode from which the cochlea will arise; however, direct activity of canonical Wnt signaling as well as its function during cochlear mechanosensory hair cell development had yet to be identified. Using TCF/Lef:H2B-GFP reporter mice and transfection of an independent TCF/Lef reporter construct, we describe the pattern of canonical Wnt activity in the developing mouse cochlea. We show that prior to terminal mitosis, canonical Wnt activity is high in early prosensory cells from which hair cells and support cells will differentiate, and activity becomes reduced as development progresses. Using an in vitro model we demonstrate that Wnt/ß-catenin signaling regulates both proliferation and hair cell differentiation within the developing cochlear duct. Inhibition of Wnt/ß-catenin signaling blocks proliferation during early mitotic phases of development and inhibits hair cell formation in the differentiating organ of Corti. Conversely, activation increases the number of hair cells that differentiate and induces proliferation in prosensory cells, causing an expansion of the Sox2-positive prosensory domain. We further demonstrate that the induced proliferation of Sox2-positive cells may be mediated by the cell cycle regulator cyclin D1. Lastly, we provide evidence that the mitotic Sox2-positive cells are competent to differentiate into hair cells. Combined, our data suggest that Wnt/ß-catenin signaling has a dual function in cochlear development, regulating both proliferation and hair cell differentiation.
Assuntos
Cóclea/embriologia , Células Ciliadas Auditivas/metabolismo , Órgão Espiral/embriologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Cóclea/citologia , Cóclea/metabolismo , Ciclina D1/metabolismo , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Órgão Espiral/metabolismo , Organogênese , Fatores de Transcrição SOXB1/metabolismoRESUMO
Paraxial mesoderm is the tissue which gives rise to the skeletal muscles and vertebral column of the body. A gene regulatory network operating in the formation of paraxial mesoderm has been described. This network hinges on three key factors, Wnt3a, Msgn1 and Tbx6, each of which is critical for paraxial mesoderm formation, since absence of any one of these factors results in complete absence of posterior somites. In this study we determined and compared the spatial and temporal patterns of expression of Wnt3a, Msgn1 and Tbx6 at a time when paraxial mesoderm is being formed. Then, we performed a comparative characterization of mutants in Wnt3a, Msgn1 and Tbx6. To determine the epistatic relationship between these three genes, and begin to decipher the complex interplay between them, we analyzed double mutant embryos and compared their phenotypes to the single mutants. Through the analysis of molecular markers in mutants, our data support the bipotential nature of the progenitor cells for paraxial mesoderm and establish regulatory relationships between genes involved in the choice between neural and mesoderm fates.
Assuntos
Embrião de Mamíferos/metabolismo , Mesoderma/citologia , Animais , Diferenciação Celular , Transição Epitelial-Mesenquimal , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Camundongos , Mutação , Somitos/metabolismoRESUMO
Primitive erythroid (EryP) progenitors are the first cell type specified from the mesoderm late in gastrulation. We used a transgenic reporter to image and purify the earliest blood progenitors and their descendants from developing mouse embryos. EryP progenitors exhibited remarkable proliferative capacity in the yolk sac immediately before the onset of circulation, when these cells comprise nearly half of all cells of the embryo. Global expression profiles generated at 24-hour intervals from embryonic day 7.5 through 2.5 revealed 2 abrupt changes in transcript diversity that coincided with the entry of EryPs into the circulation and with their late maturation and enucleation, respectively. These changes were paralleled by the expression of critical regulatory factors. Experiments designed to test predictions from these data demonstrated that the Wnt-signaling pathway is active in EryP progenitors, which display an aerobic glycolytic profile and the numbers of which are regulated by transforming growth factor-ß1 and hypoxia. This is the first transcriptome assembled for a single hematopoietic lineage of the embryo over the course of its differentiation.
Assuntos
Linhagem da Célula/genética , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/metabolismo , Eritropoese/genética , Redes Reguladoras de Genes , Animais , Sequência de Bases , Citocinas/genética , Primers do DNA/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Glicólise/genética , Proteínas de Fluorescência Verde/genética , Substâncias de Crescimento/genética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Oxigênio/metabolismo , Gravidez , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais/genética , Globinas épsilon/genéticaRESUMO
Anthrax Toxin Receptor 1 (ANTXR1; also known as Tumor Endothelial Marker 8, TEM8) is one of several genes that was recently found to be up-regulated in tumor-associated endothelial cells. In vitro, the protein can link extracellular matrix components with the actin cytoskeleton to promote cell adhesion and cell spreading. Both, ANTXR1 and the closely related ANTXR2 can bind anthrax toxin and interact with lipoprotein receptor-related protein 5 and 6, which also work as coreceptors in the WNT signaling pathway. Here, we report the cloning of chick ANTXR1 from a suppression subtractive hybridization screen for fibroblast growth factor (FGF) -inducible genes in chicken embryonic facial mesenchyme. We show that chicken ANTXR1 is dynamically expressed throughout embryogenesis, starting from Hamburger and Hamilton stage 10. Furthermore, we demonstrate that FGF signaling is sufficient, but not necessary, to induce ANTXR1 expression in chicken facial mesenchyme.
Assuntos
Desenvolvimento Embrionário , Face/embriologia , Fator 8 de Crescimento de Fibroblasto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Receptores de Peptídeos/metabolismo , Animais , Embrião de Galinha , Transdução de Sinais , Proteínas Wnt/metabolismoRESUMO
BACKGROUND: Understanding the dynamic cellular behaviors and underlying molecular mechanisms that drive morphogenesis is an ongoing challenge in biology. Live imaging provides the necessary methodology to unravel the synergistic and stereotypical cell and molecular events that shape the embryo. Genetically-encoded reporters represent an essential tool for live imaging. Reporter strains can be engineered by placing cis-regulatory elements of interest to direct the expression of a desired reporter gene. In the case of canonical Wnt signaling, also referred to as Wnt/ß-catenin signaling, since the downstream transcriptional response is well understood, reporters can be designed that reflect sites of active Wnt signaling, as opposed to sites of gene transcription, as is the case with many fluorescent reporters. However, even though several transgenic Wnt/ß-catenin reporter strains have been generated, to date, none provides the single-cell resolution favored for live imaging studies. RESULTS: We have placed six copies of a TCF/Lef responsive element and an hsp68 minimal promoter in front of a fluorescent protein fusion comprising human histone H2B to GFP and used it to generate a strain of mice that would report Wnt/ß-catenin signaling activity. Characterization of developmental and adult stages of the resulting TCF/Lef:H2B-GFP strain revealed discrete and specific expression of the transgene at previously characterized sites of Wnt/ß-catenin signaling. In support of the increased sensitivity of the TCF/Lef:H2B-GFP reporter, additional sites of Wnt/ß-catenin signaling not documented with other reporters but identified through genetic and embryological analysis were observed. Furthermore, the sub-cellular localization of the reporter minimized reporter perdurance, and allowed visualization and tracking of individual cells within a cohort, so facilitating the detailed analysis of cell behaviors and signaling activity during morphogenesis. CONCLUSION: By combining the Wnt activity read-out efficiency of multimerized TCF/Lef DNA binding sites, together with the high-resolution imaging afforded by subcellularly-localized fluorescent fusion proteins such as H2B-GFP, we have created a mouse transgenic line that faithfully recapitulates Wnt signaling activity at single-cell resolution. The TCF/Lef:H2B-GFP reporter represents a unique tool for live imaging the in vivo processes triggered by Wnt/ß-catenin signaling, and thus should help the formulation of a high-resolution understanding of the serial events that define the morphogenetic process regulated by this signaling pathway.
Assuntos
Camundongos/embriologia , Transdução de Sinais , Análise de Célula Única/métodos , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Endoderma/metabolismo , Gastrulação , Proteínas de Fluorescência Verde/metabolismo , Camundongos Transgênicos , Linha Primitiva/metabolismo , Fatores de Transcrição TCF/metabolismoRESUMO
Apoptosis signal-regulating kinase 1 (ASK1) is an important regulator of stress-induced cell death. ASK1 is activated by oxidative stress, TNF and endoplasmatic reticulum stress and activates the JNK- and p38-dependent intracellular death pathways. A number of studies have suggested that ASK1 may also have other roles in addition to its pro-apoptotic activity. Expression of ASK1 during early embryonic development has so far not been analyzed. We have identified and cloned chick ASK1 in a screen for FGF8 inducible genes in chick facial mesenchyme. Here we report the expression of chick ASK1 from the gastrulation stage (HH4) to day 4 of development, its expression in the developing inner organs and limbs, and we compare its expression to the expression of Ask1 during mouse development. Furthermore, we provide evidence that FGF signaling is required for ASK1 expression in chick nasal mesenchyme. In contrast, expression in the mouse nasal region was restricted to the epithelium and was independent of FGF signaling. Our analysis demonstrates that ASK1 has a spatially restricted and temporally dynamic expression pattern in both chick and mouse embryos, which includes conserved as well as species-specific expression domains.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , MAP Quinase Quinase Quinase 5/biossíntese , Animais , Apoptose , Embrião de Galinha , Fatores de Crescimento de Fibroblastos/metabolismo , Rim/embriologia , Pulmão/embriologia , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Crista Neural/embriologia , Timo/embriologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Reggies are plasma membrane-associated proteins and characteristic markers of lipid-raft microdomains. They are highly conserved from flies to humans and have been implicated in axon regeneration and cell process and contact formation, possibly providing functional platforms for cell-signaling in neurons and other cell types. We analyzed reggie mRNA and protein expression patterns during early zebrafish development. All three zebrafish genes, re-1a, -2a, and -2b, span a considerably diverse set of expression patterns, and their proteins are induced maternally, showing ubiquitous expression at early stages. Although re-2a mRNA can be observed in differentiating neurons in the brain, spinal cord, and neurogenic placodes, re-2b is transcribed mainly in head mesoderm, in neural crest derivates, and along somite boundaries. re-1a mRNA is present at high levels in expression domains that overlap with the combined expression pattern of both re-2 genes except at the somites, where it complements the pattern of re-2b. Immunostaining on embryos reveals reggie protein localization at the cell membrane, at cell-cell contacts, and along all early axon tracts. The early phase of reggie expression suggests a basic and ubiquitous function during the first stages of embryogenesis and into the gastrula period. Upon segmentation, a second phase of expression shows distinctly localized expression patterns, indicating tissue-specific roles and an involvement of re-1a/re-2a in neural development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Membrana/metabolismo , Peixe-Zebra/embriologia , Animais , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/metabolismo , Membrana Celular/metabolismo , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Olho/citologia , Olho/embriologia , Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Membrana/genética , Mesoderma/citologia , Mesoderma/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Mucosa Olfatória/citologia , Mucosa Olfatória/embriologia , Mucosa Olfatória/metabolismo , RNA Mensageiro/análise , Medula Espinal/citologia , Medula Espinal/embriologia , Medula Espinal/metabolismo , Distribuição Tecidual , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Congenital malformations represent approximately 3 in 100 live births within the human population. Understanding their pathogenesis and ultimately formulating effective treatments are underpinned by knowledge of the events and factors that regulate normal embryonic development. Studies in model organisms, primarily in the mouse, the most prominent genetically tractable mammalian model, have equipped us with a rudimentary understanding of mammalian development from early lineage commitment to morphogenetic processes. In this way, information provided by studies in the mouse can, in some cases, be used to draw parallels with other mammals, including human. Here, we provide an overview of our current understanding of the general sequence of developmental events from early cell cleavages to gastrulation and axis extension occurring in human embryos. We will also review some of the rare birth defects occurring at these stages, in particular those resulting in conjoined twinning or caudal dysgenesis.
Assuntos
Blastocisto , Cauda Equina/anormalidades , Ectromelia/etiologia , Desenvolvimento Embrionário , Gêmeos Unidos/embriologia , Animais , Cauda Equina/embriologia , Cauda Equina/metabolismo , Ectromelia/classificação , Ectromelia/embriologia , Ectromelia/metabolismo , Gastrulação , HumanosRESUMO
The ureteric bud is an epithelial tube that undergoes branching morphogenesis to form the renal collecting system. Although development of a normal kidney depends on proper ureteric bud morphogenesis, the cellular events underlying this process remain obscure. Here, we used time-lapse microscopy together with several genetic labeling methods to observe ureteric bud cell behaviors in developing mouse kidneys. We observed an unexpected cell behavior in the branching tips of the ureteric bud, which we term "mitosis-associated cell dispersal." Premitotic ureteric tip cells delaminate from the epithelium and divide within the lumen; although one daughter cell retains a basal process, allowing it to reinsert into the epithelium at the site of origin, the other daughter cell reinserts at a position one to three cell diameters away. Given the high rate of cell division in ureteric tips, this cellular behavior causes extensive epithelial cell rearrangements that may contribute to renal branching morphogenesis.
Assuntos
Células Epiteliais/citologia , Proteínas de Homeodomínio/fisiologia , Rim/citologia , Mitose/fisiologia , Morfogênese , Ureter/citologia , Animais , Movimento Celular , Células Epiteliais/metabolismo , Imunofluorescência , Rim/metabolismo , Camundongos , Camundongos Knockout , Ureter/metabolismoRESUMO
The gene expression, signaling, and cellular dynamics driving mouse embryo development have emerged through embryology and genetic studies. However, since mouse development is a temporally regulated three-dimensional process, any insight needs to be placed in this context of real-time visualization. Live imaging using genetically encoded fluorescent protein reporters is pushing the envelope of our understanding by uncovering unprecedented insights into mouse development and leading to the formulation of quantitative accurate models.