Nup133 belongs to the Y-complex, a key component of the nuclear pore complex (NPC) scaffold. Studies on a null mutation in mice previously revealed that Nup133 is essential for embryonic development ...but not for mouse embryonic stem cell (mESC) proliferation. Using single-pore detection and average NE-fluorescence intensity, we find that Nup133 is dispensable for interphase and postmitotic NPC scaffold assembly in pluripotent mESCs. However, loss of Nup133 specifically perturbs the formation of the nuclear basket as manifested by the absence of Tpr in about half of the NPCs combined with altered dynamics of Nup153. We further demonstrate that its central domain mediates Nup133’s role in assembling Tpr and Nup153 into a properly configured nuclear basket. Our findings thus revisit the role of the Y-complex in pore biogenesis and provide insights into the interplay between NPC scaffold architecture, nuclear basket assembly, and the generation of heterogeneity among NPCs.
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•Nup133 is dispensable for interphase NPC assembly in mESCs•Assembly of Tpr within the basket of all NPCs requires Nup133•Nup133 deficiency increases Nup153 dynamics in vivo•The central domain of Nup133 is required for proper NPC basket assembly and dynamics
Souquet et al. report that Nup133, a structural component of the nuclear pore complex (NPC), is dispensable for NPC scaffold formation during post-mitotic and interphase pore assembly in embryonic stem cells. Yet, they also find that Nup133’s central domain is required for a properly configured nuclear basket in all NPCs.
The mechanisms regulating germ line sex determination and meiosis initiation are poorly understood. Here, we provide evidence for the involvement of homeobox Msx transcription factors in foetal ...meiosis initiation in mammalian germ cells. Upon meiosis initiation, Msx1 and Msx2 genes are strongly expressed in the foetal ovary, possibly stimulated by soluble factors found there: bone morphogenetic proteins Bmp2 and Bmp4, and retinoic acid. Analysis of Msx1/Msx2 double mutant embryos revealed a majority of undifferentiated germ cells remaining in the ovary and, importantly, a decrease in the number of meiotic cells. In vivo, the Msx1/Msx2 double-null mutation prevented full activation of Stra8, a gene required for meiosis. In F9 cells, Msx1 can bind to Stra8 regulatory sequences and Msx1 overexpression stimulates Stra8 transcription. Collectively, our data demonstrate for the first time that some homeobox genes are required for meiosis initiation in the female germ line.
Besides assembling nuclear pore complexes, the conduits of nuclear transport, many nucleoporins also contribute to chromatin organization and gene expression, with critical roles in development and ...pathologies. We previously reported that Nup133 and Seh1, two components of the Y-complex subassembly of the nuclear pore scaffold, are dispensable for mouse embryonic stem cell viability but required for their survival during neuroectodermal differentiation. Here, a transcriptomic analysis revealed that Nup133 regulates a subset of genes at early stages of neuroectodermal differentiation, including Lhx1 and Nup210L, encoding a newly validated nucleoporin. These genes are also misregulated in Nup133▵Mid neuronal progenitors, in which nuclear pore basket assembly is impaired. However, a four-fold reduction of Nup133, despite also affecting basket assembly, is not sufficient to alter Nup210L and Lhx1 expression. Finally, these two genes are also misregulated in Seh1-deficient neural progenitors only showing a mild reduction in nuclear pore density. Together these data reveal a shared function of Y-complex nucleoporins in gene regulation during neuroectodermal differentiation, apparently independent of nuclear pore basket integrity.
Recently reporting in Developmental Cell, Mészáros et al. (2015) and Vollmer et al. (2015) extend the inventory of membrane-binding and -bending nuclear pore proteins to peripheral nucleoporins ...localized at the nuclear basket.
The mechanisms regulating the entry into meiosis in mammalian germ cells remain incompletely understood. We investigated the involvement of the TGF-β family members in fetal germ cell meiosis ...initiation. Nodal, a member of the TGF-β family, and its target genes are precociously expressed in embryonic gonads and show sexual dimorphism in favor of the developing testis. Nodal receptor genes, Acvr2a and Acvr2b, Alk4, and Tdgf1/Cripto, were identified in male germ cells. Nodal itself, Tdgf1, and Lefty1 and Lefty2 are targets of Nodal signaling and were all found specifically expressed in male germ cells. To elucidate the role of this signaling pathway, activin-like kinases that mediate TGF-β/Nodal/activin signaling were inhibited in 11.5 d postconception testis in organotypic culture. Activin-like kinases inhibition disrupted normal male germ cell development and induced germ cell entry into meiosis such as that observed in female germ cells at the equivalent stage. Interestingly Stra8, the gatekeeper of the mitotic/meiotic switch, was induced independently of any change of either Cyp26b1 or Fgf9 expression, the two genes currently identified as testicular meiotic inhibitors. On the other hand, recombinant Nodal significantly dampened Stra8 expression and germ cell meiosis in cultured 11.5 d postconception ovaries. Our results allowed us to propose for the first time an autocrine role of Nodal during the development of germ cells and indicate that members of the TGB-β family may reinforce the male fate and prevent meiosis in embryonic germ cells.
The fate of hematopoietic stem and progenitor cells (HSPCs) is regulated by their interaction with stromal cells in the bone marrow. However, the cellular mechanisms regulating HSPC interaction with ...these cells and their potential impact on HSPC polarity are still poorly understood. Here we evaluated the impact of cell-cell contacts with osteoblasts or endothelial cells on the polarity of HSPC. We found that an HSPC can form a discrete contact site that leads to the extensive polarization of its cytoskeleton architecture. Notably, the centrosome was located in proximity to the contact site. The capacity of HSPCs to polarize in contact with stromal cells of the bone marrow appeared to be specific, as it was not observed in primary lymphoid or myeloid cells or in HSPCs in contact with skin fibroblasts. The receptors ICAM, VCAM, and SDF1 were identified in the polarizing contact. Only SDF1 was independently capable of inducing the polarization of the centrosome-microtubule network.
Besides assembling nuclear pore complexes, the conduits of nuclear transport, many nucleoporins also contribute to chromatin organization and gene expression, with critical roles in development and ...pathologies. We previously reported that Nup133 and Seh1, two components of the Y-complex subassembly of the nuclear pore scaffold, are dispensable for mouse embryonic stem cell viability but required for their survival during neuroectodermal differentiation. Here, a transcriptomic analysis revealed that Nup133 regulates a subset of genes at early stages of neuroectodermal differentiation, including Lhx1 and Nup210l, which encodes a newly validated nucleoporin. These genes are also misregulated in Nup133ΔMid neuronal progenitors, in which nuclear pore basket assembly is impaired. However, a four-fold reduction of Nup133 levels, despite also affecting basket assembly, is not sufficient to alter Nup210l and Lhx1 expression. Finally, these two genes are also misregulated in Seh1-deficient neural progenitors, which only show a mild reduction in nuclear pore density. Together these data reveal a shared function of Y-complex nucleoporins in gene regulation during neuroectodermal differentiation, apparently independent of nuclear pore basket integrity.
Maternal IGFs regulate cytotrophoblast proliferation and, thereby, placental growth and function. IGF bioavailability is controlled by IGF-binding proteins (IGFBPs); in placenta, IGFBP-3 is ...particularly abundant. In other systems, IGFBP-3 can regulate cellular events independently of IGFs; these effects are thought to be mediated by TGFβ receptors (TβR). We have examined IGFBP-3 regulation of IGF-dependent and -independent cytotrophoblast proliferation in first-trimester placental explants and the role of TβRII in mediating these effects. In the presence of IGFBP-3 (50 nm), IGF-induced (10 nm) proliferation (monitored by immunohistochemical analysis of Ki67 expression and bromodeoxyuridine incorporation) was significantly reduced (P < 0.05). IGFBP-3 also reduced basal proliferation independently of IGF receptor signaling. Immunohistochemical analysis demonstrated that TGFβ signaling molecules TGFβ receptor I (TβRI), TβRII, TβRV, Smad-2, and ERK are expressed in syncytium and/or cytotrophoblast. TGFβ1 (10 ng/ml) enhanced cytotrophoblast proliferation and activated both Smad-2 and ERK-1/2, whereas IGFBP-3 activated only Smad-2. The function of both TGFβ1 and IGFBP-3 was attenuated by a TβRII function-blocking antibody and by small interfering RNA-mediated knockdown of TβRII (P < 0.05); this was accompanied by a reduction in Smad-2 activation. This study demonstrates that both TGFβ1 and IGFBP-3 signal through TβRI/II to influence human cytotrophoblast proliferation. However, downstream pathways are distinct, because IGFBP-3 acts only through Smad-2, whereas TGFβ1 also phosphorylates ERK, resulting in opposite effects on cytotrophoblast proliferation. The effects of maternal growth signals on placental growth and function therefore depend on the balance of ligands, receptors, and signaling molecules at the syncytiotrophoblast surface. Therapeutic manipulation of this balance might offer a strategy to optimize placental development and pregnancy outcome.
Cytotrophoblast proliferation is enhanced by TGFβ1 but inhibited by IGFBP-3; however, both signal through TβRI/II downstream, IGFBP-3 activates only Smad-2, whereas TGFβ1 also phosphorylates ERK.
We identified three doublesex and mab-3-related transcription factors (DMRT) that were sexually differentially expressed in human fetal gonads and present in the ovaries at the time of meiotic ...initiation. These were also identified in murine embryonic female germ cells. Among these, we focused on DMRTA2 (DMRT5), whose function is unknown in the developing gonads, and clarified its role in human female fetal germ cells, using an original xenograft model. Early human fetal ovaries (8-11 weeks post-fertilization) were grafted into nude mice. Grafted ovaries developed normally, with no apparent overt changes, when compared with ungrafted ovaries at equivalent developmental stages. Appropriate germ cell density, mitotic/meiotic transition, markers of meiotic progression and follicle formation were evident. Four weeks after grafting, mice were treated with siRNA, specifically targeting human DMRTA2 mRNA. DMRTA2 inhibition triggered an increase in undifferentiated FUT4-positive germ cells and a decrease in the percentage of meiotic γH2AX-positive germ cells, when compared with mice that were injected with control siRNA. Interestingly, the expression of markers associated with pre-meiotic germ cell differentiation was also impaired, as was the expression of DMRTB1 (DMRT6) and DMRTC2 (DMRT7). This study reveals, for the first time, the requirement of DMRTA2 for normal human female embryonic germ cell development. DMRTA2 appears to be necessary for proper differentiation of oogonia, prior to entry into meiosis, in the human species. Additionally, we developed a new model of organ xenografting, coupled with RNA interference, which provides a useful tool for genetic investigations of human germline development.
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