Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 139
Filtrar
1.
Biol Reprod ; 2021 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-33847353

RESUMO

Primordial follicles, a finite reservoir of eggs in mammalian ovaries, are composed of a single oocyte and its supporting somatic cells, termed granulosa cells. Although their formation may require reciprocal interplay between oocytes and pre-granulosa cells, precursors of granulosa cells, little is known about the underlying mechanisms. We addressed this issue by decoding the transcriptome of pre-granulosa cells during the formation of primordial follicles. We found that marked gene expression changes including extracellular matrix, cell adhesion and several signaling pathways, occur along with primordial follicle formation. Importantly, differentiation of Lgr5-EGFP-positive pre-granulosa cells to FOXL2-positive granulosa cells was delayed in mutant ovaries of the germ cell-specific genes Nanos3 and Figla, accompanied by perturbed gene expression in mutant pre-granulosa cells. These results suggest that proper development of oocytes is required for the differentiation of pre-granulosa cells. Our data provide a valuable resource for understanding the gene regulatory networks involved in the formation of primordial follicles. Summary sentence: Gene expression profile in mouse pre-granulosa cells alters coinciding with primordial follicle formation and oocyte development contributes to this process.

2.
Development ; 2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-33199444

RESUMO

NANOS2 and NANOS3 are evolutionarily conserved RNA-binding proteins involved in murine germ cell development. NANOS3 is required for protection from apoptosis during migration and gonadal colonization in both sexes, whereas NANOS2 is male-specific and required for the male-type differentiation of germ cells. Ectopic NANOS2 rescues the functions of NANOS3, but NANOS3 cannot rescue NANOS2 function even though its expression is up-regulated in Nanos2-null conditions. It is unknown why NANOS3 cannot rescue NANOS2 function and it is unclear whether NANOS3 plays any role in male germ cell differentiation. To address these questions, we made conditional Nanos3/Nanos2 knockout mice and chimeric mice expressing chimeric NANOS proteins. Conditional double knockout of Nanos2 and 3 led to the rapid loss of germ cells, and in vivo and in vitro experiments revealed that DND1 and NANOS2 binding is dependent on the specific NANOS2 zinc finger structure. Moreover, murine NANOS3 failed to bind CNOT1, an interactor of NANOS2 at its N-terminal. Collectively, our study suggests that the inability of NANOS3 to rescue NANOS2 function is due to poor DND1 recruitment and CNOT1 binding.

3.
Nat Commun ; 11(1): 5701, 2020 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-33177522

RESUMO

Protein knockdown using the auxin-inducible degron (AID) technology is useful to study protein function in living cells because it induces rapid depletion, which makes it possible to observe an immediate phenotype. However, the current AID system has two major drawbacks: leaky degradation and the requirement for a high dose of auxin. These negative features make it difficult to control precisely the expression level of a protein of interest in living cells and to apply this method to mice. Here, we overcome these problems by taking advantage of a bump-and-hole approach to establish the AID version 2 (AID2) system. AID2, which employs an OsTIR1(F74G) mutant and a ligand, 5-Ph-IAA, shows no detectable leaky degradation, requires a 670-times lower ligand concentration, and achieves even quicker degradation than the conventional AID. We demonstrate successful generation of human cell mutants for genes that were previously difficult to deal with, and show that AID2 achieves rapid target depletion not only in yeast and mammalian cells, but also in mice.

4.
PLoS One ; 15(4): e0232047, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32339196

RESUMO

Spontaneous testicular teratomas (STTs) derived from primordial germ cells (PGCs) in the mouse embryonic testes predominantly develop in the 129 family inbred strain. Ter (spontaneous mutation) is a single nucleotide polymorphism that generates a premature stop codon of Dead end1 (Dnd1) and increases the incidence of STTs in the 129 genetic background. We previously found that DND1 interacts with NANOS2 or NANOS3 and that these complexes play a vital role in male embryonic germ cells and adult spermatogonia. However, the following are unclear: (a) whether DND1 works with NANOS2 or NANOS3 to regulate teratoma incidence, and (b) whether Ter simply causes Dnd1 loss or produces a short mutant DND1 protein. In the current study, we newly established a conventional Dnd1-knockout mouse line and found that these mice showed phenotypes similar to those of Ter mutant mice in spermatogenesis, oogenesis, and teratoma incidence, with a slight difference in spermiogenesis. In addition, we found that the amount of DND1 in Dnd1+/Ter embryos decreased to half of that in wild-type embryos, while the expression of the short mutant DND1 was not detected. We also found that double mutants for Dnd1 and Nanos2 or Nanos3 showed synergistic increase in the incidence of STTs. These data support the idea that Ter causes Dnd1 loss, leading to an increase in STT incidence, and that DND1 acts with NANOS2 and NANOS3 to regulate the development of teratoma from PGCs in the 129 genetic background. Thus, our results clarify the role of Dnd1 in the development of STTs and provide a novel insight into its pathogenic mechanism.


Assuntos
Células Germinativas Embrionárias/patologia , Proteínas de Neoplasias/fisiologia , Proteínas de Ligação a RNA/metabolismo , Teratoma/etiologia , Neoplasias Testiculares/etiologia , Testículo/patologia , Animais , Células Germinativas Embrionárias/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Oogênese , Proteínas de Ligação a RNA/genética , Espermatogênese , Teratoma/metabolismo , Teratoma/patologia , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patologia , Testículo/metabolismo
6.
EMBO Rep ; 20(12): e48251, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31657143

RESUMO

Formation of primordial follicles is a fundamental, early process in mammalian oogenesis. However, little is known about the underlying mechanisms. We herein report that the RNA-binding proteins ELAVL2 and DDX6 are indispensable for the formation of quiescent primordial follicles in mouse ovaries. We show that Elavl2 knockout females are infertile due to defective primordial follicle formation. ELAVL2 associates with mRNAs encoding components of P-bodies (cytoplasmic RNP granules involved in the decay and storage of RNA) and directs the assembly of P-body-like granules by promoting the translation of DDX6 in oocytes prior to the formation of primordial follicles. Deletion of Ddx6 disturbs the assembly of P-body-like granules and severely impairs the formation of primordial follicles, indicating the potential importance of P-body-like granules in the formation of primordial follicles. Furthermore, Ddx6-deficient oocytes are abnormally enlarged due to misregulated PI3K-AKT signaling. Our data reveal that an ELAVL2-directed post-transcriptional network is essential for the formation of quiescent primordial follicles.


Assuntos
Proteína Semelhante a ELAV 2/metabolismo , Redes Reguladoras de Genes , Infertilidade Feminina/genética , Folículo Ovariano/metabolismo , Animais , Células Cultivadas , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Proteína Semelhante a ELAV 2/genética , Feminino , Camundongos , Oogênese , Folículo Ovariano/citologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo
7.
Sci Rep ; 9(1): 11953, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31420575

RESUMO

The endocardium is the endothelial component of the vertebrate heart and plays a key role in heart development. Where, when, and how the endocardium segregates during embryogenesis have remained largely unknown, however. We now show that Nkx2-5+ cardiac progenitor cells (CPCs) that express the Sry-type HMG box gene Sox17 from embryonic day (E) 7.5 to E8.5 specifically differentiate into the endocardium in mouse embryos. Although Sox17 is not essential or sufficient for endocardium fate, it can bias the fate of CPCs toward the endocardium. On the other hand, Sox17 expression in the endocardium is required for heart development. Deletion of Sox17 specifically in the mesoderm markedly impaired endocardium development with regard to cell proliferation and behavior. The proliferation of cardiomyocytes, ventricular trabeculation, and myocardium thickening were also impaired in a non-cell-autonomous manner in the Sox17 mutant, likely as a consequence of down-regulation of NOTCH signaling. An unknown signal, regulated by Sox17 and required for nurturing of the myocardium, is responsible for the reduction in NOTCH-related genes in the mutant embryos. Our results thus provide insight into differentiation of the endocardium and its role in heart development.


Assuntos
Diferenciação Celular , Embrião de Mamíferos/embriologia , Endocárdio/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas HMGB/biossíntese , Fatores de Transcrição SOXF/biossíntese , Transdução de Sinais , Células-Tronco/metabolismo , Animais , Embrião de Mamíferos/citologia , Endocárdio/citologia , Proteínas HMGB/genética , Mesoderma/citologia , Mesoderma/embriologia , Camundongos , Camundongos Transgênicos , Receptores Notch/genética , Receptores Notch/metabolismo , Fatores de Transcrição SOXF/genética , Células-Tronco/citologia
8.
Sci Rep ; 9(1): 515, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30679547

RESUMO

In embryonic male germ cells, the RNA-binding protein NANOS2 recruits its target RNAs to processing bodies (P-bodies), where they are repressed. This process is necessary to promote male-type germ cell differentiation. However, it remains unclear whether all NANOS2 functions depend on P-bodies. To address this question, we established ES cell lines containing a germ cell-specific inducible Cre and reporter together with the floxed Ddx6 allele. We deleted the Ddx6 gene by administering tamoxifen to chimeric embryos containing germ cells derived from recombinant ES cells. DDX6-null germ cells exhibited both similar and distinct defects from those observed in NANOS2-null germ cells. These results demonstrate that NANOS2 function is carried out via both P-body-dependent and -independent mechanisms. RNA-seq analyses further supported the phenotypic differences between DDX6-null and NANOS2-null germ cells, and indicated distinct molecular cascades involved in NANOS2-mediated gene regulation.


Assuntos
RNA Helicases DEAD-box/metabolismo , Células Germinativas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Quimera/genética , Quimera/metabolismo , RNA Helicases DEAD-box/análise , RNA Helicases DEAD-box/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/citologia , Masculino , Camundongos , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/genética , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/genética , Espermatogênese , Transcriptoma
9.
Dev Biol ; 445(1): 103-112, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30439356

RESUMO

Dead end is a vertebrate-specific RNA-binding protein implicated in germ cell development. We have previously shown that mouse Dead end1 (DND1) is expressed in male embryonic germ cells and directly interacts with NANOS2 to cooperatively promote sexual differentiation of fetal germ cells. In addition, we have also reported that NANOS2 is expressed in self-renewing spermatogonial stem cells and is required for the maintenance of the stem cell state. However, it remains to be determined whether DND1 works with NANOS2 in the spermatogonia. Here, we show that DND1 is expressed in a subpopulation of differentiating spermatogonia and undifferentiated spermatogonia, including NANOS2-positive spermatogonia. Conditional disruption of DND1 depleted both differentiating and undifferentiated spermatogonia; however, the numbers of Asingle and Apaired spermatogonia were preferentially decreased as compared with those of Aaligned spermatogonia. Finally, we found that postnatal DND1 associates with NANOS2 in vivo, independently of RNA, and interacts with some of NANOS2-target mRNAs. These data not only suggest that DND1 is a partner of NANOS2 in undifferentiated spermatogonia as well as in male embryonic germ cells, but also show that DND1 plays an essential role in the survival of differentiating spermatogonia.


Assuntos
Proteínas de Neoplasias/metabolismo , Espermatogônias/metabolismo , Animais , Diferenciação Celular , Células Germinativas Embrionárias/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/fisiologia , RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/fisiologia , Reprodução/fisiologia , Espermatogênese/genética , Espermatogônias/citologia , Espermatozoides/metabolismo , Células-Tronco/citologia , Testículo/metabolismo
11.
Sci Rep ; 8(1): 13011, 2018 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-30158674

RESUMO

Podocyte injury has been proposed to play an important role in diabetic nephropathy; however, its pathological mechanism remains unclear. We have shown that bone morphogenetic protein 4 (BMP4) signaling leads to the glomerular changes characteristic of this disorder. To analyze the molecular mechanism of podocyte injury, the effect of BMP4 was investigated using streptozotocin (STZ)-induced, Bmp4 heterozygous knockout (Bmp4+/-) and podocyte-specific Bmp4 knockout mice. Mice with STZ-induced diabetes exhibited glomerular matrix hyperplasia and decreased numbers of podocyte nucleus-specific WT1-positive cells. The number of podocytes and proteinuria were improved in both diabetic Bmp4 knockout mouse models compared to the effects observed in the control mice. The effect of BMP4 overexpression on Bmp4-induced or podocyte-specific transgenic mice was examined. Tamoxifen-induced Bmp4-overexpressing mice exhibited mesangial matrix expansion and decreased numbers of WT1-positive cells. Podocyte-specific Bmp4-overexpressing mice displayed increased kidney BMP4 expression and mesangial matrix expansion but decreased nephrin expression and numbers of WT1-positive cells. Both lines of Bmp4-overexpressing mice exhibited increased albuminuria. In cultured podocytes, BMP4 increased phospho-p38 levels. BMP4 decreased nephrin expression but increased cleaved caspase-3 levels. p38 suppression inhibited caspase-3 activation. Apoptosis was confirmed in STZ-diabetic glomeruli and Bmp4-overexpressing mice. Bmp4 +/- mice with diabetes displayed reduced apoptosis. Based on these data, the BMP4 signaling pathway plays important roles in the development of both podocyte injury and mesangial matrix expansion in diabetic nephropathy.


Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/fisiopatologia , Podócitos/patologia , Transdução de Sinais , Albuminúria , Animais , Proteína Morfogenética Óssea 4/deficiência , Células Mesangiais/patologia , Camundongos , Camundongos Knockout , Proteínas Repressoras/análise , Proteínas WT1
12.
PLoS Genet ; 14(6): e1007436, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29883445

RESUMO

Functional oocytes are produced through complex molecular and cellular processes. In particular, the contribution of post-transcriptional gene regulation mediated by RNA-binding proteins (RBPs) is crucial for controlling proper gene expression during this process. DAZL (deleted in azoospermia-like) is one of the RBPs required for the sexual differentiation of primordial germ cells and for the progression of meiosis in ovulated oocytes. However, the involvement of DAZL in the development of follicular oocytes is still unknown. Here, we show that Dazl is translationally suppressed in a 3'-UTR-dependent manner in follicular oocytes, and this suppression is required for normal pre-implantation development. We found that suppression of DAZL occurred in postnatal oocytes concomitant with the formation of primordial follicles, whereas Dazl mRNA was continuously expressed throughout oocyte development, raising the possibility that DAZL is dispensable for the survival and growth of follicular oocytes. Indeed, follicular oocyte-specific knockout of Dazl resulted in the production of normal number of pups. On the other hand, genetically modified female mice that overexpress DAZL produced fewer numbers of pups than the control due to defective pre-implantation development. Our data suggest that post-transcriptional suppression of DAZL in oocytes is an important mechanism controlling gene expression in the development of functional oocytes.


Assuntos
Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Oogênese/genética , Folículo Ovariano/crescimento & desenvolvimento , Proteínas de Ligação a RNA/genética , Regiões 3' não Traduzidas/genética , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , RNA Mensageiro/genética
13.
Elife ; 72018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29761784

RESUMO

The metameric structure in vertebrates is based on the periodic formation of somites from the anterior end of the presomitic mesoderm (PSM). The segmentation boundary is defined by the Tbx6 expression domain, whose anterior limit is determined by Tbx6 protein destabilization via Ripply2. However, the molecular mechanism of this process is poorly understood. Here, we show that Ripply2 directly binds to Tbx6 in cultured cells without changing the stability of Tbx6, indicating an unknown mechanism for Tbx6 degradation in vivo. We succeeded in reproducing in vivo events using a mouse ES induction system, in which Tbx6 degradation occurred via Ripply2. Mass spectrometry analysis of the PSM-fated ES cells revealed that proteasomes are major components of the Ripply2-binding complex, suggesting that recruitment of a protein-degradation-complex is a pivotal function of Ripply2. Finally, we identified a motif in the T-box, which is required for Tbx6 degradation independent of binding with Ripply2 in vivo.


Assuntos
Células-Tronco Embrionárias Murinas/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Repressoras/metabolismo , Somitos/embriologia , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Espectrometria de Massas , Camundongos , Ligação Proteica , Proteólise , Proteínas com Domínio T
14.
Mech Dev ; 149: 27-40, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29339164

RESUMO

In the male mouse embryos, the primordial germ cells colonize the developing testes at E11.5. These resident germ cells termed gonocytes are the predecessors of spermatogonial stem cells (SSCs) and differentiating spermatogonia. Both of which are essential for male fertility where the former maintains the continuity of spermatogenesis and the latter generates pioneering waves of spermatozoa. Therefore the timely initiation of gonocytes-to-spermatogonia transition (GST) is an important process during which the cell fates of gonocytes might be segregated. However, it is unknown whether gonocytes are composed of a heterogeneous mixture of germ cells with distinct differentiation potentials during GST. Here, we find that gonocytes exhibit heterogeneity in terms of the expression pattern of at least three early spermatogonial marker genes namely Nanos2, Stra8 and Gfra1. NANOS2 expression levels are negatively correlated with those of STRA8 and GFRA1 before GST, while positive correlation with GFRA1 is established after GST. We further find that overexpression of NANOS2 results in the repression of GFRA1 and PLZF in gonocytes, leading to a delay in GST. On the other hand, loss of NANOS2 results in the up-regulation of GFRA1 and PLZF, indicating a precocious entry of GST. Taken together, our data suggest that NANOS2 functions as an intrinsic timekeeper of GST in the mouse testes.


Assuntos
Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteínas de Ligação a RNA/genética , Testículo/crescimento & desenvolvimento , Animais , Diferenciação Celular/genética , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Masculino , Camundongos , Espermatogênese/genética , Espermatogônias/crescimento & desenvolvimento , Células-Tronco/citologia , Células-Tronco/metabolismo , Testículo/metabolismo
15.
PLoS One ; 12(11): e0187248, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29095923

RESUMO

The segmental pattern of the vertebrate body is established via the periodic formation of somites from the presomitic mesoderm (PSM). This periodical process is controlled by the cyclic and synchronized activation of Notch signaling in the PSM. Protein O-fucosyltransferase1 (Pofut1), which transfers O-fucose to the EGF domains of the Notch1 receptor, is indispensable for Notch signaling activation. The Drosophila homologue Ofut1 was reported to control Notch localization via two different mechanisms, working as a chaperone for Notch or as a regulator of Notch endocytosis. However, these were found to be independent of O-fucosyltransferase activity because the phenotypes were rescued by Ofut1 mutants lacking O-fucosyltransferase activity. Pofut1 may also be involved in the Notch receptor localization in mice. However, the contribution of enzymatic activity of Pofut1 to the Notch receptor dynamics remains to be elucidated. In order to clarify the importance of the O-fucosyltransferase activity of Pofut1 for Notch signaling activation and the protein localization in the PSM, we established mice carrying point mutations at the 245th a.a. or 370-372th a.a., highly conserved amino-acid sequences whose mutations disrupt the O-fucosyltransferase activity of both Drosophila Ofut1 and mammalian Pofut1, with the CRISPR/Cas9 mediated genome-engineering technique. Both mutants displayed the same severely perturbed somite formation and Notch1 subcellular localization defects as the Pofut1 null mutants. In the mutants, Pofut1 protein, but not RNA, became undetectable by E9.5. Furthermore, both wild-type and mutant Pofut1 proteins were degraded through lysosome dependent machinery. Pofut1 protein loss in the point mutant embryos caused the same phenotypes as those observed in Pofut1 null embryos.


Assuntos
Fucosiltransferases/metabolismo , Mutação Puntual , Receptor Notch1/metabolismo , Transdução de Sinais , Somitos/crescimento & desenvolvimento , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Regulação para Baixo , Fucosiltransferases/genética , Camundongos , Processamento Pós-Transcricional do RNA
16.
Nat Commun ; 8: 15662, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28585553

RESUMO

P bodies (PBs) and stress granules (SGs) are conserved cytoplasmic aggregates of cellular messenger ribonucleoprotein complexes (mRNPs) that are implicated in mRNA metabolism and play crucial roles in adult stem cell homeostasis and stress responses. However, the mechanisms underlying the dynamics of mRNP granules are poorly understood. Here, we report NEDD4, an E3 ubiquitin ligase, as a key regulator of mRNP dynamics that controls the size of the spermatogonial progenitor cell (SPC) pool. We find that NEDD4 targets an RNA-binding protein, NANOS2, in spermatogonia to destabilize it, leading to cell differentiation. In addition, NEDD4 is required for SG clearance. NEDD4 targets SGs and facilitates their rapid clearance through the endosomal-lysosomal pathway during the recovery period. Therefore, NEDD4 controls the turnover of mRNP components and inhibits pathological SG accumulation. Accordingly, we propose that a NEDD4-mediated mechanism regulates mRNP dynamics, and facilitates SPC homeostasis and viability under normal and stress conditions.


Assuntos
Ubiquitina-Proteína Ligases Nedd4/fisiologia , Ribonucleoproteínas/fisiologia , Espermatogônias/fisiologia , Células-Tronco/citologia , Animais , Apoptose , Diferenciação Celular , Proliferação de Células , Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Homeostase , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/fisiologia , Transdução de Sinais , Espermatogênese , Temperatura , Ubiquitina-Proteína Ligases/fisiologia , Ubiquitinação
17.
Mech Dev ; 144(Pt B): 125-139, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28341395

RESUMO

Spermatogenesis is a continuous and highly coordinated process of spermatozoa production. In mice, this process is believed to initiate shortly after birth with the emergence of nascent spermatogonia in the testes. However, because the nascent spermatogonia originated from the gonocytes are morphologically indistinguishable from their predecessors and there is no clear definition for the gonocytes-to-spermatogonia transition (GST), it remains unclear when and how spermatogenesis is initiated in the mouse testes. To address these questions, we characterized the emergence of nascent spermatogonia in ICR mice. We found that GST is initiated in a subset of gonocytes as early as E18.5. These nascent spermatogonia express markers typical of undifferentiated spermatogonia residing in testes of adult mice. In addition to markers expression, we identified FOXO1 nuclear-to-cytoplasmic translocation as a novel feature of GST distinguishing nascent spermatogonia from the gonocytes. Using those criteria, we demonstrated that GST requires FGF signaling. When FGF signaling was inhibited pharmacologically, gonocytes retained nuclear FOXO1 expression, did not express spermatogonial markers and failed to proliferate. We found that FGF signaling acts upstream of GDNF and RA signalings for the activation of the MEK/ERK and PI3K/Akt pathways in germ cells during GST. Taken together, we defined the precise timing of GST and revealed FGF signaling as a master regulator of GST in the perinatal mouse testes.


Assuntos
Fatores de Crescimento de Fibroblastos/fisiologia , Espermatogênese , Espermatogônias/fisiologia , Animais , Diferenciação Celular , Proteína Forkhead Box O1/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Fator Neurotrófico Derivado de Linhagem de Célula Glial/fisiologia , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos Endogâmicos ICR , Testículo/citologia , Testículo/embriologia , Técnicas de Cultura de Tecidos , Transcriptoma , Tretinoína/fisiologia
18.
Nat Commun ; 8: 14664, 2017 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-28287088

RESUMO

Upon acquirement of pulmonary circulation, the ancestral heart may have been remodelled coincidently with, or accompanied by, the production and rearrangement of progenitor cells. However, the progenitor populations that give rise to the left ventricle (LV) and sinus venosus (SV) are still ambiguous. Here we show that the expression of Secreted frizzled-related protein Sfrp5 in the mouse identifies common progenitors for the outflow tract (OFT), LV, atrium and SV but not the right ventricle (RV). Sfrp5 expression begins at the lateral sides of the cardiac crescent, excluding early differentiating regions, and continues in the venous pole, which gives rise to the SV. Lineage-tracing analysis revealed that descendants of Sfrp5-expressing cells at E7.5 contribute not only to the SV but also to the LV, atria and OFT and are found also in the dorsal splanchnic mesoderm accompanied by the expression of the secondary heart field marker, Islet1. These findings provide insight into the arrangement of cardiac progenitors for systemic circulation.


Assuntos
Seio Coronário/metabolismo , Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Miocárdio/metabolismo , Células-Tronco/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Biomarcadores/metabolismo , Padronização Corporal/genética , Linhagem da Célula/genética , Rastreamento de Células/métodos , Seio Coronário/citologia , Seio Coronário/crescimento & desenvolvimento , Embrião de Mamíferos , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Expressão Gênica , Átrios do Coração/citologia , Átrios do Coração/crescimento & desenvolvimento , Ventrículos do Coração/citologia , Ventrículos do Coração/crescimento & desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Mesoderma/citologia , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , Camundongos , Camundongos Transgênicos , Miocárdio/citologia , Células-Tronco/citologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
Nucleic Acids Res ; 45(9): 5387-5398, 2017 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-28115634

RESUMO

The mouse PIWI-interacting RNA (piRNA) pathway produces a class of 26-30-nucleotide (nt) small RNAs and is essential for spermatogenesis and retrotransposon repression. In oocytes, however, its regulation and function are poorly understood. In the present study, we investigated the consequences of loss of piRNA-pathway components in growing oocytes. When MILI (or PIWIL2), a PIWI family member, was depleted by gene knockout, almost all piRNAs disappeared. This severe loss of piRNA was accompanied by an increase in transcripts derived from specific retrotransposons, especially IAPs. MIWI (or PIWIL1) depletion had a smaller effect. In oocytes lacking PLD6 (or ZUCCHINI or MITOPLD), a mitochondrial nuclease/phospholipase involved in piRNA biogenesis in male germ cells, the piRNA level was decreased to 50% compared to wild-type, a phenotype much milder than that in males. Since PLD6 is essential for the creation of the 5΄ ends of primary piRNAs in males, the presence of mature piRNA in PLD6-depleted oocytes suggests the presence of compensating enzymes. Furthermore, we identified novel 21-23-nt small RNAs, termed spiRNAs, possessing a 10-nt complementarity with piRNAs, which were produced dependent on MILI and independent of DICER. Our study revealed the differences in the biogenesis and function of the piRNA pathway between sexes.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas Mitocondriais/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Fosfolipase D/metabolismo , Animais , Proliferação de Células , Feminino , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos Endogâmicos C57BL , Oócitos/ultraestrutura , Ovário/metabolismo , RNA Interferente Pequeno/metabolismo , Retroelementos/genética
20.
PLoS Biol ; 14(9): e1002553, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27606421

RESUMO

The differential programming of sperm and eggs in gonads is a fundamental topic in reproductive biology. Although the sexual fate of germ cells is believed to be determined by signaling factors from sexually differentiated somatic cells in fetal gonads, the molecular mechanism that determines germ cell fate is poorly understood. Herein, we show that mothers against decapentaplegic homolog 4 (SMAD4) in germ cells is required for female-type differentiation. Germ cells in Smad4-deficient ovaries respond to retinoic acid signaling but fail to undergo meiotic prophase I, which coincides with the weaker expression of genes required for follicular formation, indicating that SMAD4 signaling is essential for oocyte differentiation and meiotic progression. Intriguingly, germline-specific deletion of Smad4 in Stra8-null female germ cells resulted in the up-regulation of genes required for male gonocyte differentiation, including Nanos2 and PLZF, suggesting the initiation of male-type differentiation in ovaries. Moreover, our transcriptome analyses of mutant ovaries revealed that the sex change phenotype is achieved without global gene expression changes in somatic cells. Our results demonstrate that SMAD4 and STRA8 are essential factors that regulate the female fate of germ cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Oócitos/fisiologia , Proteína Smad4/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Feminino , Deleção de Genes , Expressão Gênica , Masculino , Meiose , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Ovário/citologia , Ovário/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Processos de Determinação Sexual , Transdução de Sinais , Proteína Smad4/metabolismo , Tretinoína/fisiologia , Cromossomo X/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...