Dynamic organelle localization and cytoskeletal reorganization during preimplantation mouse embryo development revealed by live imaging of genetically encoded fluorescent fusion proteins

Live imaging is one of the most powerful technologies for studying the behaviors of cells and molecules in living embryos. Previously, we established a series of reporter mouse lines in which specific organelles are labeled with various fluorescent proteins. In this study, we examined the localizations of fluorescent signals during preimplantation development of these mouse lines, as well as a newly established one, by time‐lapse imaging. Each organelle was specifically marked with fluorescent fusion proteins; fluorescent signals were clearly visible during the whole period of time‐lapse observation, and the expression of the reporters did not affect embryonic development. We found that some organelles dramatically change their sub‐cellular distributions during preimplantation stages. In addition, by crossing mouse lines carrying reporters of two distinct colors, we could simultaneously visualize two types of organelles. These results confirm that our reporter mouse lines can be valuable genetic tools for live imaging of embryonic development.     

A novel Cre‐inducible knock‐in ARL13B‐tRFP fusion cilium reporter

Cilia play a major role in the regulation of numerous signaling pathways and are essential for embryonic development. Mutations in genes affecting ciliary function can cause a variety of diseases in humans summarized as ciliopathies. To facilitate the detection and visualization of cilia in a temporal and spatial manner in mouse tissues, we generated a Cre‐inducible cilium‐specific reporter mouse line expressing an ARL13B‐tRFP fusion protein driven by a CMV enhancer/chicken β actin promotor (pCAG) from the Hprt locus. We detected bright and specific ciliary signals by immunostainings of various mono‐ and multiciliated tissues and by time‐lapse live‐cell analysis of cultured embryos and organ explant cultures. Additionally, we monitored cilium assembly and disassembly in embryonic fibroblast cells using live‐cell imaging. Thus, the ARL13B‐tRFP reporter mouse strain is a valuable tool for the investigation of ciliary structure and function in a tissue‐specific manner to understand processes, such as ciliary protein trafficking or cilium‐dependent signaling in vitro and in vivo.     

Asynchronous CDX2 expression and polarization of porcine trophoblast cells reflects a species‐specific trophoderm lineage determination progress model

Upregulation of Cdx2 expression in outer cells is a key event responsible for cell lineage segregation between the inner cell mass and the trophoderm (TE) in mouse morula‐stage embryos. In TE cells, polarization can regulate Hippo and Rho‐associated kinase (Rho‐ROCK) signaling to induce the nuclear location of YAP, which has been demonstrated to further induce the expression of Cdx2. However, we found that CDX2 expression could not be detected in the outer cells of porcine morula‐stage embryos but only in some TE cells at the early blastocyst stage. The biological significance and the regulation mechanism of this species‐specific CDX2 expression pattern have still not been determined. We show here that an asynchronous CDX2 expression pattern exists in porcine TE cells during the development of the blastocyst. We demonstrate that CDX2 expression in porcine TE cells depends on the nuclear localization of YAP and polarization of the embryo through Y27632 treatment. We found that the polarization process in the morula to the late blastocyst stage porcine embryos was asynchronous, which was revealed by the apical localization of phosphorylated EZRIN staining. Artificially enhancing the number of polarized blastomeres by culturing the separated blastomeres of four‐cell stage porcine embryos resulted in increased CDX2‐positive cell numbers. These results indicate that the mechanism of CDX2 expression regulation is conserved, but the polarization progress is not conserved between the pig and the mouse, and results in a species‐specific trophoblast determination progress model.     

A mouse reporter line to conditionally mark nuclei and cell membranes for in vivo live-imaging

Live-imaging is an essential tool to visualize live cells and monitor their behaviors during development. This technology demands a variety of mouse reporter lines, each uniquely expressing a fluorescent protein. Here, we developed an R26R-RG reporter mouse line that conditionally and simultaneously expresses mCherry and EGFP in nuclei and plasma membranes, respectively, from the Rosa26 locus. The intensity and resolution of mCherry nuclear localization and EGFP membrane localization were demonstrated to be sufficient for live-imaging with embryos that express RG (mCherry and EGFP) ubiquitously and specifically in fetal Sertoli cells. The conditional R26R-RG reporter mouse line should be a useful tool for labeling nuclei and membranes simultaneously in distinct cell populations.     

Using a histone yellow fluorescent protein fusion for tagging and tracking endothelial cells in ES cells and mice

We report the first endothelial lineage-specific transgenic mouse allowing live imaging at subcellular resolution. We generated an H2B-EYFP fusion protein which can be used for fluorescent labeling of nucleosomes and used it to specifically label endothelial cells in mice and in differentiating embryonic stem (ES) cells. A fusion cDNA encoding a human histone H2B tagged at its C-terminus with enhanced yellow fluorescent protein (EYFP) was expressed under the control of an Flk1 promoter and intronic enhancer. The Flk1::H2B-EYFP transgenic mice are viable and high levels of chromatin-localized reporter expression are maintained in endothelial cells of developing embryos and in adult animals upon breeding. The onset of fluorescence in differentiating ES cells and in embryos corresponds with the beginning of endothelial cell specification. These transgenic lines permit real-time imaging in normal and pathological vasculogenesis and angiogenesis to track individual cells and mitotic events at a level of detail that is unprecedented in the mouse.     

In vivo imaging and differential localization of lipid-modified GFP-variant fusions in embryonic stem cells and mice

The visualization of live cell behaviors operating in situ combined with the power of mouse genetics represents a major step toward understanding the mechanisms regulating embryonic development, homeostasis, and disease progression in mammals. The availability of genetically encoded fluorescent protein reporters, combined with improved optical imaging modalities, have led to advances in our ability to examine cells in vivo. We developed a series of lipid-modified fluorescent protein fusions that are targeted to and label the secretory pathway and the plasma membrane, and that are amenable for use in mice. Here we report the generation of two strains of mice, each expressing a spectrally distinct lipid-modified GFP-variant fluorescent protein fusion. The CAG::GFP-GPI strain exhibited widespread expression of a glycosylphosphatidylinositol-tagged green fluorescent protein (GFP) fusion, while the CAG::myr-Venus strain exhibited widespread expression of a myristoyl-Venus yellow fluorescent protein fusion. Imaging of live transgenic embryonic stem (ES) cells, either live or fixed embryos and postnatal tissues demonstrated that glycosylphosphatidyl inositol- and myristoyl-tagged GFP-variant fusion proteins are targeted to and serve as markers of the plasma membrane. Moreover, our data suggest that these two lipid-modified protein fusions are dynamically targeted both to overlapping as well as distinct lipid-enriched compartments within cells. These transgenic strains not only represent high-contrast reporters of cell morphology and plasma membrane dynamics, but also may be used as in vivo sensors of lipid localization. Furthermore, combining these reporters with the study of mouse mutants will be a step forward in understanding the inter- and intracellular behaviors underlying morphogenesis in both normal and mutant contexts.     

mED2—A Novel Gene Involved in Mouse Embryonic Development

Dissection of new genes underlying embryonic development is important for our understanding of the molecular mechanism of vertebrate embryonic development. In this study, the expression pattern and functional analysis of a new gene, called mED2, originally cloned from mouse embryos using subtractive hybridization was reported. mED2 expression patterns were characterized by RT-PCR-Southern hybridization and in situ hybridization. The results showed that mED2 was mainly expressed in the embryonic nervous system and mesoderm-derived tissues and its expression varied depending on the embryonic developmental stages. The knockdown of mED2 activity by antisense RNA injection inhibited zygote cleavage and blastocyst formation during pre-implantation in mice. Subcellular localization of mED2-eGFP fusion protein revealed a pattern of nuclear membrane and juxta-/perinuclear location such as in the rough endoplasmic reticulum and Golgi apparatus. This finding was supported by bioinformatics analysis, which indicated mED2 protein to be a transmembrane protein with partial homology to the thioredoxin family of proteins. It is inferred that mED2 gene can probably take part in early embryonic development in mouse and may be involved in target protein posttranslational modification, turnover, folding, and stability at the endoplasmic reticulum and/or the Golgi apparatus.     

Laser Fusion of Mouse Embryonic Cells and Intra-Embryonic Fusion of Blastomeres without Affecting the Embryo Integrity

Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo’s integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.     

Early embryonic development in the spider Achaearanea tepidariorum: Microinjection verifies that cellularization is complete before the blastoderm stage

The spider Achaearanea tepidariorum is emerging as a non-insect model for studying developmental biology. However, the availability of microinjection into early embryos of this spider has not been reported. We defined the early embryonic stages in A. tepidariorum and applied microinjection to its embryos. During the preblastoderm 16- and 32-nucleus stages, the energids were moving toward the egg periphery. When fluorochrome-conjugated dextran was microinjected into the peripheral region of 16-nucleus stage embryos, it was often incorporated into a single energid and inherited in the progeny without leaking out to surrounding energids. This suggested that 16-nucleus stage embryos consisted of compartments, each containing a single energid. These compartments were considered to be separate cells. Fluorochrome-conjugated dextran could be introduced into single cells of 16- to 128-nucleus stage embryos, allowing us to track cell fate and movement. Injection with mRNA encoding a nuclear localization signal/green fluorescent protein fusion construct demonstrated exogenous expression of the protein in live spider embryos. We propose that use of microinjection will facilitate studies of spider development. Furthermore, these data imply that in contrast to the Drosophila syncytial blastoderm embryo, the cell-based structure of the Achaearanea blastoderm embryo restricts diffusion of cytoplasmic gene products.     

Site-specific modification of genome with cell-permeable Cre fusion protein in preimplantation mouse embryo

Site-specific recombination (SSR) by Cre recombinase and its target sequence, loxP, is a valuable tool in genetic analysis of gene function. Recently, several studies reported successful application of Cre fusion protein containing protein transduction peptide for inducing gene modification in various mammalian cells including ES cell as well as in the whole animal. In this study, we show that a short incubation of preimplantation mouse embryos with purified cell-permeable Cre fusion protein results in efficient SSR. X-Gal staining of preimplantation embryos, heterozygous for Gtrosa26^tm1Sor, revealed that treatment of 1-cell or 2-cell embryos with 3 μM of Cre fusion protein for 2 h leads to Cre-mediated excision in 70-85% of embryos. We have examined the effect of the concentration of the Cre fusion protein and the duration of the treatment on embryonic development, established a condition for full term development and survival to adulthood, and demonstrated the germ line transmission of excised Gtrosa26 allele. Potential applications and advantages of the highly efficient technique described here are discussed.     

Matrix stiffness controls cardiac fibroblast activation through regulating YAP via AT1R

Cardiac fibrosis is a common pathway leading to heart failure and involves continued activation of cardiac fibroblasts (CFs) into myofibroblasts during myocardium damage, causing excessive deposition of the extracellular matrix (ECM) and thus increases matrix stiffness. Increasing evidence has shown that stiffened matrix plays an important role in promoting CF activation and cardiac fibrosis, and several signaling factors mediating CF mechanotransduction have been identified. However, the key molecules that perceive matrix stiffness to regulate CF activation remain to be further explored. Here, we detected significantly increased expression and nuclear localization of Yes-associated protein (YAP) in native fibrotic cardiac tissues. By using mechanically regulated in vitro cell culture models, we found that a stiff matrix-induced high expression and nuclear localization of YAP in CFs, accompanied by enhanced cell activation. We also demonstrated that YAP knockdown decreased fibrogenic response of CFs and that YAP overexpression promoted CF activation, indicating that YAP plays an important role in mediating matrix stiffness-induced CF activation. Further mechanistic studies revealed that the YAP pathway is an important signaling branch downstream of angiotensin II type 1 receptor in CF mechanotransduction. The findings help elucidate the mechanism of fibrotic mechanotransduction and may contribute to the development of new approaches for treating fibrotic diseases.     

Subcellular translocation signals regulate Geminin activity during embryonic development

BACKGROUND INFORMATION: Geminin (Gem) is a protein with roles in regulating both the fidelity of DNA replication and cell fate during embryonic development. The distribution of Gem is predominantly nuclear in cells undergoing the cell cycle. Previous studies have demonstrated that Gem performs multiple activities in the nucleus and that regulation of Gem activation requires nuclear import in at least one context. In the present study, we defined structural and mechanistic features underlying subcellular localization of Gem and tested whether regulation of the subcellular localization of Gem has an impact on its activity in cell fate specification during embryonic development. RESULTS: We determined that nuclear localization of Gem is dependent on a bipartite NLS (nuclear localization signal) in the N-terminus of Xenopus Gem protein. This bipartite motif mapped to a Gem N-terminal region previously shown to regulate neural cell fate acquisition. Microinjection into Xenopus embryos demonstrated that import-deficient Gem was incapable of modulating ectodermal cell fate, but that this activity was rescued by fusion to a heterologous NLS. Cross-species comparison of Gem protein sequences revealed that the Xenopus bipartite signal is conserved in many non-mammalian vertebrates, but not in mammalian species assessed. Instead, we found that human Gem employs an alternative N-terminal motif to regulate the protein's nuclear localization. Finally, we found that additional mechanisms contributed to regulating the subcellular localization of Gem. These included a link to Crm1-dependent nuclear export and the observation that Cdt1, a protein in the pre-replication complex, could also mediate nuclear import of Gem. CONCLUSIONS: We have defined new structural and regulatory features of Gem, and showed that the activity of Gem in regulating cell fate, in addition to its cell-cycle-regulatory activity, requires control of its subcellular localization. Our data suggest that rather than being constitutively nuclear, Gem may undergo nucleocytoplasmic shuttling through several mechanisms involving distinct protein motifs. The use of multiple mechanisms for modulating Gem subcellular localization is congruent with observations that Gem levels and activity must be stringently controlled during cell-cycle progression and embryonic development.     

Cwc15家族同源基因mED1在小鼠早期胚胎发育中的表达模式(英文)

RNA选择性剪接机制涉及基因表达模式的多样性、胚胎发育的调控和疾病的发展与转归.Cwf15/Cwc15蛋白家族与RNA剪接体的功能相关,其基因序列在很多物种之间是十分保守的.然而,在哺乳类,Cwf15/Cwc15基因的表达模式和生物学功能研究至今未见实验性研究报道.本文首次报道了Cwc15家族同源基因mED1在小鼠早期胚胎发育过程中的表达规律.RT-PCR结果表明,mED1基因的转录水平从小鼠桑葚胚到器官形成期呈逐渐上升趋势;整体原位杂交结果显示mED1基因主要在小鼠6.5-dpc胚胎的ICM、8.5-dpc胚胎的神经褶衍生物和10.5-dpc的头部、鳃弓和体节中表达.说明mED1基因参与了小鼠胚胎的早期发育.此外,GFP-融合蛋白的亚细胞定位实验表明,mED1蛋白具有核定位的功能(剪接体蛋白的必要特性),验证了其核定位序列(NLS)的预测.本文是关于Cwc15家族基因的首次实验研究报道.     

Live Imaging of Whole Mouse Embryos during Gastrulation: Migration Analyses of Epiblast and Mesodermal Cells

During gastrulation in the mouse embryo, dynamic cell movements including epiblast invagination and mesodermal layer expansion lead to the establishment of the three-layered body plan. The precise details of these movements, however, are sometimes elusive, because of the limitations in live imaging. To overcome this problem, we developed techniques to enable observation of living mouse embryos with digital scanned light sheet microscope (DSLM). The achieved deep and high time-resolution images of GFP-expressing nuclei and following 3D tracking analysis revealed the following findings: (i) Interkinetic nuclear migration (INM) occurs in the epiblast at embryonic day (E)6 and 6.5. (ii) INM-like migration occurs in the E5.5 embryo, when the epiblast is a monolayer and not yet pseudostratified. (iii) Primary driving force for INM at E6.5 is not pressure from neighboring nuclei. (iv) Mesodermal cells migrate not as a sheet but as individual cells without coordination.