RESUMEN
Early embryonic development is a finely orchestrated process that requires precise regulation of gene expression coordinated with morphogenetic events. TATA-box binding protein-associated factors (TAFs), integral components of transcription initiation coactivators like TFIID and SAGA, play a crucial role in this intricate process. Here we show that disruptions in TAF5, TAF12 and TAF13 individually lead to embryonic lethality in the mouse, resulting in overlapping yet distinct phenotypes. Taf5 and Taf12 mutant embryos exhibited a failure to implant post-blastocyst formation, and Taf5 mutants have aberrant lineage specification within the inner cell mass. In contrast, Taf13 mutant embryos successfully implant and form egg-cylinder stages but fail to initiate gastrulation. Strikingly, we observed a depletion of pluripotency factors in TAF13-deficient embryos, including OCT4, NANOG and SOX2, highlighting an indispensable role of TAF13 in maintaining pluripotency. Transcriptomic analysis revealed distinct gene targets affected by the loss of TAF5, TAF12 and TAF13. Thus, we propose that TAF5, TAF12 and TAF13 convey locus specificity to the TFIID complex throughout the mouse genome.
Asunto(s)
Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Factores Asociados con la Proteína de Unión a TATA , Animales , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factores Asociados con la Proteína de Unión a TATA/genética , Ratones , Desarrollo Embrionario/genética , Factor de Transcripción TFIID/metabolismo , Factor de Transcripción TFIID/genética , Femenino , Blastocisto/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Gastrulación/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción SOXB1/genética , Proteína Homeótica Nanog/metabolismo , Proteína Homeótica Nanog/genética , Embrión de Mamíferos/metabolismoRESUMEN
Mammalian preimplantation development culminates in the formation of a blastocyst which undergoes extensive gene expression regulation to successfully implant into the maternal endometrium. Zinc-finger HIT domain-containing (ZNHIT) 1 and 2 are members of a highly conserved family, yet they have been identified as subunits of distinct complexes. Here we report that knockout of either Znhit1 or Znhit2 results in embryonic lethality during peri-implantation stages. Znhit1 and Znhit2 mutant embryos have overlapping phenotypes, including reduced proportion of SOX2-positive ICM cells, a lack of Fgf4 expression and aberrant expression of NANOG and SOX17. Furthermore, we find that the similar phenotypes are caused by distinct mechanisms. Specifically, embryos lacking ZNHIT1 likely fail to incorporate sufficient H2A.Z at the promoter region of Fgf4 and other genes involved in cell projection organization resulting in impaired invasion of trophoblast cells during implantation. In contrast, Znhit2 mutant embryos display a complete lack of nuclear EFTUD2, a key component of U5 spliceosome, indicating a global splicing deficiency. Our findings unveil the indispensable yet distinct roles of ZNHIT1 and ZNHIT2 in early mammalian embryonic development.
RESUMEN
Retinoic acid (RA), a metabolite of vitamin A, is a small molecule and morphogen that is required for embryonic development. While normal RA signals are required for hepatic development in a variety of vertebrates, a role for RA during mammalian hepatic specification has yet to be defined. To examine the requirement for RA in murine liver induction, we performed whole embryo culture with the small molecule RA inhibitor, BMS493, to attenuate RA signaling immediately prior to hepatic induction and through liver bud formation. BMS493 treated embryos demonstrated a significant loss of hepatic specification that was confined to the prospective dorsal anterior liver bud. Examination of RA attenuated embryos demonstrates that while the liver bud displays normal expression of foregut endoderm markers and the hepato-pancreatobiliary domain marker, PROX1, the dorsal/anterior liver bud excludes the critical hepatic marker, HNF4α, indicating that RA signals are required for dorsal/anterior hepatic induction. These results were confirmed and extended by careful examination of Rdh10<sup>trex/trex</sup> embryos, which carry a genetic perturbation in RA synthesis. At E9.5 Rdh10<sup>trex/trex</sup> embryos display a similar yet more significant loss of the anterior/dorsal liver bud. Notably the anterior/dorsal liver bud loss correlates with the known dorsal-ventral gradient of the RA synthesis enzyme, Aldh1a2. In addition to altered hepatic specification, the mesoderm surrounding the liver bud is disorganized in RA abrogated embryos. Analysis of E10.5 Rdh10<sup>trex/trex</sup> embryos reveals small livers that appear to lack the dorsal/caudal lobes. Finally, addition of exogenous RA prior to hepatic induction results in a liver bud that has failed to thicken and is largely unspecified. Taken together our ex vivo and in vivo evidence demonstrate that the generation of normal RA gradients is required for hepatic patterning, specification, and growth.
Asunto(s)
Tretinoina , Vitamina A , Animales , Endodermo/metabolismo , Femenino , Hígado , Mamíferos/metabolismo , Ratones , Embarazo , Estudios Prospectivos , Tretinoina/metabolismo , Tretinoina/farmacología , Vitamina A/metabolismoRESUMEN
Mitochondria are essential for energy production and although they have their own genome, many nuclear-encoded mitochondrial ribosomal proteins (MRPs) are required for proper function of the organelle. Although mutations in MRPs have been associated with human diseases, little is known about their role during development. Presented here are the null phenotypes for 21 nuclear-encoded mitochondrial proteins and in-depth characterization of mouse embryos mutant for the Mrp genes Mrpl3, Mrpl22, Mrpl44, Mrps18c and Mrps22 Loss of each MRP results in successful implantation and egg-cylinder formation, followed by severe developmental delay and failure to initiate gastrulation by embryonic day 7.5. The robust and similar single knockout phenotypes are somewhat surprising given there are over 70 MRPs and suggest little functional redundancy. Metabolic analysis reveals that Mrp knockout embryos produce significantly less ATP than controls, indicating compromised mitochondrial function. Histological and immunofluorescence analyses indicate abnormal organelle morphology and stalling at the G2/M checkpoint in Mrp null cells. The nearly identical pre-gastrulation phenotype observed for many different nuclear-encoded mitochondrial protein knockouts hints that distinct energy systems are crucial at specific time points during mammalian development.
Asunto(s)
Desarrollo Embrionario/genética , Gastrulación/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Ribosomas Mitocondriales/metabolismo , Proteínas Ribosómicas/genética , Animales , Puntos de Control del Ciclo Celular/genética , Femenino , Técnicas de Inactivación de Genes , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FenotipoRESUMEN
Early development and differentiation require precise control of cellular functions. Lysosomal degradation is a critical component of normal cellular homeostasis, allowing for degradation of signaling molecules, proteins, and other macromolecules for cellular remodeling and signaling. Little is known about the role of lysosomal function in mammalian embryos before gastrulation. Borcs6 is a protein involved in lysosomal trafficking as well as endo-lysosomal and autophagosome fusion. Here, we show that Borcs6 is necessary for efficient endo-lysosomal degradation in the early embryo. Although embryos lacking Borcs6 are developmentally comparable to control littermates at E5.5, they are characterized by large cells containing increased levels of late endosomes and abnormal nuclei. Furthermore, these embryos display a skewed ratio of extraembryonic and embryonic cell lineages, are delayed by E6.5, and do not undergo normal gastrulation. These results demonstrate the essential functions of lysosomal positioning and fusion with endosomes during early embryonic development and indicate that the early lethality of BORCS6 mutant embryos is primarily due to defects in the HOPS-related function of BORC rather than lysosomal positioning.
Asunto(s)
Endosomas , Lisosomas , Animales , Autofagia , Endosomas/metabolismo , Homeostasis , Lisosomas/metabolismo , Mamíferos , Fusión de Membrana , Proteínas/metabolismoRESUMEN
Protein phosphatases regulate a wide array of proteins through post-translational modification and are required for a plethora of intracellular events in eukaryotes. While some core components of the protein phosphatase complexes are well characterized, many subunits of these large complexes remain unstudied. Here we characterize a loss-of-function allele of the protein phosphatase 1 regulatory subunit 35 (Ppp1r35) gene. Homozygous mouse embryos lacking Ppp1r35 are developmental delayed beginning at embryonic day (E) 7.5 and have obvious morphological defects at later stages. Mutants fail to initiate turning and do not progress beyond the size or staging of normal E8.5 embryos. Consistent with recent in vitro studies linking PPP1R35 with the microcephaly protein Rotatin and with a role in centrosome formation, we show that Ppp1r35 mutant embryos lack primary cilia. Histological and molecular analysis of Ppp1r35 mutants revealed that notochord development is irregular and discontinuous and consistent with a role in primary cilia, that the floor plate of the neural tube is not specified. Similar to other mutant embryos with defects in centriole function, Ppp1r35 mutants displayed increased cell death that is prevalent in the neural tube and an increased number of proliferative cells in prometaphase. We hypothesize that loss of Ppp1r35 function abrogates centriole homeostasis, resulting in a failure to produce functional primary cilia, cell death and cell cycle delay/stalling that leads to developmental failure. Taken together, these results highlight the essential function of Ppp1r35 during early mammalian development and implicate this gene as a candidate for human microcephaly.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Cilios/metabolismo , Notocorda/enzimología , Organogénesis , Animales , Proteínas de Ciclo Celular/genética , Cilios/genética , Ratones , Ratones NoqueadosRESUMEN
The dynein axonemal assembly factor (Dnaaf) protein family is involved in preassembly and stability of dynein arms before they are transported into the cilia. In humans, mutations in DNAAF genes lead to several diseases related to cilia defects such as primary ciliary dyskinesia (PCD; OMIM: 612518). Patients with PCD experience malfunctions in cilia motility, which can result in inflammation and infection of the respiratory tract among other defects. Previous studies have identified that a mutation in DNAAF2 results in PCD and that 40% of these patients also experience laterality defects. In an outbred genetic background, Dnaaf2 homozygotes die after birth and have left/right defects among other phenotypes. Here we characterize a novel null allele of Dnaaf2 obtained from the International Mouse Phenotyping Consortium. Our data indicate that on a defined C57bl/6NJ genetic background, homozygous Dnaaf2 mouse embryos fail to progress beyond organogenesis stages with many abnormalities including left-right patterning defects. These findings support studies indicating that hypomorphic mutations of human DNAAF2 can result in ciliary dyskinesia and identify Dnaaf2 as an essential component of cilia function in vivo.
Asunto(s)
Trastornos de la Motilidad Ciliar/etiología , Genes Letales , Proteínas Asociadas a Microtúbulos/deficiencia , Mutación , Alelos , Animales , Tipificación del Cuerpo , Trastornos de la Motilidad Ciliar/metabolismo , Modelos Animales de Enfermedad , Desarrollo Embrionario/genética , Expresión Génica , Genotipo , Ratones , FenotipoRESUMEN
The definitive endoderm (DE) is the embryonic germ layer that forms the gut tube and associated organs, including thymus, lungs, liver and pancreas. To understand how individual DE cells furnish gut organs, genetic fate mapping was performed using the Rosa26lacZ Cre-reporter paired with a tamoxifen-inducible DE-specific Cre-expressing transgene. We established a low tamoxifen dose that infrequently induced heritable lacZ expression in a single cell of individual E8.5 mouse embryos and identified clonal cell descendants at E16.5. As expected, only a fraction of the E16.5 embryos contained lacZ-positive clonal descendants and a subset of these contained descendants in multiple organs, revealing novel ontogeny. Furthermore, immunohistochemical analysis was used to identify lacZ-positive hepatocytes and biliary epithelial cells, which are the cholangiocyte precursors, in each clonally populated liver. Together, these data not only uncover novel and suspected lineage relationships between DE-derived organs, but also illustrate the bipotential nature of individual hepatoblasts by demonstrating that single hepatoblasts contribute to both the hepatocyte and the cholangiocyte lineage in vivo.
Asunto(s)
Mapeo Cromosómico , Endodermo/citología , Hepatocitos/citología , Especificidad de Órganos/genética , Análisis de la Célula Individual , Células Madre/citología , Animales , Conductos Biliares/citología , Linaje de la Célula , Células Clonales , Embrión de Mamíferos/citología , Células Epiteliales/citología , Femenino , Hepatocitos/metabolismo , Masculino , Ratones Endogámicos C57BL , Células Madre/metabolismoRESUMEN
BACKGROUND AND AIMS: During liver development, bipotent progenitor cells differentiate into hepatocytes and biliary epithelial cells to ensure a functional liver required to maintain organismal homeostasis. The developmental cues controlling the differentiation of committed progenitors into these cell types, however, are incompletely understood. Here, we discover an essential role for estrogenic regulation in vertebrate liver development to affect hepatobiliary fate decisions. APPROACH AND RESULTS: Exposure of zebrafish embryos to 17ß-estradiol (E2) during liver development significantly decreased hepatocyte-specific gene expression, liver size, and hepatocyte number. In contrast, pharmacological blockade of estrogen synthesis or nuclear estrogen receptor (ESR) signaling enhanced liver size and hepatocyte marker expression. Transgenic reporter fish demonstrated nuclear ESR activity in the developing liver. Chemical inhibition and morpholino knockdown of nuclear estrogen receptor 2b (esr2b) increased hepatocyte gene expression and blocked the effects of E2 exposure. esr2b-/- mutant zebrafish exhibited significantly increased expression of hepatocyte markers with no impact on liver progenitors, other endodermal lineages, or vasculature. Significantly, E2-stimulated Esr2b activity promoted biliary epithelial differentiation at the expense of hepatocyte fate, whereas loss of esr2b impaired biliary lineage commitment. Chemical and genetic epistasis studies identified bone morphogenetic protein (BMP) signaling as a mediator of the estrogen effects. The divergent impact of estrogen on hepatobiliary fate was confirmed in a human hepatoblast cell line, indicating the relevance of this pathway for human liver development. CONCLUSIONS: Our studies identify E2, esr2b, and downstream BMP activity as important regulators of hepatobiliary fate decisions during vertebrate liver development. These results have significant clinical implications for liver development in infants exposed to abnormal estrogen levels or estrogenic compounds during pregnancy.
Asunto(s)
Sistema Biliar/embriología , Estradiol/metabolismo , Receptor beta de Estrógeno/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hígado/embriología , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Sistema Biliar/citología , Sistema Biliar/metabolismo , Diferenciación Celular/genética , Línea Celular , Embrión no Mamífero , Estradiol/administración & dosificación , Receptor beta de Estrógeno/genética , Femenino , Técnicas de Silenciamiento del Gen , Hepatocitos/fisiología , Hígado/citología , Hígado/metabolismo , Masculino , Modelos Animales , Morfolinos/administración & dosificación , Morfolinos/genética , Transducción de Señal/genética , Células Madre/fisiología , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Microspherule protein 1 (MCRS1, also known as MSP58) is an evolutionarily conserved protein that has been implicated in various biological processes. Although a variety of functions have been attributed to MCRS1 in vitro, mammalian MCRS1 has not been studied in vivo. Here we report that MCRS1 is essential during early murine development. Mcrs1 mutant embryos exhibit normal morphology at the blastocyst stage but cannot be recovered at gastrulation, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts do not form a typical inner cell mass (ICM) colony, the source of embryonic stem cells (ESCs). Surprisingly, cell death and histone H4 acetylation analysis reveal that apoptosis and global H4 acetylation are normal in mutant blastocysts. However, analysis of lineage specification reveals that while the trophoblast and primitive endoderm are properly specified, the epiblast lineage is compromised and exhibits a severe reduction in cell number. In summary, our study demonstrates the indispensable role of MCRS1 in epiblast development during early mammalian embryogenesis.
Asunto(s)
Masa Celular Interna del Blastocisto/citología , Embrión de Mamíferos/citología , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Estratos Germinativos/citología , Mutación , Proteínas de Unión al ARN/fisiología , Animales , Masa Celular Interna del Blastocisto/metabolismo , Diferenciación Celular , Linaje de la Célula , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Femenino , Estratos Germinativos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
The murine pancreas buds from the ventral embryonic endoderm at approximately 8.75 dpc and a second pancreas bud emerges from the dorsal endoderm by 9.0 dpc. Although it is clear that secreted signals from adjacent mesoderm-derived sources are required for both the appropriate emergence and further refinement of the pancreatic endoderm, neither the exact signals nor the requisite tissue sources have been defined in mammalian systems. Herein we use DiI fate mapping of cultured murine embryos to identify the embryonic sources of both the early inductive and later condensed pancreatic mesenchyme. Despite being capable of supporting pancreas induction from dorsal endoderm in co-culture experiments, we find that in the context of the developing embryo, the dorsal aortae as well as the paraxial, intermediate, and lateral mesoderm derivatives only transiently associate with the dorsal pancreas bud, producing descendants that are decidedly anterior to the pancreas bud. Unlike these other mesoderm derivatives, the axial (notochord) descendants maintain association with the dorsal pre-pancreatic endoderm and early pancreas bud. This fate mapping data points to the notochord as the likely inductive source in vivo while also revealing dynamic morphogenetic movements displayed by individual mesodermal subtypes. Because none of the mesoderm examined above produced the pancreatic mesenchyme that condenses around the induced bud to support exocrine and endocrine differentiation, we also sought to identify the mesodermal origins of this mesenchyme. We identify a portion of the coelomic mesoderm that contributes to the condensed pancreatic mesenchyme. In conclusion, we identify a portion of the notochord as a likely source of the signals required to induce and maintain the early dorsal pancreas bud, demonstrate that the coelomic mesothelium contributes to the dorsal and ventral pancreatic mesenchyme, and provide insight into the dynamic morphological rearrangements of mesoderm-derived tissues during early organogenesis stages of mammalian development.
Asunto(s)
Embrión de Mamíferos/embriología , Mesodermo/embriología , Organogénesis/fisiología , Páncreas/embriología , Animales , Embrión de Mamíferos/citología , Mesodermo/citología , Ratones , Páncreas/citologíaRESUMEN
During development, the endoderm initiates organ-restricted gene expression patterns in a spatiotemporally controlled manner. This process, termed induction, requires signals from adjacent mesodermal derivatives. Fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) emanating from the cardiac mesoderm and the septum transversum mesenchyme (STM), respectively, are believed to be simultaneously and uniformly required to directly induce hepatic gene expression from the murine endoderm. Using small molecule inhibitors of BMP signals during liver bud induction in the developing mouse embryo, we found that BMP signaling was not uniformly required to induce hepatic gene expression. Although BMP inhibition caused an overall reduction in the number of induced hepatoblasts, the STM-bounded posterior liver bud demonstrated the most severe loss of the essential hepatic transcription factor, hepatocyte nuclear factor 4-α (HNF4α), whereas the sinus venosus-lined anterior liver bud was less affected. We found that the posterior liver bud progenitors were anteriorly displaced and aberrantly activated pancreatobiliary markers, including sex-determining region Y-box 9 (SOX9). Additionally, we found that ectopically expressed SOX9 inhibited HNF4α and that BMP was indirectly required for hepatoblast induction. Finally, because previous studies have demonstrated that FGF signals are essential for anterior but not posterior liver bud induction, we examined synchronous BMP and FGF inhibition and found this led to a nearly complete loss of hepatoblasts. CONCLUSION: BMP signaling is required to maintain the hepato-pancreatobiliary boundary, at least in part, by indirectly repressing SOX9 in the hepatic endoderm. BMP and FGF signals are each required for the induction of spatially complementary subsets of hepatoblasts. These results underscore the importance of studying early inductive processes in the whole embryo. (Hepatology 2018;68:274-288).
Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Inducción Embrionaria , Hígado/embriología , Animales , Receptores de Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proliferación Celular , Factores de Crecimiento de Fibroblastos/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Ratones , Factor de Transcripción SOX9/metabolismoRESUMEN
Mediator is an evolutionarily conserved multi-subunit complex, bridging transcriptional activators and repressors to the general RNA polymerase II (Pol II) initiation machinery. Though the Mediator complex is crucial for the transcription of almost all Pol II promoters in eukaryotic organisms, the phenotypes of individual Mediator subunit mutants are each distinct. Here, we report for the first time, the essential role of subunit MED20 in early mammalian embryo development. Although Med20 mutant mouse embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at early post-gastrulation stages. Outgrowth assays show that mutant blastocysts cannot hatch from the zona pellucida, indicating impaired blastocyst function. Assessments of cell death and cell lineage specification reveal that apoptosis, inner cell mass, trophectoderm and primitive endoderm markers are normal in mutant blastocysts. However, the epiblast marker NANOG is ectopically expressed in the trophectoderm of Med20 mutants, indicative of defects in trophoblast specification. These results suggest that MED20 specifically, and the Mediator complex in general, are essential for the earliest steps of mammalian development and cell lineage specification.
Asunto(s)
Blastocisto/citología , Embrión de Mamíferos/citología , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Complejo Mediador/fisiología , Proteína Homeótica Nanog/genética , Animales , Blastocisto/metabolismo , Linaje de la Célula , Embrión de Mamíferos/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Homeótica Nanog/metabolismoRESUMEN
The primary cilium is a sensory organelle, defects in which cause a wide range of human diseases including retinal degeneration, polycystic kidney disease and birth defects. The sensory functions of cilia require specific receptors to be targeted to the ciliary subdomain of the plasma membrane. Arf4 has been proposed to sort cargo destined for the cilium at the Golgi complex and deemed a key regulator of ciliary protein trafficking. In this work, we show that Arf4 binds to the ciliary targeting sequence (CTS) of fibrocystin. Knockdown of Arf4 indicates that it is not absolutely required for trafficking of the fibrocystin CTS to cilia as steady-state CTS levels are unaffected. However, we did observe a delay in delivery of newly synthesized CTS from the Golgi complex to the cilium when Arf4 was reduced. Arf4 mutant mice are embryonic lethal and die at mid-gestation shortly after node formation. Nodal cilia appeared normal and functioned properly to break left-right symmetry in Arf4 mutant embryos. At this stage of development Arf4 expression is highest in the visceral endoderm but we did not detect cilia on these cells. In the visceral endoderm, the lack of Arf4 caused defects in cell structure and apical protein localization. This work suggests that while Arf4 is not required for ciliary assembly, it is important for the efficient transport of fibrocystin to cilia, and also plays critical roles in non-ciliary processes.
Asunto(s)
Factores de Ribosilacion-ADP/genética , Desarrollo Embrionario/genética , Riñón Poliquístico Autosómico Recesivo/genética , Receptores de Superficie Celular/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Animales , Membrana Celular , Cilios/genética , Cilios/ultraestructura , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Humanos , Ratones , Riñón Poliquístico Autosómico Recesivo/metabolismo , Riñón Poliquístico Autosómico Recesivo/patología , Receptores de Superficie Celular/genéticaRESUMEN
BACKGROUND: The definitive endoderm arises as a naive epithelial sheet that produces the entire gut tube and associated organs including the liver, pancreas and lungs. Murine explant studies demonstrate that fibroblast growth factor (FGF) signaling from adjacent tissues is required to induce hepatic gene expression from isolated foregut endoderm. The requirement of FGF signaling during liver development is examined by means of small molecule inhibition during whole embryo culture. RESULTS: Loss of FGF signaling before hepatic induction results in morphological defects and gene expression changes that are confined to the anterior liver bud. In contrast the posterior portion of the liver bud remains relatively unaffected. Because FGF is thought to act as a morphogen during endoderm organogenesis, the ventral pancreas was also examined after FGF inhibition. Although the size of the ventral pancreas is not affected, loss of FGF signaling results in a significantly higher density of ventral pancreas cells. CONCLUSIONS: The requirement for FGF-mediated induction of hepatic gene expression differs across the anterior/posterior axis of the developing liver bud. These results underscore the importance of studying tissue differentiation in the context of the whole embryo.
Asunto(s)
Endodermo/embriología , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Hígado/embriología , Organogénesis/fisiología , Transducción de Señal/fisiología , Animales , Endodermo/citología , Hígado/citología , Ratones , Especificidad de Órganos/fisiologíaRESUMEN
BACKGROUND: Laser-mediated cell ablation is a powerful tool that has been used to understand cell fate in a variety of externally developing organisms but has not been used during mammalian post-implantation development. RESULTS: We describe a method pairing laser ablation with murine embryo culture and establish parameters that can be used to precisely ablate cells in the selected field with minimal disruption to adjacent cells or the underlying cell matrix. Ablation of a large domain of endoderm, followed by ~1 day of culture results in a phenotypically normal embryo and gut tube, indicating that laser ablation is compatible with normal development. We next focused on one of the three precursor populations that have been shown to produce the liver bud. Ablations of a single progenitor domain result in a unilateral delay in the liver bud while the contralateral side is unaffected. CONCLUSIONS: We demonstrate that laser ablation is a specific and useful technique for studying cell fate in the mouse embryo. This method represents a powerful advance in developmental studies in the mouse and can be used to provide information on the specification of organs, differentiation, cell migration, and vital tissue interactions during development.
Asunto(s)
Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Desarrollo Embrionario/fisiología , Endodermo , Rayos Láser , Hígado , Animales , Endodermo/citología , Endodermo/embriología , Hígado/citología , Hígado/embriología , RatonesRESUMEN
The ubiquitously expressed Polycomb Group protein Yin-Yang1 (YY1) is believed to regulate gene expression through direct binding to DNA elements found in promoters or enhancers of target loci. Additionally, YY1 contains diverse domains that enable a plethora of protein-protein interactions, including association with the Oct4/Sox2 pluripotency complex and Polycomb Group silencing complexes. To elucidate the in vivo role of YY1 during gastrulation, we generated embryos with an epiblast specific deletion of Yy1. Yy1 conditional knockout (cKO) embryos initiate gastrulation, but both primitive streak formation and ingression through the streak is severely impaired. These streak descendants fail to repress E-Cadherin and are unable to undergo an appropriate epithelial to mesenchymal transition (EMT). Intriguingly, overexpression of Nodal and concomitant reduction of Lefty2 are observed in Yy1 cKO embryos, suggesting that YY1 is normally required for proper Nodal regulation during gastrulation. Furthermore, definitive endoderm is specified but fails to properly integrate into the outer layer. Although anterior neuroectoderm is specified, mesoderm production is severely restricted. We show that YY1 directly binds to the Lefty2 locus in E7.5 embryos and that pharmacological inhibition of Nodal signaling partially restores mesoderm production in Yy1 cKO mutant embryos. Our results reveal critical requirements for YY1 during several important developmental processes, including EMT and regulation of Nodal signaling. These results are the first to elucidate the diverse role of YY1 during gastrulation in vivo.
Asunto(s)
Transición Epitelial-Mesenquimal/genética , Gastrulación/genética , Proteína Nodal/genética , Transducción de Señal/genética , Factor de Transcripción YY1/genética , Animales , Secuencia de Bases , Benzodioxoles/farmacología , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Estratos Germinativos/embriología , Estratos Germinativos/metabolismo , Imidazoles/farmacología , Inmunohistoquímica , Hibridación in Situ , Factores de Determinación Derecha-Izquierda/genética , Factores de Determinación Derecha-Izquierda/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Microscopía Fluorescente , Proteína Nodal/metabolismo , Unión Proteica , Piridinas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Factor de Transcripción YY1/metabolismoRESUMEN
Fluorescent proteins have revolutionized modern biology with their ability to report the presence of tagged proteins in living systems. Although several fluorescent proteins have been described in which the excitation and emission properties can be modulated by external triggers, no fluorescent proteins have been described that can be activated from a silent dark state to a bright fluorescent state directly by the activity of an enzyme. We have developed a version of GFP in which fluorescence is completely quenched by appendage of a hydrophobic quenching peptide that tetramerizes GFP and prevents maturation of the chromophore. The fluorescence can be fully restored by catalytic removal of the quenching peptide, making it a robust reporter of proteolysis. We have demonstrated the utility of this uniquely dark state of GFP as a genetically encoded apoptosis reporter that monitors the function of caspases, which catalyze the fate-determining step in programmed cell death. Caspase Activatable-GFP (CA-GFP) can be activated both in vitro and in vivo, resulting in up to a 45-fold increase in fluorescent signal in bacteria and a 3-fold increase in mammalian cells. We used CA-GFP successfully to monitor real-time apoptosis in mammalian cells. This dark state of GFP may ultimately serve as a useful platform for probes of other enzymatic processes.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Proteolisis , Animales , Caspasas/genética , Catálisis , Proteínas Fluorescentes Verdes/genética , Ratones , Microscopía Fluorescente/métodos , Células 3T3 NIHRESUMEN
The endoderm emerges as an epithelial sheet that covers the surface of the developing murine embryo. This tissue will produce the entire gut tube as well as associated digestive and respiratory organs including the thyroid, thymus, lung, liver, and pancreas. The emergence of each endodermal organ occurs in a temporally distinct manner that is dependant upon reciprocal inductive interactions between the endoderm and the underlying mesoderm. The emergence of the hepatic endoderm, which occurs using a morphological process termed liver budding, initiates during early somitogenesis in the mouse at approximately 8.25 days post-coitum (dpc). Explant and transplant studies performed in chicken and mouse have demonstrated that secreted signals from adjacent mesodermal tissues initiate the hepatic gene program from ventral-fated endoderm. Here, we review the data in support of the roles of members of the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), and Wnt signaling pathways in liver budding and discover that little is known about the precise endogenous signals involved in the molecular and morphological induction of liver budding in the mouse.
Asunto(s)
Hígado/metabolismo , Transducción de Señal , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Endodermo/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Edad Gestacional , Hígado/embriología , Ratones , Morfogénesis , Proteínas Wnt/metabolismoRESUMEN
While particular combinations of mesodermal signals are known to induce distinct tissue-specific programs in the endoderm, there is little information about the response pathways within endoderm cells that control their specification. We have used signaling inhibitors on embryo tissue explants and whole-embryo cultures as well as genetic approaches to reveal part of an intracellular network by which FGF signaling helps induce hepatic genes and stabilize nascent hepatic cells within the endodermal epithelium. Specifically, we found that hepatic gene induction is elicited by an FGF/MAPK pathway. Although the PI3K pathway is activated in foregut endoderm cells, its inhibition does not block hepatic gene induction in explants; however, it does block tissue growth. We also found that at the onset of hepatogenesis, the FGF/MAPK and PI3K pathways do not crossregulate in the endoderm. The finding of separate pathways for endoderm tissue specification and growth provides insights for guiding cellular regeneration and stem cell differentiation.