RESUMEN
BACKGROUND: Proteins of the TGFß family, which are largely studied as homodimers, are also known to form heterodimers with biological activity distinct from their component homodimers. For instance, heterodimers of bone morphogenetic proteins, including BMP2/BMP7, BMP2/BMP6, and BMP9/BMP10, among others, have illustrated the importance of these heterodimeric proteins within the context of TGFß signaling. RESULTS: In this study, we have determined that mature GDF5 can be combined with mature BMP2 or BMP4 to form BMP2/GDF5 and BMP4/GDF5 heterodimer. Intriguingly, this combination of a BMP2 or BMP4 monomer, which exhibit high affinity to heparan sulfate characteristic to the BMP class, with a GDF5 monomer with low heparan sulfate affinity produces a heterodimer with an intermediate affinity. Using heparin affinity chromatography to purify the heterodimeric proteins, we then determined that both the BMP2/GDF5 and BMP4/GDF5 heterodimers consistently signaled potently across an array of cellular and in vivo systems, while the activities of their homodimeric counterparts were more context dependent. These differences were likely driven by an increase in the combined affinities for the type 1 receptors, Alk3 and Alk6. Furthermore, the X-ray crystal structure of BMP2/GDF5 heterodimer was determined, highlighting the formation of two asymmetric type 1 receptor binding sites that are both unique relative to the homodimers. CONCLUSIONS: Ultimately, this method of heterodimer production yielded a signaling molecule with unique properties relative to the homodimeric ligands, including high affinity to multiple type 1 and moderate heparan binding affinity.
Asunto(s)
Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas , Proteínas Morfogenéticas Óseas/metabolismo , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Unión Proteica , Proteínas Portadoras/metabolismo , Heparitina SulfatoRESUMEN
Trachea-esophageal defects (TEDs), including esophageal atresia (EA), tracheoesophageal fistula (TEF), and laryngeal-tracheoesophageal clefts (LTEC), are a spectrum of life-threatening congenital anomalies in which the trachea and esophagus do not form properly. Up until recently, the developmental basis of these conditions and how the trachea and esophagus arise from a common fetal foregut was poorly understood. However, with significant advances in human genetics, organoids, and animal models, and integrating single cell genomics with high resolution imaging, we are revealing the molecular and cellular mechanisms that orchestrate tracheoesophageal morphogenesis and how disruption in these processes leads to birth defects. Here we review the current understanding of the genetic and developmental basis of TEDs. We suggest future opportunities for integrating developmental mechanisms elucidated from animals and organoids with human genetics and clinical data to gain insight into the genotype-phenotype basis of these heterogeneous birth defects. Finally, we envision how this will enhance diagnosis, improve treatment, and perhaps one day, lead to new tissue replacement therapy.
Asunto(s)
Esófago/anomalías , Tráquea/anomalías , Animales , Anomalías del Sistema Digestivo/diagnóstico , Anomalías del Sistema Digestivo/etiología , Anomalías del Sistema Digestivo/genética , Modelos Animales de Enfermedad , Esófago/embriología , Humanos , Organoides/embriología , Tráquea/embriologíaRESUMEN
Characterization of the pluripotent "ground state" has led to a greater understanding of species-specific stem cell differences and has imparted an appreciation of the pluripotency continuum that exists in stem cells in vitro. Pluripotent stem cells are functionally coupled via connexins that serve in gap junctional intercellular communication (GJIC) and here we report that the level of connexin expression in pluripotent stem cells depends upon the state in which stem cells exist in vitro. Human and mouse pluripotent stem cells stabilized in a developmentally primitive or "naïve" state exhibit significantly less connexin expression compared with stem cells which are "primed" for differentiation. This dynamic connexin expression pattern may be governed, in part, by differential regulation by pluripotency transcription factors expressed in each cell state. Species-specific differences do exist, however, with mouse stem cells expressing several additional connexin transcripts not found in human pluripotent stem cells. Moreover, pharmacological inhibition of GJIC shows limited impact on naïve human stem cell survival, self-renewal, and pluripotency but plays a more significant role in primed human pluripotent stem cells. However, CRISPR-Cas9 gene ablation of Cx43 in human and mouse primed and naïve pluripotent stem cells reveals that Cx43 is dispensable in each of these four pluripotent stem cell types.
Asunto(s)
Conexinas/metabolismo , Células Madre Pluripotentes/metabolismo , Animales , Comunicación Celular , Diferenciación Celular , Humanos , RatonesRESUMEN
STUDY QUESTION: What is the impact of adenosine monophosphate-activated protein kinase (AMPK) activation on blastocyst formation, gene expression, and tight junction formation and function? SUMMARY ANSWER: AMPK activity must be tightly controlled for normal preimplantation development and blastocyst formation to occur. WHAT IS KNOWN ALREADY: AMPK isoforms are detectable in oocytes, cumulus cells and preimplantation embryos. Cultured embryos are subject to many stresses that can activate AMPK. STUDY DESIGN, SIZE, DURATION: Two primary experiments were carried out to determine the effect of AICAR treatment on embryo development and maintenance of the blastocoel cavity. Embryos were recovered from superovulated mice. First, 2-cell embryos were treated with a concentration series (0-2000 µM) of AICAR for 48 h until blastocyst formation would normally occur. In the second experiment, expanded mouse blastocysts were treated for 9 h with 1000 µM AICAR. PARTICIPANTS/MATERIALS, SETTING, METHODS: Outcomes measured included development to the blastocyst stage, cell number, blastocyst volume, AMPK phosphorylation, Cdx2 and blastocyst formation gene family expression (mRNAs and protein measured using quantitative RT-PCR, immunoblotting, immunofluorescence), tight junction function (FITC dextran dye uptake assay), and blastocyst ATP levels. The reversibility of AICAR treatment was assessed using Compound C (CC), a well-known inhibitor of AMPK, alone or in combination with AICAR. MAIN RESULTS AND THE ROLE OF CHANCE: Prolonged treatment with AICAR from the 2-cell stage onward decreases blastocyst formation, reduces total cell number, embryo diameter, leads to loss of trophectoderm cell contacts and membrane zona occludens-1 staining, and increased nuclear condensation. Treatment with CC alone inhibited blastocyst development only at concentrations that are higher than normally used. AICAR treated embryos displayed altered mRNA and protein levels of blastocyst formation genes. Treatment of blastocysts with AICAR for 9 h induced blastocyst collapse, altered blastocyst formation gene expression, increased tight junction permeability and decreased CDX2. Treated blastocysts displayed three phenotypes: those that were unaffected by treatment, those in which treatment was reversible, and those in which effects were irreversible. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Our study investigates the effects of AICAR treatment on early development. While AICAR does increase AMPK activity and this is demonstrated in our study, AICAR is not a natural regulator of AMPK activity and some outcomes may result from off target non-AMPK AICAR regulated events. To support our results, blastocyst developmental outcomes were confirmed with two other well-known small molecule activators of AMPK, metformin and phenformin. WIDER IMPLICATIONS OF THE FINDINGS: Metformin, an AMPK activator, is widely used to treat type II diabetes and polycystic ovarian disorder (PCOS). Our results indicate that early embryonic AMPK levels must be tightly regulated to ensure normal preimplantation development. Thus, use of metformin should be carefully considered during preimplantation and early post-embryo transfer phases of fertility treatment cycles. STUDY FUNDING AND COMPETING INTEREST(S): Canadian Institutes of Health Research (CIHR) operating funds. There are no competing interests.
Asunto(s)
Aminoimidazol Carboxamida/análogos & derivados , Blastocisto/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hipoglucemiantes/farmacología , Ribonucleótidos/farmacología , Uniones Estrechas/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Trifosfato/biosíntesis , Aminoimidazol Carboxamida/farmacología , Animales , Blastocisto/metabolismo , Blastocisto/ultraestructura , Factor de Transcripción CDX2/genética , Factor de Transcripción CDX2/metabolismo , Diferenciación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Técnicas de Cultivo de Embriones , Femenino , Ratones , Oxazinas/farmacología , Fosforilación/efectos de los fármacos , Transducción de Señal , Uniones Estrechas/metabolismo , Uniones Estrechas/ultraestructuraRESUMEN
STUDY QUESTION: Do high oxygen tension and high glucose concentrations dysregulate p66Shc (Src homologous-collagen homologue adaptor protein) expression during mouse preimplantation embryo culture? SUMMARY ANSWER: Compared with mouse blastocysts in vivo, P66Shc mRNA and protein levels in blastocysts maintained in vitro increased under high oxygen tension (21%), but not high glucose concentration. WHAT IS KNOWN ALREADY: Growth in culture adversely impacts preimplantation embryo development and alters the expression levels of the oxidative stress adaptor protein p66Shc, but it is not known if p66Shc expression is linked to metabolic changes observed in cultured embryos. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: We used a standard wild-type CD1 mouse model of preimplantation embryo development and embryo culture with different atmospheric oxygen tension and glucose media concentrations. Changes to p66Shc expression in mouse blastocysts were measured using quantitative RT-PCR, immunoblotting and immunofluorescence followed by confocal microscopy. Changes to oxidative phosphorylation metabolism were measured by total ATP content and superoxide production. Statistical analyses were performed on a minimum of three experimental replicates using Students' t-test or one-way ANOVA. MAIN RESULTS AND THE ROLE OF CHANCE: P66Shc is basally expressed during in vivo mouse preimplantation development. Within in vivo blastocysts, p66Shc is primarily localized to the cell periphery of the trophectoderm. Blastocysts cultured under atmospheric oxygen levels have significantly increased p66Shc mRNA transcript and protein abundances compared to in vivo controls (P < 0.05). However, the ratio of phosphorylated serine 36 (S36) p66Shc to total p66Shc decreased in culture regardless of O2 atmosphere used, supporting a shift in the mitochondrial fraction of p66Shc. Total p66Shc localized to the cell periphery of the blastocyst trophectoderm and phosphorylated S36 p66Shc displayed nuclear and cytoplasmic immunoreactivity, suggesting distinct compartmentalization of phosphorylated S36 p66Shc and the remaining p66Shc fraction. Glucose concentration in the culture medium did not significantly change p66Shc mRNA or protein abundance or its localization. Blastocysts cultured under low or high oxygen conditions exhibited significantly decreased cellular ATP and increased superoxide production compared to in vivo derived embryos (P < 0.05). LIMITATIONS/REASONS FOR CAUTION: This study associates embryonic p66Shc expression levels with metabolic abnormalities but does not directly implicate p66Shc in metabolic changes. Additionally, we used one formulation of embryo culture medium that differs from that used in other mouse model studies and from clinical media used to support human blastocyst development. Our findings may, therefore, be limited to this media, or may be a species-specific phenomenon. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to show distinct immunolocalization of p66Shc to the trophectoderm of mouse blastocysts and that its levels are abnormally increased in embryos exposed to culture conditions. Changes in p66Shc expression and/or localization could possibly serve as a molecular marker of embryo viability for clinical applications. The outcomes provide insight into the potential metabolic role of p66Shc. Metabolic anomalies are induced even under the current optimal culture conditions, which could negatively impact trophectoderm and placental development. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: Canadian Institutes of Health Research (CIHR) operating funds, Ontario Graduate Scholarship (OGS). There are no competing interests.
Asunto(s)
Especies Reactivas de Oxígeno/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/metabolismo , Animales , Blastocisto/metabolismo , Western Blotting , Línea Celular , Células Cultivadas , Modelos Animales de Enfermedad , Técnicas de Cultivo de Embriones , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Femenino , Técnica del Anticuerpo Fluorescente , Glucosa/farmacología , Masculino , Ratones , Microscopía Confocal , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/genéticaRESUMEN
Disruptions in foregut morphogenesis can result in life-threatening conditions where the trachea and esophagus fail to separate properly, such as esophageal atresia (EA) and tracheoesophageal fistulas (TEF). The developmental basis of these congenital anomalies is poorly understood, but recent genome sequencing reveals that de novo variants in intracellular trafficking genes are enriched in EA/TEF patients. Here we show that mutation of orthologous genes in Xenopus disrupts trachea-esophageal separation similar to EA/TEF patients. We show that the Rab11a recycling endosome pathway is required to localize Vangl-Celsr polarity complexes at the cell surface where opposite sides of the common foregut tube fuse. Partial loss of endosome trafficking or the Vangl/Celsr complex disrupts epithelial polarity and cell division orientation. Mutant cells accumulate at the fusion point, fail to downregulate cadherin, and do not separate into distinct trachea and esophagus. These data provide new insights into the mechanisms of congenital anomalies and general paradigms of tissue fusion during organogenesis.
RESUMEN
The ShcA adapter protein is necessary for early embryonic development. The role of ShcA in development is primarily attributed to its 52 and 46 kDa isoforms that transduce receptor tyrosine kinase signaling through the extracellular signal regulated kinase (ERK). During embryogenesis, ERK acts as the primary signaling effector, driving fate acquisition and germ layer specification. P66Shc, the largest of the ShcA isoforms, has been observed to antagonize ERK in several contexts; however, its role during embryonic development remains poorly understood. We hypothesized that p66Shc could act as a negative regulator of ERK activity during embryonic development, antagonizing early lineage commitment. To explore the role of p66Shc in stem cell self-renewal and differentiation, we created a p66Shc knockout murine embryonic stem cell (mESC) line. Deletion of p66Shc enhanced basal ERK activity, but surprisingly, instead of inducing mESC differentiation, loss of p66Shc enhanced the expression of core and naive pluripotency markers. Using pharmacologic inhibitors to interrogate potential signaling mechanisms, we discovered that p66Shc deletion permits the self-renewal of naive mESCs in the absence of conventional growth factors, by increasing their responsiveness to leukemia inhibitory factor (LIF). We discovered that loss of p66Shc enhanced not only increased ERK phosphorylation but also increased phosphorylation of Signal transducer and activator of transcription in mESCs, which may be acting to stabilize their naive-like identity, desensitizing them to ERK-mediated differentiation cues. These findings identify p66Shc as a regulator of both LIF-mediated ESC pluripotency and of signaling cascades that initiate postimplantation embryonic development and ESC commitment.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular , Células Madre Embrionarias de Ratones , Animales , Ratones , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/genética , Factor Inhibidor de Leucemia/genética , Factor Inhibidor de Leucemia/farmacología , Factor Inhibidor de Leucemia/metabolismo , Diferenciación Celular , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
[This corrects the article DOI: 10.1016/j.xhgg.2022.100107.].
RESUMEN
Esophageal atresias/tracheoesophageal fistulas (EA/TEF) are rare congenital anomalies caused by aberrant development of the foregut. Previous studies indicate that rare or de novo genetic variants significantly contribute to EA/TEF risk, and most individuals with EA/TEF do not have pathogenic genetic variants in established risk genes. To identify the genetic contributions to EA/TEF, we performed whole genome sequencing of 185 trios (probands and parents) with EA/TEF, including 59 isolated and 126 complex cases with additional congenital anomalies and/or neurodevelopmental disorders. There was a significant burden of protein-altering de novo coding variants in complex cases (p = 3.3 × 10-4), especially in genes that are intolerant of loss-of-function variants in the population. We performed simulation analysis of pathway enrichment based on background mutation rate and identified a number of pathways related to endocytosis and intracellular trafficking that as a group have a significant burden of protein-altering de novo variants. We assessed 18 variants for disease causality using CRISPR-Cas9 mutagenesis in Xenopus and confirmed 13 with tracheoesophageal phenotypes. Our results implicate disruption of endosome-mediated epithelial remodeling as a potential mechanism of foregut developmental defects. Our results suggest significant genetic heterogeneity of EA/TEF and may have implications for the mechanisms of other rare congenital anomalies.
RESUMEN
The endoderm is the innermost germ layer that forms the linings of the respiratory and gastrointestinal tracts, and their associated organs, during embryonic development. Xenopus embryology experiments have provided fundamental insights into how the endoderm develops in vertebrates, including the critical role of TGFß-signaling in endoderm induction,elucidating the gene regulatory networks controlling germ layer development and the key molecular mechanisms regulating endoderm patterning and morphogenesis. With new genetic, genomic, and imaging approaches, Xenopus is now routinely used to model human disease, discover mechanisms underlying endoderm organogenesis, and inform differentiation protocols for pluripotent stem cell differentiation and regenerative medicine applications. In this chapter, we review historical and current discoveries of endoderm development in Xenopus, then provide examples of modeling human disease and congenital defects of endoderm-derived organs using Xenopus.
Asunto(s)
Modelos Animales de Enfermedad , Endodermo/embriología , Morfogénesis , Xenopus laevis/embriología , Animales , Diferenciación Celular , Endodermo/citología , Endodermo/metabolismo , Humanos , Morfogénesis/genética , Organogénesis/genética , Xenopus laevis/genéticaRESUMEN
CRISPR/Cas9 genome editing has revolutionized functional genomics in vertebrates. However, CRISPR/Cas9 edited F0 animals too often demonstrate variable phenotypic penetrance due to the mosaic nature of editing outcomes after double strand break (DSB) repair. Even with high efficiency levels of genome editing, phenotypes may be obscured by proportional presence of in-frame mutations that still produce functional protein. Recently, studies in cell culture systems have shown that the nature of CRISPR/Cas9-mediated mutations can be dependent on local sequence context and can be predicted by computational methods. Here, we demonstrate that similar approaches can be used to forecast CRISPR/Cas9 gene editing outcomes in Xenopus tropicalis, Xenopus laevis, and zebrafish. We show that a publicly available neural network previously trained in mouse embryonic stem cell cultures (InDelphi-mESC) is able to accurately predict CRISPR/Cas9 gene editing outcomes in early vertebrate embryos. Our observations can have direct implications for experiment design, allowing the selection of guide RNAs with predicted repair outcome signatures enriched towards frameshift mutations, allowing maximization of CRISPR/Cas9 phenotype penetrance in the F0 generation.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Penetrancia , Xenopus laevis/embriología , Xenopus laevis/genética , Pez Cebra/embriología , Pez Cebra/genética , Animales , Proteína 9 Asociada a CRISPR/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Mutación del Sistema de Lectura , Frecuencia de los Genes , Células HEK293 , Humanos , Ratones , Células Madre Embrionarias de Ratones/metabolismo , ARN Guía de Kinetoplastida/genéticaRESUMEN
The trachea and esophagus arise from the separation of a common foregut tube during early fetal development. Mutations in key signaling pathways such as Hedgehog (HH)/Gli can disrupt tracheoesophageal (TE) morphogenesis and cause life-threatening birth defects (TEDs); however, the underlying cellular mechanisms are unknown. Here, we use mouse and Xenopus to define the HH/Gli-dependent processes orchestrating TE morphogenesis. We show that downstream of Gli the Foxf1+ splanchnic mesenchyme promotes medial constriction of the foregut at the boundary between the presumptive Sox2+ esophageal and Nkx2-1+ tracheal epithelium. We identify a unique boundary epithelium co-expressing Sox2 and Nkx2-1 that fuses to form a transient septum. Septum formation and resolution into distinct trachea and esophagus requires endosome-mediated epithelial remodeling involving the small GTPase Rab11 and localized extracellular matrix degradation. These are disrupted in Gli-deficient embryos. This work provides a new mechanistic framework for TE morphogenesis and informs the cellular basis of human TEDs.
Asunto(s)
Endosomas/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Hedgehog/metabolismo , Morfogénesis/fisiología , Animales , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Sistema Digestivo/metabolismo , Endodermo/metabolismo , Endosomas/genética , Esófago/embriología , Factores de Transcripción Forkhead/metabolismo , Humanos , Mesodermo/metabolismo , Mutación/genética , XenopusRESUMEN
The p66Shc adaptor protein regulates apoptosis and senescence during early mammalian development. However, p66Shc expression during mouse preimplantation development is upregulated at the blastocyst stage. Our objective was to determine the biological function of p66Shc during mouse blastocyst development. In this study, we demonstrate that a reduced p66Shc transcript abundance following its short interfering RNA (siRNA)-mediated knockdown alters the spatiotemporal expression of cell lineage-associated transcription factors in the inner cell mass (ICM) of the mouse blastocyst. P66Shc knockdown blastocysts restrict OCT3/4 earlier to the inner cells of the early blastocyst and have ICMs containing significantly higher OCT3/4 levels, more GATA4-positive cells, and fewer NANOG-positive cells. P66Shc knockdown blastocysts also show a significantly reduced ability to form ICM-derived outgrowths when explanted in vitro. The increase in cells expressing primitive endoderm markers may be due to increased ERK1/2 activity, as it is reversed by ERK1/2 inhibition. These results suggest that p66Shc may regulate the relative abundance and timing of lineage-associated transcription factor expression in the blastocyst ICM.
Asunto(s)
Masa Celular Interna del Blastocisto/metabolismo , Blastocisto/metabolismo , Linaje de la Célula/genética , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/genética , Animales , Blastocisto/citología , Masa Celular Interna del Blastocisto/citología , Diferenciación Celular/genética , Proliferación Celular/genética , Desarrollo Embrionario , Endodermo/crecimiento & desarrollo , Endodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Sistema de Señalización de MAP Quinasas/genética , Ratones , Proteína Homeótica Nanog/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , ARN Interferente Pequeño/genética , Factores de Transcripción/genéticaRESUMEN
CD-1 mice are commonly employed as a research model for defining mechanisms controlling early mammalian development and for understanding environmental impacts on mammalian fertility. CD-1 female mice were kept four to eight months under conventional animal care housing, and were fed ad libitum with normal laboratory mouse chow. Female weight, mating success, oocyte morphology, blastocyst development in vivo and in vitro, and RT-qPCR analysis of trophectoderm cell markers (Cdx2, Slc2a1, and Atp1a1 transcript abundance, and CDX2 localization) were assessed and contrasted with outcomes from four-week-old control CD-1 mice. Embryo development in vivo in four to eight-month-old mice was significantly reduced compared to four-week-old controls. Oocytes and blastocysts from four to eight-month-old CD-1 mice displayed high levels of fragmentation and degradation, significantly reduced embryo cell counts, decreased Cdx2 transcript abundance, and number of CDX2 positive cells in morulae. We have discovered that female CD-1 mice housed under conventional conditions display a rapid loss of fecundity as they age over a few months. Paradoxically, embryo loss can be avoided by placing early embryos collected from four to eight-month-old mice into culture to support development to the blastocyst stage. We conclude that oocyte quality rapidly declines in CD-1 female mice housed under conventional animal care conditions. Thus, four to eight-month-old female CD-1 mice represent a very distinct research model from that of younger mice and this older research animal model may be preferred for understanding environmental and physiological influences limiting fertility in women.