RESUMO
To date, no studies have examined the tooth formation during developmental stages of brush-tailed mice (Calomyscidae) and true hamsters (Cricetidae). Herein, we compared the timing of tooth morphogenesis and FGF4 expression pattern during development of the first lower molar in Goodwin's brush-tailed mouse, Calomyscus elburzensis with two other muroid rodents; the house mouse, Mus musculus (Muridae), model organism for tooth morphogenesis, and the golden hamster, Mesocricetus auratus which shares great similarities in cusp pattern with brush-tailed mice. All three species were bred in captivity and developing embryos were isolated at different embryonic days (E). Histological evaluation of lower molars was performed and spatiotemporal pattern of FGF4 expression was determined by immunohistochemistry. Results indicated that morphogenesis of the tooth cusps starts at the beginning of the cap stage of the first lower molar (E14 in house mouse, about E11.5 in golden hamster and E22 in Goodwin's brush-tailed mouse). During the cap to bell stage (E15 in house mouse, E12 in golden hamster and at about E24 in Goodwin's brush-tailed mouse), a decrease in the expression of FGF4 was observed in the mesenchyme, except for the cusp tips. According to our observations, the developmental process of the first lower molar formation in Goodwin's brush-tailed mouse began much later as compared with the other two species. Despite the differences in the temporal pattern of molar development between these three members of the same superfamily (Muroidea), the correlation in the expression of FGF4 with specific stages of tooth morphogenesis supported its regulatory function. Anat Rec, 300:2138-2149, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Fator 4 de Crescimento de Fibroblastos/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Dente Molar/embriologia , Dente Molar/crescimento & desenvolvimento , Morfogênese/fisiologia , Animais , Animais Recém-Nascidos , Arvicolinae , Cricetinae , Feminino , Fator 4 de Crescimento de Fibroblastos/genética , Masculino , Mesocricetus , Camundongos , Dente Molar/metabolismo , Muridae , Especificidade da Espécie , Dente/metabolismoRESUMO
Fibroblast growth factor 4 (FGF4) is a crucial growth factor for the development of mammalian embryos. We previously produced hexahistidine-tagged, bovine and porcine FGF4 (Pro(32) to Leu(206) ) proteins without a secretory signal peptide at the aminoterminus in Escherichia coli. Here, we found that these were unstable; site-specific cleavage between Ser(54) and Leu(55) in both FGF4 derivatives was identified. In order to generate stable FGF4 derivatives and to investigate their biological activities, aminoterminally truncated and hexahistidine-tagged bovine and porcine FGF4 (Leu(55) to Leu(206) ) proteins, termed HisbFGF4L and HispFGF4L, respectively, were produced in E. coli. These FGF4 derivatives were sufficiently stable and exerted mitogenic activities in fibroblasts. Treatment with the FGF4 derivatives promoted the phosphorylation of ERK1/2, which are crucial kinases in the FGF signaling pathway. In the presence of PD173074, an FGF receptor inhibitor, the phosphorylation of ERK1/2 was inhibited and resulted in abolition of the growth-promoting activity of FGF4 derivatives. Taken together, we demonstrate that HisbFGF4L and HispFGF4L are capable of promoting the proliferation of bovine- and porcine-derived cells, respectively, via an authentic FGF signaling pathway. These FGF4 derivatives may be applicable for dissecting the roles of FGF4 during embryogenesis in cattle and pigs.
Assuntos
Escherichia coli/metabolismo , Fator 4 de Crescimento de Fibroblastos/biossíntese , Fator 4 de Crescimento de Fibroblastos/química , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Animais , Bovinos , Células Cultivadas , Estabilidade de Medicamentos , Escherichia coli/genética , Dados de Sequência Molecular , Proteínas Recombinantes/genética , SuínosRESUMO
The development of the craniofacial muscles requires reciprocal interactions with surrounding craniofacial tissues that originate from cranial neural crest cells (CNCCs). However, the molecular mechanism involved in the tissue-tissue interactions between CNCCs and muscle progenitors during craniofacial muscle development is largely unknown. In the current study, we address how CNCCs regulate the development of the tongue and other craniofacial muscles using Wnt1-Cre; Alk5(fl/fl) mice, in which loss of Alk5 in CNCCs results in severely disrupted muscle formation. We found that Bmp4 is responsible for reduced proliferation of the myogenic progenitor cells in Wnt1-Cre; Alk5(fl/fl) mice during early myogenesis. In addition, Fgf4 and Fgf6 ligands were reduced in Wnt1-Cre; Alk5(fl/fl) mice and are critical for differentiation of the myogenic cells. Addition of Bmp4 or Fgf ligands rescues the proliferation and differentiation defects in the craniofacial muscles of Alk5 mutant mice in vitro. Taken together, our results indicate that CNCCs play critical roles in controlling craniofacial myogenic proliferation and differentiation through tissue-tissue interactions.
Assuntos
Músculos Faciais/embriologia , Desenvolvimento Muscular/genética , Crista Neural/metabolismo , Proteínas Serina-Treonina Quinases/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Animais , Apoptose/genética , Proteína Morfogenética Óssea 4/biossíntese , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Diferenciação Celular/genética , Proliferação de Células , Células Cultivadas , Fator 4 de Crescimento de Fibroblastos/biossíntese , Fator 4 de Crescimento de Fibroblastos/genética , Fator 4 de Crescimento de Fibroblastos/metabolismo , Fator 6 de Crescimento de Fibroblastos/biossíntese , Fator 6 de Crescimento de Fibroblastos/genética , Fator 6 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Transgênicos , Crista Neural/citologia , Técnicas de Cultura de Órgãos , Receptor do Fator de Crescimento Transformador beta Tipo I , Transdução de Sinais/genética , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Proteína Smad8/metabolismo , Língua/embriologia , Doenças da Língua/genética , Fator de Crescimento Transformador beta/genética , Proteína Wnt1/genéticaRESUMO
The epiblast (EPI) and the primitive endoderm (PE), which constitute foundations for the future embryo body and yolk sac, build respectively deep and surface layers of the inner cell mass (ICM) of the blastocyst. Before reaching their target localization within the ICM, the PE and EPI precursor cells, which display distinct lineage-specific markers, are intermingled randomly. Since the ICM cells are produced in two successive rounds of asymmetric divisions at the 8â16 (primary inner cells) and 16â32 cell stage (secondary inner cells) it has been suggested that the fate of inner cells (decision to become EPI or PE) may depend on the time of their origin. Our method of dual labeling of embryos allowed us to distinguish between primary and secondary inner cells contributing ultimately to ICM. Our results show that the presence of two generations of inner cells in the 32-cell stage embryo is the source of heterogeneity within the ICM. We found some bias concerning the level of Fgf4 and Fgfr2 expression between primary and secondary inner cells, resulting from the distinct number of cells expressing these genes. Analysis of experimental aggregates constructed using different ratios of inner cells surrounded by outer cells revealed that the fate of cells does not depend exclusively on the timing of their generation, but also on the number of cells generated in each wave of asymmetric division. Taking together, the observed regulatory mechanism adjusting the proportion of outer to inner cells within the embryo may be mediated by FGF signaling.
Assuntos
Massa Celular Interna do Blastocisto/metabolismo , Divisão Celular/fisiologia , Embrião de Mamíferos/metabolismo , Endoderma/embriologia , Trofoblastos/metabolismo , Animais , Massa Celular Interna do Blastocisto/citologia , Diferenciação Celular , Linhagem da Célula/genética , Embrião de Mamíferos/citologia , Endoderma/citologia , Endoderma/crescimento & desenvolvimento , Feminino , Fator 4 de Crescimento de Fibroblastos/biossíntese , Fator 4 de Crescimento de Fibroblastos/metabolismo , Fator de Transcrição GATA6/biossíntese , Proteínas de Homeodomínio/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/biossíntese , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/biossíntese , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Fatores de Transcrição SOXB1/biossíntese , Transdução de Sinais , Trofoblastos/citologiaRESUMO
This study was undertaken to establish rat embryonic stem (ES) cells from parthenogenetically developing blastocysts. Ten blastocysts were prepared by treatment of ovulated rat oocytes with ionomycin and cycloheximide, and three alkaline phosphatase-positive ES cell lines were established using the N2B27 medium supplemented with mitogen activated protein kinase kinase inhibitor PD0325901, glycogen synthase kinase 3 inhibitor CHIR99021, rat leukemia inhibitory factor, and forskolin. Expression of stem cell marker genes (Oct-4, rNanog, Fgf-4, and Rex-1) was confirmed in all three ES cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR). Combined bisulfite restriction analysis showed that the differentially methylated region locus of five imprinted genes (H19, Meg3IG, Igf2r, Peg5, and Peg10) in these ES cells remained to be demethylated or was hypomethylated, which was similar to that in control ES cells established from normal blastocysts. Characteristics of the parthenogenetic blastocyst-derived ES cells were successfully transmitted to the next generation through a chimeric rat for one of the three ES cell lines. This is the first report on germline-competent (genuine) ES cells derived from parthenogenetically developing rat blastocysts.
Assuntos
Blastocisto/metabolismo , Células-Tronco Embrionárias/metabolismo , Oócitos/metabolismo , Partenogênese , Adjuvantes Imunológicos/farmacologia , Animais , Benzamidas/farmacologia , Ionóforos de Cálcio/farmacologia , Diferenciação Celular , Células Cultivadas , Colforsina/farmacologia , Cicloeximida/farmacologia , Metilação de DNA , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Feminino , Fator 4 de Crescimento de Fibroblastos/biossíntese , Marcadores Genéticos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Ionomicina/farmacologia , Fator Inibidor de Leucemia/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/biossíntese , Oócitos/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , Ratos , Fatores de Transcrição/biossínteseRESUMO
In mice, fibroblast growth factor 4 (Fgf4) is a crucial gene for the generation of trophectoderm, progenitor cells of the placenta. Therefore, exogenous FGF4 promotes the isolation and maintenance of trophoblast stem cells from preimplantation embryos. We previously produced a 6× histidine (His)-tagged, mouse FGF4 (Pro(31)-Leu(202)) without a secretory signal peptide at the amino-terminus, referred to as HismFGF4, in Escherichia coli. Here, we found that HismFGF4 was unstable, such as in phosphate-buffered saline. In these conditions, site-specific cleavage between Ser(50) and Leu(51) was identified. In order to generate stable mouse FGF4 derivatives, a 6× His-tagged mouse FGF4 (Leu(51)-Leu(202)), termed HismFGF4L, was expressed in E. coli. HismFGF4L could be purified from the supernatant of cell lysates by heparin column chromatography. In phosphate-buffered saline, HismFGF4L was relatively stable. HismFGF4L exerted significant mitogenic activities at concentrations as low as 0.01 nM (P < 0.01) in mouse embryonic fibroblast Balb/c 3T3 cells expressing FGF receptor 2. In the presence of PD173074, an FGF receptor inhibitor, the growth-promoting activity of HismFGF4L was abolished. Taken together, we suggest that aminoterminally truncated HismFGF4L is capable of promoting the proliferation of mouse-derived cells via an authentic FGF signaling pathway. We consider that HismFGF4L is useful as a derivative of mouse FGF4 protein for analyzing the effects of mouse FGF4 and for stimulating cell growth of mouse-derived cells, such as trophoblast stem cells. Our study provides a simple method for the production of a bioactive, stable mouse FGF4 derivative in E. coli.
Assuntos
Escherichia coli/metabolismo , Fator 4 de Crescimento de Fibroblastos/biossíntese , Fator 4 de Crescimento de Fibroblastos/química , Células 3T3 , Animais , Proliferação de Células/efeitos dos fármacos , Cromatografia de Afinidade , Escherichia coli/genética , Fator 4 de Crescimento de Fibroblastos/genética , Fator 4 de Crescimento de Fibroblastos/isolamento & purificação , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Heparina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Células-Tronco/citologia , Trofoblastos/citologiaRESUMO
In mammals, breeding is preceded by species-specific mating behaviours. In this study, we investigated whether parthenogenetic embryo quality could be improved by mating behaviours in mice. To investigate this hypothesis, female mice were mated with vasectomized Kunming white male mice after superovulation. Oocytes were collected and counted at 16 h after superovulation. The oocytes were then artificially activated by medium containing 10 mM strontium chloride and 5 µg/ml cytochalasin B. Blastocysts were obtained by cultivating activated oocytes in vitro. Expression levels of reprogramming transcription factors (i.e. Oct4, Sox2, Klf4 and c-Myc) in oocytes, apoptosis-related genes (i.e. Bax, Bcl2 and c-Myc) in cumulus cells and pluripotency-related transcription factors (i.e. Oct4, Nanog and FGF4) in blastocysts were analysed in samples collected from mated and unmated mice. Additionally, developmental competence of parthenogenetic embryos was used to assess following fibroblast growth factor 4 (FGF4) treatment. The results showed that the formation rate of blastocysts in unmated mice was significantly higher than that in mated mice (p < 0.05). Embryo development was primarily blocked at the eight-cell stage in mated mice; however, the blastocyst formation rate did not differ significantly between groups after the addition of 25 ng/ml FGF4 to the medium at the four-cell stage (p > 0.05). Moreover, the expression of the reprogramming factor Sox2 was significantly different in oocytes collected from mated versus unmated mice. Taken together, our results demonstrated that mating behaviours influenced embryonic development in vitro by decreasing FGF4 expression.
Assuntos
Blastocisto/metabolismo , Fator 4 de Crescimento de Fibroblastos/biossíntese , Oócitos/metabolismo , RNA Mensageiro/biossíntese , Comportamento Sexual Animal , Animais , Desenvolvimento Embrionário , Feminino , Fator 4 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fator 4 Semelhante a Kruppel , Masculino , Camundongos , Oócitos/crescimento & desenvolvimento , Gravidez , Fatores de Transcrição SOXB1/biossíntese , Fatores de Transcrição SOXB1/metabolismoRESUMO
Cyclic adenosine diphosphoribose (cADPR) is an endogenous Ca(2+) mobilizing messenger that is formed by ADP-ribosyl cyclases from nicotinamide adenine dinucleotide (NAD). The main ADP-ribosyl cyclase in mammals is CD38, a multi-functional enzyme and a type II membrane protein. Here we explored the role of CD38-cADPR-Ca(2+) in the cardiomyogenesis of mouse embryonic stem (ES) cells. We found that the mouse ES cells are responsive to cADPR and possess the key components of the cADPR signaling pathway. In vitro cardiomyocyte (CM) differentiation of mouse ES cells was initiated by embryoid body (EB) formation. Interestingly, beating cells appeared earlier and were more abundant in CD38 knockdown EBs than in control EBs. Real-time RT-PCR and Western blot analyses further showed that the expression of several cardiac markers, including GATA4, MEF2C, NKX2.5, and α-MLC, were increased markedly in CD38 knockdown EBs than those in control EBs. Similarly, FACS analysis showed that more cardiac Troponin T-positive CMs existed in CD38 knockdown or 8-Br-cADPR, a cADPR antagonist, treated EBs compared with that in control EBs. On the other hand, overexpression of CD38 in mouse ES cells significantly inhibited CM differentiation. Moreover, CD38 knockdown ES cell-derived CMs possess the functional properties characteristic of normal ES cell-derived CMs. Last, we showed that the CD38-cADPR pathway negatively modulated the FGF4-Erks1/2 cascade during CM differentiation of ES cells, and transiently inhibition of Erk1/2 blocked the enhanced effects of CD38 knockdown on the differentiation of CM from ES cells. Taken together, our data indicate that the CD38-cADPR-Ca(2+) signaling pathway antagonizes the CM differentiation of mouse ES cells.
Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Sinalização do Cálcio/fisiologia , Diferenciação Celular/fisiologia , ADP-Ribose Cíclica/metabolismo , Células-Tronco Embrionárias/enzimologia , Glicoproteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Miócitos Cardíacos/enzimologia , ADP-Ribosil Ciclase 1/genética , Animais , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/genética , Linhagem Celular , ADP-Ribose Cíclica/genética , Corpos Embrioides/citologia , Corpos Embrioides/enzimologia , Células-Tronco Embrionárias/citologia , Fator 4 de Crescimento de Fibroblastos/biossíntese , Fator 4 de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Glicoproteínas de Membrana/genética , Camundongos , Proteínas Musculares/genética , Miócitos Cardíacos/citologiaRESUMO
Fibroblast growth factor 4 (FGF4) promotes isolation of trophoblast stem (TS) cells from mouse blastocysts and maintenance of TS cells in an undifferentiated state in vitro. To date, commercially available, bacterially expressed human FGF4 (RhFGF4) has been used generally for this purpose. In this study, HismFGF4, a 6x histidine-tagged mouse FGF4, was produced in E. coli and purified using heparin column chromatography. We demonstrated that HismFGF4 (25 ng/ml) more efficiently generates mouse TS cells from a single blastocyst than RhFGF4 (25 ng/ml) and that TS cells isolated and maintained with HismFGF4 retained their ability to differentiate into the trophoblast cell lineage in vitro. In addition, TS cells cultured with HismFGF4 (25 ng/ml) were maintained in an undifferentiated state better than with RhFGF4 (25 ng/ml). To the best of our knowledge, this is the first application of a mouse FGF4 derivative for isolation and maintenance of mouse TS cells.
Assuntos
Escherichia coli/genética , Fator 4 de Crescimento de Fibroblastos/biossíntese , Fator 4 de Crescimento de Fibroblastos/farmacologia , Cultura Primária de Células/métodos , Células-Tronco/citologia , Trofoblastos/citologia , Animais , Linhagem da Célula/efeitos dos fármacos , Separação Celular/métodos , Células Cultivadas , Clonagem Molecular/métodos , Escherichia coli/metabolismo , Feminino , Fator 4 de Crescimento de Fibroblastos/genética , Fator 4 de Crescimento de Fibroblastos/metabolismo , Humanos , Camundongos , Gravidez , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismoRESUMO
Cardiomyocyte loss in the ischaemic heart can be the reason of many complications, eventually being even the cause of patient's death. Despite many promises, cell therapy with the use of skeletal muscle stem cells (SMSC) still remains to be modified and improved. Combined cell and gene therapy seems to be a promising strategy to heal damaged myocardium. In the present study we have investigated the influence of a simultaneous overexpression of two potent pro-angiogenic genes encoding the fibroblast growth factor-4 (FGF-4) and the vascular endothelial growth factor-A (VEGF-A) on a myogenic murine C2C12 cell line. We have demonstrated in in vitro conditions that myoblasts which overexpressed these factors exhibited significant changes in the cell cycle and pro-angiogenic potential with only slight differences in the expression of the myogenic genes. There was not observed the influence of transient or stable overexpression of FGF-4 and VEGF on cell apoptosis/necrosis in standard or oxidative stress conditions comparing to non transfected controls. Overall, our results suggest that the possible transplantation of myoblasts overexpressing pro-angiogenic factors may potentially improve the functionality of the injured myocardium although the definite proof must originate from in situ conducted pre-clinical studies.
Assuntos
Fator 4 de Crescimento de Fibroblastos/genética , Mioblastos/fisiologia , Neovascularização Fisiológica/genética , Transfecção/métodos , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Linhagem Celular , Proliferação de Células , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Fator 4 de Crescimento de Fibroblastos/biossíntese , Humanos , Camundongos , Mioblastos/citologia , Neovascularização Fisiológica/fisiologia , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
PURPOSE: To optimize In vitro maturation (IVM) of quality oocytes for embryo production through IVF and SCNT. METHODS: Buffalo oocytes were in vitro matured in the presence of the pokeweed lectin (Phytolacca americana), a potent lymphocyte mitogen. Lectin was supplemented in TCM + 10% FBS at the doses of 0, 1, 5, 10, 15, 20 and 40 microg/ml and cumulus expansion and gene expression patterns were characterized. RESULTS: The degree of cumulus expansion in different lectin treatment levels improved from 1.1 at 1 Ag/ml level to 3.60 at 10 microg/ml level and then decreased in higher concentration 20 microg/ml (1.66) and 40 microg/ml (0.64). IVF embryos showed highest cleavage rate (88.8%) in 10 microg/ml lectin treatment. Expression of all mRNA transcript studied (Cx43, GDF 9, FGF-4 and Fibronectin) was positively correlated with cumulus expansion and polar body extrusion. CONCLUSIONS: Mitogenic lectin supplemented maturation media improves oocyte quality for in vitro embryo production.
Assuntos
Proteína Morfogenética Óssea 15/biossíntese , Búfalos , Conexina 43/biossíntese , Fator 4 de Crescimento de Fibroblastos/biossíntese , Fibronectinas/biossíntese , Fator 9 de Diferenciação de Crescimento/metabolismo , Oócitos/efeitos dos fármacos , Mitógenos de Phytolacca americana/farmacologia , Animais , Proteína Morfogenética Óssea 15/genética , Conexina 43/genética , Fertilização in vitro/veterinária , Fator 4 de Crescimento de Fibroblastos/genética , Fibronectinas/genética , Meiose , Oócitos/metabolismo , Oogênese/efeitos dos fármacos , RNA Mensageiro/biossínteseRESUMO
By means of the tumorigenicity assay applying DNA from a patient with a gastric carcinoma (MA) we have already reported the identification of the putative oncogene myeov. In addition we have shown its involvement in t(11;14)-positive multiple myelomas and amplifications of breast tumours and esophageal carcinomas. The failure of myeov cDNA to induce tumour formation in NIH/3T3 cells prompted us to analyze the human sequences present in our MA-T1a1 tertiary transfectants. Sequence analysis revealed the presence of the human oncogene hst (fgf4) at a distance of approximately 9kb from the myeov gene in our MA-T1a1 tertiary transfectants. Both myeov and hst (fgf4) are normally situated approximately 475-kb apart at band 11q13, a region that is frequently amplified and overexpressed in various tumours. Southern and Northern blot analyses confirmed our sequence data and showed rearrangement of hst sequences during the transfection process and its expression in our MA-T1a1 tertiary transfectants.