RESUMEN
FGF signaling is known to play a critical role in the specification of primitive endoderm (PrE) and epiblast (Epi) from the inner cell mass (ICM) during mouse preimplantation development, but how FGFs synergize with other growth factor signaling pathways is unknown. Because PDGFRα signaling has also been implicated in the PrE, we investigated the coordinate functions of PDGFRα together with FGFR1 or FGFR2 in PrE development. PrE development was abrogated in Pdgfra; Fgfr1 compound mutants, or significantly reduced in Pdgfra; Fgfr2 or PdgfraPI3K; Fgfr2 compound mutants. We provide evidence that both Fgfr2 and Pdgfra play roles in PrE cell survival while Fgfr1 controls PrE cell specification. Our results suggest a model where FGFR1-engaged ERK1/2 signaling governs PrE specification while PDGFRα- and by analogy possibly FGFR2- engaged PI3K signaling regulates PrE survival and positioning in the embryo. Together, these studies indicate how multiple growth factors and signaling pathways can cooperate in preimplantation development.
Asunto(s)
Factor 4 de Crecimiento de Fibroblastos/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Blastocisto/metabolismo , Masa Celular Interna del Blastocisto/metabolismo , Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , Endodermo/metabolismo , Factor 4 de Crecimiento de Fibroblastos/fisiología , Factores de Crecimiento de Fibroblastos/metabolismo , Estratos Germinativos/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Transgénicos , Fosfatidilinositol 3-Quinasas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/fisiología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Embryonic morphogenesis of a complex organism requires proper regulation of patterning and directional growth. Planar cell polarity (PCP) signaling is emerging as a crucial evolutionarily conserved mechanism whereby directional information is conveyed. PCP is thought to be established by global cues, and recent studies have revealed an instructive role of a Wnt signaling gradient in epithelial tissues of both invertebrates and vertebrates. However, it remains unclear whether Wnt/PCP signaling is regulated in a coordinated manner with embryonic patterning during morphogenesis. Here, in mouse developing limbs, we find that apical ectoderm ridge-derived Fgfs required for limb patterning regulate PCP along the proximal-distal axis in a Wnt5a-dependent manner. We demonstrate with genetic evidence that the Wnt5a gradient acts as a global cue that is instructive in establishing PCP in the limb mesenchyme, and that Wnt5a also plays a permissive role to allow Fgf signaling to orient PCP. Our results indicate that limb morphogenesis is regulated by coordination of directional growth and patterning through integration of Wnt5a and Fgf signaling.
Asunto(s)
Tipificación del Cuerpo/fisiología , Polaridad Celular/fisiología , Factor 4 de Crecimiento de Fibroblastos/fisiología , Factor 8 de Crecimiento de Fibroblastos/fisiología , Proteína Wnt-5a/fisiología , Animales , Tipificación del Cuerpo/genética , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Extremidades/embriología , Factor 4 de Crecimiento de Fibroblastos/deficiencia , Factor 4 de Crecimiento de Fibroblastos/genética , Factor 8 de Crecimiento de Fibroblastos/deficiencia , Factor 8 de Crecimiento de Fibroblastos/genética , Mesodermo/embriología , Ratones , Ratones Noqueados , Ratones Transgénicos , Transducción de Señal , Proteína Wnt-5a/deficiencia , Proteína Wnt-5a/genéticaRESUMEN
The apical ectodermal ridge (AER), located at the distal end of each limb bud, is a key signaling center which controls outgrowth and patterning of the proximal-distal axis of the limb through secretion of various molecules. Fibroblast growth factors (FGFs), particularly Fgf8 and Fgf4, are representative molecules produced by AER cells, and essential to maintain the AER and cell proliferation in the underlying mesenchyme, meanwhile Jag2-Notch pathway negatively regulates the AER and limb development. p63, a transcription factor of the p53 family, is expressed in the AER and indispensable for limb formation. However, the underlying mechanisms and specific roles of p63 variants are unknown. Here, we quantified the expression of p63 variants in mouse limbs from embryonic day (E) 10.5 to E12.5, and found that ΔNp63γ was strongly expressed in limbs at all stages, while TAp63γ expression was rapidly increased in the later stages. Fluorescence-activated cell sorting analysis of limb bud cells from reporter mouse embryos at E11.5 revealed that all variants were abundantly expressed in AER cells, and their expression was very low in mesenchymal cells. We then generated AER-specific p63 knockout mice by mating mice with a null and a flox allele of p63, and Msx2-Cre mice (Msx2-Cre;p63Δ/fl). Msx2-Cre;p63Δ/fl neonates showed limb malformation that was more obvious in distal elements. Expression of various AER-related genes was decreased in Msx2-Cre;p63Δ/fl limb buds and embryoid bodies formed by p63-knockdown induced pluripotent stem cells. Promoter analyses and chromatin immunoprecipitation assays demonstrated Fgf8 and Fgf4 as transcriptional targets of ΔNp63γ, and Jag2 as that of TAp63γ. Furthermore, TAp63γ overexpression exacerbated the phenotype of Msx2-Cre;p63Δ/fl mice. These data indicate that ΔNp63 and TAp63 control limb development through transcriptional regulation of different target molecules with different roles in the AER. Our findings contribute to further understanding of the molecular network of limb development.
Asunto(s)
Esbozos de los Miembros/crecimiento & desarrollo , Fosfoproteínas/fisiología , Transactivadores/fisiología , Animales , Animales Recién Nacidos , Factor 4 de Crecimiento de Fibroblastos/fisiología , Factor 8 de Crecimiento de Fibroblastos/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Esbozos de los Miembros/fisiología , Ratones/embriología , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfoproteínas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transactivadores/genéticaRESUMEN
Tumor-associated endothelial cells (TECs) regulate tumor cell aggressiveness. However, the core mechanism by which TECs confer stem cell-like activity to indolent tumors is unknown. Here, we used in vivo murine and human tumor models to identify the tumor-suppressive checkpoint role of TEC-expressed insulin growth factor (IGF) binding protein-7 (IGFBP7/angiomodulin). During tumorigenesis, IGFBP7 blocks IGF1 and inhibits expansion and aggresiveness of tumor stem-like cells (TSCs) expressing IGF1 receptor (IGF1R). However, chemotherapy triggers TECs to suppress IGFBP7, and this stimulates IGF1R+ TSCs to express FGF4, inducing a feedforward FGFR1-ETS2 angiocrine cascade that obviates TEC IGFBP7. Thus, loss of IGFBP7 and upregulation of IGF1 activates the FGF4-FGFR1-ETS2 pathway in TECs and converts naive tumor cells to chemoresistant TSCs, thereby facilitating their invasiveness and progression.
Asunto(s)
Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/fisiología , Células Madre Neoplásicas/efectos de los fármacos , Animales , Transformación Celular Neoplásica , Resistencia a Antineoplásicos , Células Endoteliales/fisiología , Factor 4 de Crecimiento de Fibroblastos/fisiología , Humanos , Factor I del Crecimiento Similar a la Insulina/fisiología , Ratones , Proteína Proto-Oncogénica c-ets-2/fisiología , Receptor IGF Tipo 1/fisiologíaRESUMEN
Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are defined as a small population of cells within cancer that contribute to cancer initiation and progression. Cancer-associated fibroblasts (CAFs) are stromal fibroblasts surrounding tumor cells, and they have important roles in tumor growth and tumor progression. It has been suggested that stromal fibroblasts and CSCs/CICs might mutually cooperate to enhance their growth and tumorigenic capacity. In this study, we investigated the effects of fibroblasts on tumor-initiating capacity and stem-like properties of ovarian CSCs/CICs. CSCs/CICs were isolated from the ovarian carcinoma cell line HTBoA as aldehyde dehydrogenase 1 high (ALDH1(high)) population by the ALDEFLUOR assay. Histological examination of tumor tissues derived from ALDH1(high) cells revealed few fibrous stroma, whereas those derived from fibroblast-mixed ALDH1(high) cells showed abundant fibrous stroma formation. In vivo tumor-initiating capacity and in vitro sphere-forming capacity of ALDH1(high) cells were enhanced in the presence of fibroblasts. Gene expression analysis revealed that fibroblast-mixed ALDH1(high) cells had enhanced expression of fibroblast growth factor 4 (FGF4) as well as stemness-associated genes such as SOX2 and POU5F1. Sphere-forming capacity of ALDH1(high) cells was suppressed by small-interfering RNA (siRNA)-mediated knockdown of FGFR2, the receptor for FGF4 which was expressed preferentially in ALDH1(high) cells. Taken together, the results indicate that interaction of fibroblasts with ovarian CSCs/CICs enhanced tumor-initiating capacity and stem-like properties through autocrine and paracrine FGF4-FGFR2 signaling.
Asunto(s)
Factor 4 de Crecimiento de Fibroblastos/fisiología , Fibroblastos/fisiología , Células Madre Neoplásicas/patología , Neoplasias Ováricas/patología , Familia de Aldehído Deshidrogenasa 1 , Animales , Células Cultivadas , Femenino , Factor 4 de Crecimiento de Fibroblastos/genética , Humanos , Isoenzimas/análisis , Ratones , Ratones SCID , Células Madre Neoplásicas/metabolismo , Neoplasias Ováricas/mortalidad , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/fisiología , Retinal-Deshidrogenasa/análisis , Transducción de Señal/fisiología , Microambiente Tumoral , Regulación hacia ArribaRESUMEN
In embryonic stem (ES) cells and in early mouse embryos, the transcription factor Oct4 is an essential regulator of pluripotency. Oct4 transcriptional targets have been described in ES cell lines; however, the molecular mechanisms by which Oct4 regulates establishment of pluripotency in the epiblast (EPI) have not been fully elucidated. Here, we show that neither maternal nor zygotic Oct4 is required for the formation of EPI cells in the blastocyst. Rather, Oct4 is first required for development of the primitive endoderm (PE), an extraembryonic lineage. EPI cells promote PE fate in neighboring cells by secreting Fgf4, and Oct4 is required for expression of Fgf4, but we show that Oct4 promotes PE development cell-autonomously, downstream of Fgf4 and Mapk. Finally, we show that Oct4 is required for the expression of multiple EPI and PE genes as well as multiple metabolic pathways essential for the continued growth of the preimplantation embryo.
Asunto(s)
Blastocisto/fisiología , Células Madre Embrionarias/fisiología , Endodermo/embriología , Regulación del Desarrollo de la Expresión Génica , Factor 3 de Transcripción de Unión a Octámeros/fisiología , Animales , Animales no Consanguíneos , Blastocisto/citología , Linaje de la Célula/fisiología , Células Madre Embrionarias/citología , Endodermo/citología , Femenino , Factor 4 de Crecimiento de Fibroblastos/fisiología , Factor de Transcripción GATA6/genética , Factor de Transcripción GATA6/fisiología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Ratones , Ratones Transgénicos , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , EmbarazoRESUMEN
Stem cells in the developing embryo proliferate and differentiate while maintaining genomic integrity, failure of which may lead to accumulation of mutations and subsequent damage to the embryo. Embryonic stem cells (ESCs), the in vitro counterpart of embryo stem cells are highly sensitive to genotoxic stress. Defective ESCs undergo either efficient DNA damage repair or apoptosis, thus maintaining genomic integrity. However, the genotoxicity- and apoptosis-related processes in germ-line derived pluripotent cells, multipotent adult germ-line stem cells (maGSCs), are currently unknown. Here, we analyzed the expression of apoptosis-related genes using OligoGEArray in undifferentiated maGSCs and ESCs and identified a similar set of genes expressed in both cell types. We detected the expression of intrinsic, but not extrinsic, apoptotic pathway genes in both cell types. Further, we found that apoptosis-related gene expression patterns of differentiated ESCs and maGSCs are identical to each other. Comparative analysis revealed that several pro- and anti-apoptotic genes are expressed specifically in pluripotent cells, but markedly downregulated in the differentiated counterparts of these cells. Activation of the intrinsic apoptotic pathway cause approximately â¼35% of both ESCs and maGSCs to adopt an early-apoptotic phenotype. Moreover, we performed transcriptome studies using early-apoptotic cells to identify novel pluripotency- and apoptosis-related genes. From these transcriptome studies, we selected Fgf4 (Fibroblast growth factor 4) and Mnda (Myeloid cell nuclear differentiating antigen), which are highly downregulated in early-apoptotic cells, as novel candidates and analyzed their roles in apoptosis and genotoxicity responses in ESCs. Collectively, our results show the existence of common molecular mechanisms for maintaining the pristine stem cell pool of both ESCs and maGSCs.
Asunto(s)
Antígenos de Diferenciación Mielomonocítica/fisiología , Antígenos Nucleares/fisiología , Apoptosis/genética , Células Madre Embrionarias/citología , Factor 4 de Crecimiento de Fibroblastos/fisiología , Células Germinativas/citología , Células Madre Multipotentes/citología , Transcriptoma , Animales , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Diferenciación Celular , Línea Celular , Citrinina , Daño del ADN/genética , Regulación hacia Abajo , Células Madre Embrionarias/metabolismo , Factor 4 de Crecimiento de Fibroblastos/genética , Factor 4 de Crecimiento de Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Células Germinativas/metabolismo , Ratones , Células Madre Multipotentes/metabolismoRESUMEN
Somites form along the embryonic axis by sequential segmentation from the presomitic mesoderm (PSM) and differentiate into the segmented vertebral column as well as other unsegmented tissues. Somites are thought to form via the intersection of two activities known as the clock and the wavefront. Previous work has suggested that fibroblast growth factor (FGF) activity may be the wavefront signal, which maintains the PSM in an undifferentiated state. However, it is unclear which (if any) of the FGFs expressed in the PSM comprise this activity, as removal of any one gene is insufficient to disrupt early somitogenesis. Here we show that when both Fgf4 and Fgf8 are deleted in the PSM, expression of most PSM genes is absent, including cycling genes, WNT pathway genes, and markers of undifferentiated PSM. Significantly, markers of nascent somite cell fate expand throughout the PSM, demonstrating the premature differentiation of this entire tissue, a highly unusual phenotype indicative of the loss of wavefront activity. When WNT signaling is restored in mutants, PSM progenitor markers are partially restored but premature differentiation of the PSM still occurs, demonstrating that FGF signaling operates independently of WNT signaling. This study provides genetic evidence that FGFs are the wavefront signal and identifies the specific FGF ligands that encode this activity. Furthermore, these data show that FGF action maintains WNT signaling, and that both signaling pathways are required in parallel to maintain PSM progenitor tissue.
Asunto(s)
Desarrollo Embrionario/fisiología , Factor 4 de Crecimiento de Fibroblastos/fisiología , Factor 8 de Crecimiento de Fibroblastos/fisiología , Somitos , Animales , Factor 4 de Crecimiento de Fibroblastos/genética , Factor 8 de Crecimiento de Fibroblastos/genética , Ratones , Transducción de SeñalRESUMEN
Differentiated trophoblast cell lineages arise from trophoblast stem (TS) cells. To date such a stem cell population has only been established in the mouse. The objective of this investigation was to establish TS cell populations from rat blastocysts. Blastocysts were cultured individually on a feeder layer of rat embryonic fibroblasts (REFs) in fibroblast growth factor-4 (FGF4) and heparin supplemented culture medium. Once cell colonies were established REF feeder layers could be replaced with REF conditioned medium. The blastocyst-derived cell lines, in either proliferative or differentiated states, did not express genes indicative of ICM-derived tissues. In the proliferative state the cells expressed established stem cell-associated markers of TS cells. Cells ceased proliferation and differentiated when FGF4, heparin, and REF conditioned medium were removed. Differentiation was characterized by a decline of stem cell-associated marker gene expression, the appearance of large polyploid cells (trophoblast giant cells), and the expression of trophoblast differentiation-associated genes. Collectively, the data indicate that the rat blastocyst-derived cell lines not only possess many features characteristic of mouse TS cells but also possess some distinct properties. These rat TS cell lines represent valuable new in vitro models for analyses of mechanisms controlling TS cell renewal and differentiation.
Asunto(s)
Blastocisto/citología , Diferenciación Celular , Linaje de la Célula , Factor 4 de Crecimiento de Fibroblastos/fisiología , Células Madre/citología , Trofoblastos/citología , Animales , Femenino , Masculino , Ratones , Fenotipo , Ratas , Ratas Sprague-DawleyRESUMEN
AIM: To identify the key cytokines involved in hepatic differentiation of mouse bone marrow mesenchymal stem cells (mBM-MSCs) under liver-injury conditions. METHODS: Abdominal injection of CCl(4) was adopted to duplicate a mouse acute liver injury model. Global gene expression analysis was performed to evaluate the potential genes involved in hepatic commitment under liver-injury conditions. The cytokines involved in hepatic differentiation of mBM-MSCs was functionally examined by depletion experiment using specific antibodies, followed by rescue experiment and direct inducing assay. The hepatic differentiation was characterized by the expression of hepatic lineage genes and proteins, as well as functional features. RESULTS: Cytokines potentially participating in hepatic fate commitment under liver-injury conditions were initially measured by microarray. Among the up-regulated genes determined, 18 cytokines known to closely relate to liver growth, repair and development, were selected for further identification. The fibroblast growth factor-4 (FGF-4), hepatocyte growth factor (HGF) and oncostatin M (OSM) were finally found to be involved in hepatic differentiation of mBM-MSCs under liver-injury conditions. Hepatic differentiation could be dramatically decreased after removing FGF-4, HGF and OSM from the liver-injury conditioned medium, and could be rescued by supplementing these cytokines. The FGF-4, HGF and OSM play different roles in the hepatic differentiation of mBM-MSCs, in which FGF-4 and HGF are essential for the initiation of hepatic differentiation, while OSM is critical for the maturation of hepatocytes. CONCLUSION: FGF-4, HGF and OSM are the key cytokines involved in the liver-injury conditioned medium for the hepatic differentiation of mBM-MSCs.
Asunto(s)
Citocinas/fisiología , Hepatocitos/patología , Hepatocitos/fisiología , Hígado/lesiones , Células Madre Mesenquimatosas/patología , Enfermedad Aguda , Animales , Secuencia de Bases , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/patología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Citocinas/antagonistas & inhibidores , Citocinas/genética , Citocinas/farmacología , Cartilla de ADN/genética , Factor 4 de Crecimiento de Fibroblastos/genética , Factor 4 de Crecimiento de Fibroblastos/farmacología , Factor 4 de Crecimiento de Fibroblastos/fisiología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/patología , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/farmacología , Factor de Crecimiento de Hepatocito/fisiología , Hepatocitos/efectos de los fármacos , Hígado/patología , Hígado/fisiopatología , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Oncostatina M/genética , Oncostatina M/farmacología , Oncostatina M/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacología , Regulación hacia ArribaRESUMEN
Pluripotency is defined as the capacity of individual cells to initiate all lineages of the mature organism in response to signals from the embryo or cell culture environment. A pluripotent cell has no predetermined programme; it is a blank slate. This is the foundation of mammalian development and of ES (embryonic stem) cell biology. What are the design principles of this naïve cell state? How is pluripotency acquired and maintained? Suppressing activation of ERKs (extracellular-signal-regulated kinases) is critical to establishing and sustaining ES cells. Inhibition of GSK3 (glycogen synthase kinase 3) reinforces this effect. We review the effect of selective kinase inhibitors on pluripotent cells and consider how these effects are mediated. We propose that ES cells represent a ground state, meaning a basal proliferative state that is free of epigenetic restriction and has minimal requirements for extrinsic stimuli. The stability of this state is reflected in the homogeneity of ES cell populations cultured in the presence of small-molecule inhibitors of MEK (mitogen-activated protein kinase/ERK kinase) and GSK3.
Asunto(s)
Células Madre Pluripotentes/fisiología , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular/fisiología , Proliferación Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Factor 4 de Crecimiento de Fibroblastos/genética , Factor 4 de Crecimiento de Fibroblastos/metabolismo , Factor 4 de Crecimiento de Fibroblastos/fisiología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3/fisiología , Humanos , Células Madre Pluripotentes/metabolismo , Transducción de Señal/fisiologíaRESUMEN
We have developed a novel three-dimensional (3D) cellular microarray platform to enable the rapid and efficient tracking of stem cell fate and quantification of specific stem cell markers. This platform consists of a miniaturized 3D cell culture array on a functionalized glass slide for spatially addressable high-throughput screening. A microarray spotter was used to deposit cells onto a modified glass surface to yield an array consisting of cells encapsulated in alginate gel spots with volumes as low as 60 nL. A method based on an immunofluorescence technique scaled down to function on a cellular microarray was also used to quantify specific cell marker protein levels in situ. Our results revealed that this platform is suitable for studying the expansion of mouse embryonic stem (ES) cells as they retain their pluripotent and undifferentiated state. We also examined neural commitment of mouse ES cells on the microarray and observed the generation of neuroectodermal precursor cells characterized by expression of the neural marker Sox-1, whose levels were also measured in situ using a GFP reporter system. In addition, the high-throughput capacity of the platform was tested using a dual-slide system that allowed rapid screening of the effects of tretinoin and fibroblast growth factor-4 (FGF-4) on the pluripotency of mouse ES cells. This high-throughput platform is a powerful new tool for investigating cellular mechanisms involved in stem cell expansion and differentiation and provides the basis for rapid identification of signals and conditions that can be used to direct cellular responses.
Asunto(s)
Análisis por Micromatrices/métodos , Alginatos , Animales , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Proliferación Celular , Medios de Cultivo/química , Factor 4 de Crecimiento de Fibroblastos/fisiología , Geles , Vidrio , Ratones , Factores de Transcripción SOXB1/biosíntesis , Células MadreRESUMEN
Dimethylations of histone H3 lysine 9 and lysine 27 are important epigenetic marks associated with transcription repression. Here, we identified KIAA1718 (KDM7A) as a novel histone demethylase specific for these two repressing marks. Using mouse embryonic stem cells, we demonstrated that KIAA1718 expression increased at the early phase of neural differentiation. Knockdown of the gene blocked neural differentiation and the effect was rescued by the wild-type human gene, and not by a catalytically inactive mutant. In addition, overexpression of KIAA1718 accelerated neural differentiation. We provide the evidence that the pro-neural differentiation effect of KDM7A is mediated through direct transcriptional activation of FGF4, a signal molecule implicated in neural differentiation. Thus, our study identified a dual-specificity histone demethylase that regulates neural differentiation through FGF4.
Asunto(s)
Diferenciación Celular/genética , Factor 4 de Crecimiento de Fibroblastos/genética , Histona Demetilasas/fisiología , Histona Demetilasas con Dominio de Jumonji/fisiología , Neuronas/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/fisiología , Factor 4 de Crecimiento de Fibroblastos/farmacología , Factor 4 de Crecimiento de Fibroblastos/fisiología , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Ratones , Modelos Biológicos , Neurogénesis/genética , Neuronas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad por SustratoRESUMEN
BACKGROUND: Retinoic acid (RA) and fibroblast growth factor 4 (FGF4) signaling control endoderm patterning and pancreas induction/expansion. Based on these findings, RA and FGFs, excluding FGF4, have frequently been used in differentiation protocols to direct differentiation of hESCs into endodermal and pancreatic cell types. In vivo, these signaling pathways act in a temporal and concentration-dependent manner. However, in vitro, the underlying basis for the time of addition of growth and differentiation factors (GDFs), including RA and FGFs, as well as the concentration is lacking. Thus, in order to develop robust and reliable differentiation protocols of ESCs into mature pancreatic cell types, including insulin-producing beta cells, it will be important to mechanistically understand each specification step. This includes differentiation of mesendoderm/definitive endoderm into foregut endoderm--the origin of pancreatic endoderm. METHODOLOGY/PRINCIPAL FINDINGS: Here, we provide data on the individual and combinatorial role of RA and FGF4 in directing differentiation of ActivinA (AA)-induced hESCs into PDX1-expressing cells. FGF4's ability to affect endoderm patterning and specification in vitro has so far not been tested. By testing out the optimal concentration and timing of addition of FGF4 and RA, we present a robust differentiation protocol that on average generates 32% PDX1(+) cells. Furthermore, we show that RA is required for converting AA-induced hESCs into PDX1(+) cells, and that part of the underlying mechanism involves FGF receptor signaling. Finally, further characterization of the PDX1(+) cells suggests that they represent foregut endoderm not yet committed to pancreatic, posterior stomach, or duodenal endoderm. CONCLUSION/SIGNIFICANCE: In conclusion, we show that RA and FGF4 jointly direct differentiation of PDX1(+) foregut endoderm in a robust and efficient manner. RA signaling mediated by the early induction of RARbeta through AA/Wnt3a is required for PDX1 expression. Part of RA's activity is mediated by FGF signaling.
Asunto(s)
Células Madre Embrionarias/efectos de los fármacos , Endodermo/efectos de los fármacos , Factor 4 de Crecimiento de Fibroblastos/fisiología , Proteínas de Homeodominio/biosíntesis , Transducción de Señal/fisiología , Transactivadores/biosíntesis , Tretinoina/farmacología , Activinas/farmacología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Endodermo/citología , Endodermo/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/genética , Humanos , Páncreas/citología , Páncreas/embriología , Pirroles/farmacología , ARN Mensajero/biosíntesis , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/biosíntesis , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/fisiología , Receptores de Ácido Retinoico/biosíntesis , Receptores de Ácido Retinoico/genética , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transactivadores/genética , Regulación hacia Arriba/efectos de los fármacos , Proteínas Wnt/fisiología , Proteína Wnt3 , Proteína Wnt3ARESUMEN
Fibroblast growth factors (FGFs) were initially recognized as fibroblast-specific growth factor, and it is now apparent that these growth factors regulate multiple biological functions. The diversity of FGFs function is paralleled by the emerging diversity of interactions between FGF ligands and their receptors. FGF-4 is a member of the FGF superfamily and is a mitogen exhibiting strong action on numerous different cell types. It plays a role in various stages of development and morphogenesis, as well as in a variety of biological processes. Recent studies reveal the molecular mechanisms of FGF-4 gene regulation in mammalian cells, which is involved in the developmental process. Furthermore, FGF-4 also acts on the regulation of proliferation and differentiation in embryonic stem cells and tissue stem cells. In this review, we focus on the diverse biological functions of FGF-4 in the developmental process and also discuss its putative roles in stem cell biology.
Asunto(s)
Diferenciación Celular/fisiología , Factor 4 de Crecimiento de Fibroblastos/fisiología , Morfogénesis , Células Madre/fisiología , Células Madre Adultas/citología , Células Madre Adultas/fisiología , Animales , Proliferación Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Humanos , Transducción de Señal/fisiología , Células Madre/citologíaRESUMEN
Genome endoreduplication during mammalian development is a rare event for which the mechanism is unknown. It first appears when fibroblast growth factor 4 (FGF4) deprivation induces differentiation of trophoblast stem (TS) cells into the nonproliferating trophoblast giant (TG) cells required for embryo implantation. Here we show that RO3306 inhibition of cyclin-dependent protein kinase 1 (CDK1), the enzyme required to enter mitosis, induced differentiation of TS cells into TG cells. In contrast, RO3306 induced abortive endoreduplication and apoptosis in embryonic stem cells, revealing that inactivation of CDK1 triggers endoreduplication only in cells programmed to differentiate into polyploid cells. Similarly, FGF4 deprivation resulted in CDK1 inhibition by overexpressing two CDK-specific inhibitors, p57/KIP2 and p21/CIP1. TS cell mutants revealed that p57 was required to trigger endoreduplication by inhibiting CDK1, while p21 suppressed expression of the checkpoint protein kinase CHK1, thereby preventing induction of apoptosis. Furthermore, Cdk2(-/-) TS cells revealed that CDK2 is required for endoreduplication when CDK1 is inhibited. Expression of p57 in TG cells was restricted to G-phase nuclei to allow CDK activation of S phase. Thus, endoreduplication in TS cells is triggered by p57 inhibition of CDK1 with concomitant suppression of the DNA damage response by p21.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Diferenciación Celular/fisiología , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/fisiología , Células Gigantes/citología , Trofoblastos/citología , Animales , Proteína Quinasa CDC2/antagonistas & inhibidores , Ciclo Celular/fisiología , Células Cultivadas , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Factor 4 de Crecimiento de Fibroblastos/fisiología , Células Gigantes/fisiología , Ratones , Ratones Mutantes , Quinolinas/farmacología , Tiazoles/farmacología , Trofoblastos/fisiologíaRESUMEN
BACKGROUND: Cell proliferation in multicellular organisms must be coordinated with pattern formation. The major signaling pathways directing pattern formation in the vertebrate limb are well characterized, and we have therefore chosen this organ to examine the interaction between proliferation and patterning. Two important signals for limb development are members of the Hedgehog (Hh) and Fibroblast Growth Factor (Fgf) families of secreted signaling proteins. Sonic hedgehog (Shh) directs pattern formation along the anterior/posterior axis of the limb, whereas several Fgfs in combination direct pattern formation along the proximal/distal axis of the limb. RESULTS: We used the genetic and pharmacological amenability of the zebrafish model system to dissect the relative importance of Shh and Fgf signaling in regulating proliferation during development of the pectoral fin buds. In zebrafish mutants disrupting the shh gene, proliferation in the pectoral fin buds is initially normal, but later is strongly reduced. Correlating with this reduction, Fgf signaling is normal at early stages, but is later lost in shh mutants. Furthermore, pharmacological inhibition of Hh signaling for short periods has little effect on either Fgf signaling, or on expression of G1- and S-phase cell-cycle genes, whereas long periods of inhibition lead to the downregulation of both. In contrast, even short periods of pharmacological inhibition of Fgf signaling lead to strong disruption of proliferation in the fin buds, without affecting Shh signaling. To directly test the ability of Fgf signaling to regulate proliferation in the absence of Shh signaling, we implanted beads soaked with Fgf protein into shh mutant fin buds. We find that Fgf-soaked beads rescue proliferation in the pectoral find buds of shh mutants, indicating that Fgf signaling is sufficient to direct proliferation in zebrafish fin buds in the absence of Shh. CONCLUSION: Previous studies have shown that both Shh and Fgf signaling are crucial for outgrowth of the vertebrate limb. The results presented here show that the role of Shh in this process is indirect, and is mediated by its effect on Fgf signaling. By contrast, the activity of the Fgf pathway affects proliferation directly and independently of its effect on Shh. These results show that Fgf signaling is of primary importance in directing outgrowth of the limb bud, and clarify the role of the Shh-Fgf feedback loop in regulating proliferation.
Asunto(s)
Proliferación Celular , Extremidades/embriología , Factor 4 de Crecimiento de Fibroblastos/fisiología , Proteínas Hedgehog/fisiología , Transducción de Señal/fisiología , Pez Cebra/embriología , Animales , Extremidades/fisiología , Fase G1/genética , Fase G1/fisiología , Proteínas Hedgehog/genética , Humanos , Esbozos de los Miembros/embriología , Esbozos de los Miembros/fisiología , Mutación , Proteínas Recombinantes/farmacología , Fase S/genética , Fase S/fisiologíaRESUMEN
Hepatocytes derived from human embryonic stem cells (hESCs) are a potential cell source for regenerative medicine. However, the definitive factors that are responsible for hepatic differentiation of hESCs remain unclear. We aimed to evaluate the effects of various extracellular matrixes and growth factors on endodermal differentiation and to optimize the culture conditions to induce hepatic differentiation of hESCs. The transgene vector that contained enhanced green fluorescent protein (EGFP) under the control of human alpha-fetoprotein (AFP) enhancer/promoter was transfected into hESC lines. The transgenic hESCs were cultured on extracellular matrixes (collagen type I, laminin, and Matrigel) in the presence or absence of growth factors including hepatocyte growth factor (HGF), bone morphogenetic protein 4, fibroblast growth factor 4, all-trans-retinoic acid, and activin A. The expression of AFP-EGFP was measured by flow cytometry. The culture on Matrigel-coated dishes with 100 ng/ml activin A showed 19.5% of EGFP-positive proportions. Moreover, the sequential addition of 100 ng/ml activin A and 20 ng/ml HGF resulted in 21.7% and produced a higher yield of EGFP-positive cells than the group stimulated by activin A alone. RT-PCR and immunocytochemical staining revealed these EGFP-positive cells to differentiate into mesendoderm-like cells by use of activin A and then into hepatic endoderm cells by use of HGF. Two other hESC lines also differentiated into endoderm on the hepatic lineage by our method. In conclusion, we therefore found this protocol to effectively differentiate multiple hESC lines to early hepatocytes using activin A and HGF on Matrigel.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/citología , Matriz Extracelular/fisiología , Transgenes/genética , alfa-Fetoproteínas/genética , Activinas/fisiología , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/fisiología , Carcinoma Hepatocelular , Endodermo/citología , Factor 4 de Crecimiento de Fibroblastos/fisiología , Vectores Genéticos , Proteínas Fluorescentes Verdes/biosíntesis , Factor de Crecimiento de Hepatocito/fisiología , Humanos , Regiones Promotoras Genéticas/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tretinoina/fisiologíaRESUMEN
Megakaryocytopoiesis is a continuous developmental process of platelet production. In this process, a complex network of hemopoietic growth factors are involved, among which TPO (thrombopoietin) is the most thoroughly investigated regulator of MKs (megakaryocytes). In addition to TPO, other regulators also have non-negligible effects on megakaryocytopoiesis. The majority of their effects are independent of TPO signaling. To date, TPO-independent megakaryocytopoiesis forms a regulatory system that includes four signals and (an) unknown signaling pathway(s). These four pathways are the gp 130 (glycoprotein 130)-dependent signaling pathway, the Notch pathway, NMDA (N-methyl-d-aspartate) receptor-mediated signaling, and the SDF-1 (stromal cell-derived factor-1)/FGF-4 (fibroblast growth factor-4) paradigm. Understanding of the TPO-independent regulatory system is important because the system may offer additional opportunities to understand the developmental process and the mechanisms of disorders characterized by abnormal MK and platelet production, such as thrombocytopenia and thrombocythemia, and to advance the development of therapeutics.
Asunto(s)
Trombopoyesis , Trombopoyetina/fisiología , Animales , Quimiocina CXCL12/fisiología , Receptor gp130 de Citocinas/fisiología , Factor 4 de Crecimiento de Fibroblastos/fisiología , Humanos , Receptores de N-Metil-D-Aspartato/fisiología , Receptores Notch/fisiología , Transducción de SeñalRESUMEN
Human bone marrow-derived mesenchymal stem cells (MSCs) exhibit limited in vitro growth. Fibroblast growth factors (FGFs) elicit a variety of biological responses, such as cell proliferation, differentiation and migration. FGF-4 represents one of the FGFs with the highest cell mitogenic activity. We studied the effect of FGF-4 on MSCs growth and pluripotency. MSCs duplication time (Td) was significantly reduced with FGF-4 compared to controls (2.2 +/- 0.2 vs. 4.1 +/- 0.2 days, respectively; p = 0.03) while BMP-2 and SCF-1 did not exert a significant growth effect. MSC expression of surface markers, differentiation into adipogenic and osteogenic lineages, and baseline expression of cardiomyogenic genes were unaffected by FGF-4. In summary, exogenous FGF-4 increases the rate at which MSC proliferate and has no significant effect on MSC pluripotency.