Mouse Embryo Cells Research Articles

The Impact of Commercialisation on Public Perceptions of Stem Cell Research: Exploring Differences Across the Use of Induced Pluripotent Cells, Human and Animal Embryos

The development of pluripotent cells that enable stem cell research (SCR) without destroying human embryos is now a leading priority for science. Public and political controversies associated with human embryonic SCR experienced in the recent past should be alleviated if scientists no longer need to harvest cells from human embryos. This research suggests however additional issues needing attention in order to gain the public's trust and support: the use of mouse embryos and the commercialisation of research. Using a representative sample of 2,800 Australians, and an experimental telephone survey design, this research compared levels and predictors of public support for stem cell research across three cell source conditions: human embryo (HE), mouse embryo (ME) and induced pluripotent cells (iPSCs). The results revealed that the public were significantly more likely to support research using iPSCs than HE and ME cells and public compared to private research (regardless of the cell source). There was no significant difference in support for HE compared to ME research, but the former was viewed as more likely to lead to accessible health care benefits and to be associated with more trustworthy scientists. The results of a multimediation structural equation model showed that the primary reason support for SCR significantly dropped in a private compared to public context (i.e., the commercialisation effect) was because public scientists were trusted more than private scientists. This effect was consistent across all three SCR materials, suggesting that the use of mouse embryos or even iPSCs will not reduce the publics' concern with commercialised science. The implications these results have for public acceptance of stem cell and animal research are discussed in relation to possible solutions such as increasing public awareness of the regulation of animal research and benefit sharing.

A microfluidic system to study the cytotoxic effect of drugs: the combined effect of celecoxib and 5-fluorouracil on normal and cancer cells

We have investigated the response of normal and cancer cells to exposure a combination of celecoxib (Celbx) and 5-fluorouracil (5-FU) using a lab-on-a-chip microfluidic device. Specifically, we have tested the cytotoxic effect of Celbx on normal mouse embryo cells (Balb/c 3T3) and human lung carcinoma cells (A549). The single drugs or their combinations were adjusted to five different concentrations using a concentration gradient generator (CGG) in a single step. The results suggest that Celbx can enhanced the anticancer activity of 5-FU by stronger inhibition of cancer cell growth. We also show that the A549 cancer cells are more sensitive to Celbx than the Balb/c 3T3 normal cells. The results obtained with the microfluidic system were compared to those obtained with a macroscale in vitro cell culture method. In our opinion, the microfluidic system represents a unique approach for an evaluation of cellular response to multidrug exposure that also is more simple than respective microwell plate assays. Figureᅟ Electronic supplementary materialThe online version of this article (doi:10.1007/s00604-013-1009-4) contains supplementary material, which is available to authorized users.

Epiblast Ground State Is Controlled by Canonical Wnt/β-Catenin Signaling in the Postimplantation Mouse Embryo and Epiblast Stem Cells

Epiblast stem cells (EpiSCs) are primed pluripotent stem cells and can be derived from postimplantation mouse embryos. We now show that the absence of canonical Wnt/β-catenin signaling is essential for maintenance of the undifferentiated state in mouse EpiSCs and in the epiblast of mouse embryos. Attenuation of Wnt signaling with the small-molecule inhibitor XAV939 or deletion of the β-catenin gene blocked spontaneous differentiation of EpiSCs toward mesoderm and enhanced the expression of pluripotency factor genes, allowing propagation of EpiSCs as a homogeneous population. EpiSCs were efficiently established and propagated from single epiblast cells in the presence of both XAV939 and the Rho kinase (ROCK) inhibitor Y27632. Cell transplantation revealed that EpiSCs were able to contribute to primordial germ cells and descendants of all three germ layers in a host embryo, suggesting that they maintained pluripotency, even after prolonged culture with XAV939. Such an improvement in the homogeneity of pluripotency achieved with the use of a Wnt inhibitor should prove advantageous for manipulation of primed pluripotent stem cells.

Akt/PKB plays role of apoptosis relay on entry into first mitosis of mouse embryo

The cell-cycle regulators that control meiotic divisions also regulate the events that accompany the oocyte-to-zygote transition. Thus, the meiotic machinery functions as an internal pacemaker that propels the oocyte toward embryogenesis. The preimplantation embryo expresses a number of receptors that are important for initial activity of the phosphatidylinositol 3-kinase-protein kinase B (PI3K-Akt/PKB) pathway. The complete PI3K-Akt/PKB-CDK1 cascade is implicated as a key regulator of a number of cellular functions. Selective inhibition of protein kinase B (Akt/PKB) with inhibitor SH6 and cyclin-dependent kinase 1 (CDK1) with inhibitor roscovitine arrest development of the 1-cell preimplantation mouse embryo before entry into the first mitosis. The pronuclei of these inhibited embryos migrate to one another, but do not progress to pronuclei envelope breakdown and pronuclear fusion running immediately before the onset of mitosis. SH6-treated 1-cell mouse embryos showed a high occurrence of apoptosis features (nuclear fragmentation, positive terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), active caspase-3 in both cytoplasm and nucleoplasm). In the Akt/PKB-inhibited embryos, the active phosphorylated form Ser473Akt/PKB was not detected in pronuclear areas when compared with inhibitor-free controls. Although CDK1-inhibited 1-cell embryos also failed to enter into the first mitosis, the presence of apoptotic cell death features was not observed. In the roscovitine-treated embryos, Ser473Akt/PKB was detected in the pronuclei independently of CDK1 activity. We conclude that Akt/PKB plays an important role during entry of the 1-cell mouse embryo into the first mitosis, and probably functions as a relay in the cell-cycle stage. We assume that Akt/PKB is the primary target responsible for mediating anti-apoptotic signals in the 1-cell mouse embryo.

Transcript analysis of Heat shock protein 72 and protein 53 of 4-cell mouse embryos following Cryotop vitrification

The effects of two different concentrations of cryoprotectants on survival and developmental capacity of four-cell mouse embryos were compared by Cryotop vitrification to demonstrate that lower concentrations provide the same results as higher previously reported concentrations with lesser negative molecular impact on embryo cells. For this latest, embryos were compared via transcript analyses of Heat shock protein 72 (Hsp72) and protein 53 (p53). Four-cell embryos were obtained from superovulated female mice and randomly assignedto one of three following groups: (i) control (non-vitrified), (ii) vit<sub>1</sub> (15% v/v: 7.5% ethylene glycol(EG) and 7.5% dimethyl sulfoxide (DMSO), and (iii) vit<sub>2</sub> (30% v/v: 15% EG + 15% DMSO).The cells vitrified by Cryotop were thawed and side-by-side to the control group divided into two parts: one part was used to analyze the morphological traits, survival rate, and embryo cleavage ability to form blastocysts, and the other part was examined for changes in transcript levels of Hsp72 (Hspa1a + Hspa1b), p53, and Hprt1 (reference gene) by quantitative Real-Time polymerase chain reaction (qPCR). The results were analyzed by One Way Analysis of Variance and the mean values compared with LSD (P < 0.05). The relative expression of p53 in vit<sub>2</sub> (30% v/v) was significantly higher than in vit<sub>1</sub> (15% v/v) and in vit<sub>1</sub> it was higher than in the control. The relative expression of Hsp72 was the same in vit<sub>1</sub> and vit<sub>2 </sub>and significantly higher than in the control.The survival, cleavage, and blastocyst rates were the same for both vitrification treatments and significantly lower than in the control group. The up-regulations of Hsp72 and p53 following vitrification were suggestive of imposed heat shock, cold stress, and DNA damage to the mouse 4-cell embryos. 

Differential expression of microRNAs in 2-cell and 4-cell mouse embryos

In vitro fertilized (IVF) human embryos have a high incidence of developmental arrest before the blastocyst stage, therefore characterization of the molecular mechanisms that regulate embryo development is urgently required. Post-transcriptional control by microRNAs (miRNAs) is one of the most investigated RNA control mechanisms, and is hypothesized to be involved actively in developmental arrest in preimplantation embryos. In this study, we extracted total RNA from mouse 2-cell and 4-cell embryos. Using a miRNA microarray, 192 miRNAs were found to be differentially expressed in 4-cell embryos and 2-cell embryos; 122 miRNAs were upregulated and 70 were downregulated in 4-cell embryos. The microarray results were confirmed by real-time quantitative RT-PCR for six miRNAs (mmu-miR-467h, mmu-miR-466d-3p, mmu-miR-292-5p, mmu-miR-154, mmu-miR-2145, and mmu-miR-706). Cdca4 and Tcf12 were identified as miR-154 target genes by target prediction analysis. This study provides a developmental map for a large number of miRNAs in 2-cell and 4-cell embryos. The function of these miRNAs and the mechanisms by which they modulate embryonic developmental arrest require further study. The results of this study have potential applications in the field of reproductive medicine.

Expression and localization of creatine kinase in the preimplantation embryo

Creatine Kinase (CK) catalyses the "creatine shuttle," the reversible conversion of creatine phosphate to creatine with the liberation of ATP. This article examines the potential role of the creatine shuttle in the provision of ATP during mouse preimplantation embryo development. Using quantitative PCR, transcripts of four subunit isoforms of CK--CKM, CKB, CKMT1, and CKMT2--were detectable at all developmental stages, from the presumptive zygote to late blastocyst, but there was no obvious pattern in gene expression. By contrast, total CK biochemical activity, measured by a novel method, was relatively constant from the 2- to 8-cell stage, before exhibiting a significant decrease in activity at the blastocyst stage. Immunocytochemical studies revealed a marked association of CKB with the mitotic spindle in 2- and 4-cell mouse embryos, consistent with the proposition that the creatine shuttle plays a key role in local delivery of ATP during cytokinesis. Endogenous creatine was detected in the blastocyst at a level of 0.53 pmol/embryo. In conclusion, we believe that creatine phosphate can now be added to the list of potential sources of ATP during preimplantation development.

Bivalent promoter marks and a latent enhancer may prime the leukaemia oncogene LMO1 for ectopic expression in T-cell leukaemia

LMO1 is a transcriptional regulator and a T-acute lymphoblastic leukaemia (T-ALL) oncogene. Although first identified in association with a chromosomal translocation in T-ALL, the ectopic expression of LMO1 occurs far more frequently in the absence of any known mutation involving its locus. Given that LMO1 is barely expressed in any haematopoietic lineage, and activation of transcriptional drivers in leukaemic cells is not well described, we investigated the regulation of this gene in normal haematopoietic and leukaemic cells. We show that LMO1 has two promoters that drive reporter gene expression in transgenic mice to neural tissues known to express endogenous LMO1. The LMO1 promoters display bivalent histone marks in multiple blood lineages including T-cells, and a 3' flanking region at LMO1 +57 contains a transcriptional enhancer that is active in developing blood cells in transgenic mouse embryos. The LMO1 promoters become activated in T-ALL together with the 3' enhancer, which is bound in primary T-ALL cells by SCL/TAL1 and GATA3. Taken together, our results show that LMO1 is poised for expression in normal progenitors, where activation of SCL/TAL1 together with a breakdown of epigenetic repression of LMO1 regulatory elements induces ectopic LMO1 expression that contributes to the development and maintenance of T-ALL.

Development: Do Mouse Embryos Play Dice?

Cells of early mouse embryo were considered for a long time to acquire cell fate at random. Recent analyses argue against this simple model.

Expression of Aquaporins in Human Embryos and Potential Role of AQP3 and AQP7 in Preimplantation Mouse Embryo Development

Background/Aims: Water channels, also named aquaporins (AQPs), play crucial roles in cellular water homeostasis. Methods: RT-PCR indicated the mRNA expression of AQPs 1-5, 7, 9, and 11-12, but not AQPs 0, 6, 8, and 10 in the 2∼8-cell stage human embryos. AQP3 and AQP7 were further analyzed for their mRNA expression and protein expression in the oocyte, zygote, 2-cell embryo, 4-cell embryo, 8-cell embryo, morula, and blastocyst from both human and mouse using RT-PCR and immunofluorescence, respectively. Results: AQP3 and AQP7 were detected in all these stages. Knockdown of either AQP3 or AQP7 by targeted siRNA injection into 2-cell mouse embryos significantly inhibited preimplantation embryo development. However, knockdown of AQP3 in JAr spheroid did not affect its attachment to Ishikawa cells. Conclusion: These data demonstrate that multiple aquaporins are expressed in the early stage human embryos and that AQP3 and AQP7 may play a role in preimplantation mouse embryo development.

2S09 力学的伸張刺激の付与による未分化細胞の分化と機能の調節(OS3-2:組織・細胞工学とバイオエンジニアリング(2))

2S09 力学的伸張刺激の付与による未分化細胞の分化と機能の調節(OS3-2:組織・細胞工学とバイオエンジニアリング(2))

Abnormality of maternal‐to‐embryonic transition contributes to MEHP‐induced mouse 2‐cell block

Mono (2-ethylhexyl) phthalate (MEHP), an environmental contaminant, is known to cause many serious diseases, especially in reproductive system. However, little is known about the effect of MEHP on preimplantation embryo development. In this study, we found that the development of mouse 2-cell embryo was blocked by 10(-3) M MEHP. A significant increase in the level of reactive oxygen species (ROS) was observed in arrested 2-cell embryo following 10(-3) M MEHP treatment for 24 h. However, antioxidants, catalase (CAT), and superoxide dismutase (SOD), reduced intracellular ROS and protected MEHP-exposed embryos from death but failed to return the arrested embryos. Further experiments demonstrated that the level of apoptosis was not altered in live arrested 2-cell embryo and increased in dead arrested 2-cell embryo after MEHP treatment, which implied that ROS and apoptosis were not related with 2-cell block. During analysis of the indicators of embryonic genome activation (EGA) initiation (Hsc70, MuERV-L, Hsp70.1, eIF-1A, and Zscan4) and maternal-effect genes (OCT4 and SOX2), we found that MEHP treatment could significantly decline Hsc70, MuERV-L mRNA level and SOX2 protein level, and markedly enhance Hsp70.1, eIF-1A, Zscan4 mRNA level, and OCT4 protein level at 2-cell to 4-cell stage. Supplementation of CAT and SOD did not reverse the expression tendency of EGA related genes. Collectively, this study demonstrates for the first time that MEHP-induced 2-cell block is mediated by the failure of EGA onset and maternal-effect genes, not oxidative stress and apoptosis.

Functional Dissection of Pax3 in Paraxial Mesoderm Development and Myogenesis

The paired box transcription factor Pax3 is well-known as a major regulator of embryonic myogenesis. Before Pax3 expression becomes restricted to the dermomyotome, this transcription factor is also expressed in the developing somites. The role of Pax3 at this early stage is unclear, in particular because of the scarce frequency of Pax3-positive cells in the early mouse embryo. Inducible gene expression in embryonic stem cells (ESCs) represents an excellent tool to overcome this limitation, since it can provide large quantities of otherwise rare embryonic populations expressing a factor of interest. Here we used engineered mouse ESCs to perform a functional analysis of Pax3 with the aim to identify the molecular determinants involved in the early functions of this transcription factor. We find that Pax3 induction during embryoid body differentiation results in the upregulation of genes expressed in the presomitic and somitic mesoderm. Moreover, we show that paraxial mesoderm induced by transient expression of Pax3 is not irreversibly committed to myogenesis rather requires sustained Pax3 expression. Using a series of deletion mutants of Pax3, which differentially affect its transcriptional activity, we map protein domains necessary for induction of paraxial mesoderm and induction of the myogenic program. The paired, homeo-, and transcriptional activation domains were each required for both processes, however, the paired-c-terminal RED domain showed a paraxial mesoderm-specific activity that was dispensable for myogenesis. These findings demonstrate and provide mechanistic insight into an early role for Pax3 in the generation of paraxial mesoderm.

150 IN VITRO AND IN VIVO DEVELOPMENT OF EMBRYO FOLLOWING BIOPSY WITH DIFFERENT TECHNIQUES IN MOUSE EMBRYOS

In vitro development ratios, quality evaluation, in vivo implantation, and fetal development ratios were investigated following aspiration biopsy in 8-cell mouse embryos and trophectoderm biopsy in blastocyst developed from 8-cell stage embryos in vitro. Superovulated CB6F1 hybrid female mice (5–6 weeks) were sacrificed 68 to 72 hours after hCG administration. Eight-cell embryos were flushed from oviducts of the sacrificed mouse with HTF medium supplemented with HEPES and 3 mg mL–1 BSA. Embryos were randomly divided into two groups. In the first group, embryos at 8-cell stage were used for a single cell blastomer aspiration; in the second group, embryos were cultured in vitro until blastocyst stage. Trophectoderm cells (15% of trophoblastic cells) were biopsied from developing blastocysts. There were also control groups for both groups. Biopsy procedures for both groups were applied in 50 µL drops of Ca2+/Mg2+ free HTF medium containing HEPES+3 mg mL–1 BSA+5 µg mL–1 cytochalasine B. After biopsy, embryos were cultured in Quinn’s blastocyst medium supplemented with 4 mg mL–1 BSA and incubated in 5% CO2 and 5% O2 incubator at 37°C for 48 and 24 hours for blastomer aspiration and trophectoderm biopsy groups, respectively. While some developing blastocysts were used for determining total cell number, some of them were transferred to the recipients. Results were evaluated by independent t-test and ANOVA of SPSS 17.0 statistic program (SPSS Inc., Chicago, IL, USA). In blastomere biopsy and control groups, development rates were determined as 81.02% (121/152) and 96.37% (62/63), while the total cell numbers were determined as 50 and 50, respectively. There was no significant difference between groups in terms of development ratios and total cells. In blastomer biopsy and control groups, the implantation sites and fetal development rates were found as 25% (9/36) and 26% (8/30), and 19.44% (7/36) and 20% (6/30), respectively. No significant difference was observed between groups in terms of implantation sites and fetal development rates. In trophectoderm biopsy and control groups, while the development rates were found as 86.96% (69/79) and 93.33% (23/28), the total cell numbers were 26.66 and 55.33, respectively. Although there was not any significant difference between groups in terms of development rates, there was a significant difference between groups in terms of total cell numbers (P < 0.05). In trophectoderm biopsy and control groups, the implantation sites and fetal development rates were determined as 21.88% (7/32) and 59.09% (13/22), and 0% (0/32) and 18.18% (4/22), respectively. Although there was not any significant difference between groups in terms of implantation sites, there was a significant difference between groups in terms of fetal development rates (P < 0.05). Therefore, it was concluded that biopsy applied at early stage of embryonic development does not affect embryo development negatively and biopsy procedures applied at early developmental stages have more advantages especially in embryos developing faster with low total cell numbers such as mouse species. Supported by TUBITAK KAMAG-107G027).

125 INTRACYTOPLASMIC SPERM INJECTION (ICSI)-BASED MOUSE EMBRYO ASSAY: CHOICE OF EMBRYO CULTURE SYSTEM OUTWEIGHS THE EFFECT OF FERTILIZATION PROCEDURE ON EMBRYO DEVELOPMENT

Human embryo culture media, intended for assisted reproductive technologies (ARTs), are released for clinical use if they pass the mouse embryo assay (MEA). This assay prescribes that at least 70% of in vivo fertilized mouse 1-cell embryos form blastocysts, in order to grant the culture medium approval. In the fertility clinic, however, human embryos undergo more manipulation than their MEA counterparts through, for example, fertilization by intracytoplasmic sperm injection (ICSI); further, only a minority of the embryos transferred to the uterus goes on to establish gestations. In this context, we asked if the results of the MEA only depend on the type of in vitro culture, or are also affected by the method of fertilization. Superovulated B6C3F1 mouse oocytes were fertilized by ICSI using C57Bl/6 sperm. Pronuclear-stage eggs were allocated to four developmental environments: two ART culture protocols (HTF/MultiBlast, Irvine Scientific; ISM1/ISM2, Origio), standard mouse culture medium (KSOM(aa), made in-house) and the oviduct of pseudopregnant CD1 mice. As control for the invasive manipulation, pronuclear-stage eggs were generated by mating (B6C3F1 × C57Bl/6) and cultured in KSOM(aa) medium. Embryos were recovered from culture or from the CD1 uterus and scored for blastocyst formation at 96 h of development (Table 1). For these blastocysts, we determined the number of total, inner cell mass (ICM), and trophectoderm (TE) cells (Table 1) by confocal immunofluorescence microscopy (Schwarzer et al. 2012 doi:10.1093/humrep/des223). Our results show that ART culture protocols applied to mouse ICSI embryos are not equivalent in supporting blastocyst formation. Based on blastocyst rates, the ranking observed here after ICSI, reflects the ranking reported by us for IVF embryos (Schwarzer et al. 2012); that is, KSOM(aa) > HTF/MultiBlast > oviduct > ISM1/2. This similarity suggests that the effect of in vitro culture on mouse development exceeds the effect of ICSI, provided gametes are of good quality. From the analysis of cell numbers, we note that while the ICM/TE ratios are not of easy interpretation, the absolute numbers of cells in the ICM draw a clear line between the environment of the oviduct and those of culture media. Irrespective of the ICM/TE ratio, only the oviduct environment secures 8 cells in the ICM (Table 1). Soriano and Jaenisch (1986 Cell 46, 19–29) reported that 8 cells of the ICM are set aside to give rise to the body of a mouse. In summary, the current MEA is a valuable assay to assess the quality of culture medium, however, its refinement is necessary to better model the adaptive properties of embryo culture when different methods of fertilization are applied. Until the MEA is extended into postimplantation development, as we advocate (Schwarzer et al. 2012), the absolute numbers of cells in the ICM may be a better gauge of embryo quality than the blastocyst rates. Table 1.Mouse embryo assay outcomes after ICSI

JAK2 mediates the acute response to decreased cell volume in mouse preimplantation embryos by activating NHE1

Preimplantation mouse embryos are particularly sensitive to increased osmolarity within their normal physiological range. The detrimental effects can be alleviated by organic osmolytes such as glycine transported into early embryos, an effect thought to be due to the organic osmolyte replacing a portion of intracellular inorganic ions accumulated during acute cell volume regulation. However, no mechanism of cell volume regulation dependent on inorganic ions has been identified in preimplantation embryos. We found that decreased cell volume rapidly activated Na(+)/H(+) exchange in preimplantation mouse embryos. This activity was likely mediated by the NHE1 (Slc9a1) isoform, since it was blocked by the highly selective NHE1 inhibitor, cariporide, which also inhibited the ability of the 1-cell embryo to maintain cell volume. How NHE1 is activated by decreased cell volume is not generally well understood. Full activation of NHE1 by decreased cell volume in 2-cell mouse embryos required the activity of the tyrosine kinase Janus kinase 2 (Jak2), since NHE1 activation was inhibited by the general tyrosine kinase inhibitor genistein, several selective inhibitors of Jak2, and dominant negative Jak2 expressed in 2-cell embryos. Decreased cell volume furthermore resulted in increased tyrosine phosphorylation of proteins in 2-cell embryos detected both by anti-phosphotyrosine antibody and an antibody directed against active phospho-Jak2. Thus, Jak2 apparently serves as a cell volume sensor in embryos. Evidence from pharmacological inhibitors further indicated that NHE1 activation by decreased cell volume was dependent on calmodulin activity, likely downstream of Jak2, and required active phospholipase C.

Equilibrium vitrification of mouse embryos at various developmental stages

Previously, we developed a new method by which 2-cell mouse embryos can be vitrified in liquid nitrogen in a near-equilibrium state, and then kept at -80°C for several days. In the present study, we examined whether or not the method was effective for mouse embryos at other developmental stages. Eight-cell embryos, morulae, and expanded blastocysts of ICR mice were vitrified with ethylene glycol-based solutions, named EFSc because of their composition of ethylene glycol (30-40%, v/v) and FSc solution. The FSc solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus 1.5 M sucrose. The extent of equilibrium was assessed by examining how well vitrified embryos survived after being kept at -80°C. When 8-cell embryos and morulae were vitrified with EFS35c or EFS40c and then kept at -80°C, the survival rate was high even after 4 days in storage and remained high after re-cooling in liquid nitrogen. On the other hand, the survival of vitrified-expanded blastocysts kept at -80°C was low. Therefore, 8-cell embryos and morulae can be vitrified in a near-equilibrium state using the same method as for 2-cell embryos. A high proportion of C57BL/6J embryos at the 2-cell, 8-cell, and morula stages vitrified with EFS35c developed to term after transportation on dry ice, re-cooling in liquid nitrogen, and transfer to recipients. In conclusion, the near-equilibrium vitrification method, which is effective for 2-cell mouse embryos, is also effective for embryos at the 8-cell and morula stages. The method would enable handy transportation of vitrified embryos using dry ice.

Complex invasion pattern of the cerebral cortex bymicroglial cells during development of the mouse embryo

Microglia are the immune cells of the central nervous system. They are suspected to play important roles in adult synaptogenesis and in the development of the neuronal network. Microglial cells originate from progenitors in the yolk sac. Although it was suggested that they invade the cortex at early developmental stages in the embryo, their invasion pattern remains largely unknown. To address this issue we analyzed the pattern of cortical invasion by microglial cells in mouse embryos at the onset of neuronal cell migration using in vivo immunohistochemistry and ex vivo time-lapse analysis of microglial cells. Microglial cells begin to invade the cortex at 11.5 days of embryonic age (E11.5). They first accumulate at the pial surface and within the lateral ventricles, after which they spread throughout the cortical wall, avoiding the cortical plate region in later embryonic ages. The invasion of the cortical parenchyma occurs in different phases. First, there is a gradual increase of microglial cells between E10.5 and E14.5. From E14.5 to E15.5 there is a rapid phase with a massive increase in microglia, followed by a slow phase again from E15.5 until E17.5. At early stages, many peripheral microglia are actively proliferating before entering the parenchyma. Remarkably, activated microglia accumulate in the choroid plexus primordium, where they are in the proximity of dying cells. Time-lapse analysis shows that embryonic microglia are highly dynamic cells.

Evaluation of OPEN Zinc Finger Nucleases for Direct Gene Targeting of the ROSA26 Locus in Mouse Embryos

Zinc finger nucleases (ZFNs) enable precise genome modification in a variety of organisms and cell types. Commercial ZFNs were reported to enhance gene targeting directly in mouse zygotes, whereas similar approaches using publicly available resources have not yet been described. Here we report precise targeted mutagenesis of the mouse genome using Oligomerized Pool Engineering (OPEN) ZFNs. OPEN ZFN can be constructed using publicly available resources and therefore provide an attractive alternative for academic researchers. Two ZFN pairs specific to the mouse genomic locus gt(ROSA26)Sor were generated by OPEN selections and used for gene disruption and homology-mediated gene replacement in single cell mouse embryos. One specific ZFN pair facilitated non-homologous end joining (NHEJ)-mediated gene disruption when expressed in mouse zygotes. We also observed a single homologous recombination (HR)-driven gene replacement event when this ZFN pair was co-injected with a targeting vector. Our experiments demonstrate the feasibility of achieving both gene ablation through NHEJ and gene replacement by HR by using the OPEN ZFN technology directly in mouse zygotes.

How a Blood Cell Intracellular Signal Keeps Blood Flowing

‘‘Epo,’’ or erythropoietin, is not just a performance-enhancing drug for endurance athletes. It is an essential hormone that regulates the rate of erythropoiesis, or creation of new red blood cells. Basal Epo signaling ensures that such cells are made constantly at a low level, to replace wornout cells. Certain stresses, such as hypoxia, increase Epo and lead to a steep increase in erythropoiesis, a phenomenon known to mountain climbers, who adapt to the low oxygen at high altitudes by packing more red blood cells into their blood. The Epo signaling cascade begins when the hormone binds to the Epo receptor. This ultimately leads to phosphorylation of a transcription factor called Stat5, which turns on a suite of genes controlling cell proliferation. A key question in understanding the Epo-Stat5 system is whether and how basal and stress-induced signaling differ. In a new study in this issue of PLOS Biology, Emelinda Porpiglia, Merav Socolovsky, and colleagues set out to answer that question. The authors used fluorescently labeled antibodies to tag phosphorylated Stat5 in erythropoeitic liver cells of mouse embryos. They also used a variety of cell surface markers to distinguish early versus late erythroblasts (cells that differentiate into red blood cells). They characterized three aspects of Stat5 signaling within Epostimulated cells: the median fluorescence from all cells within a subpopulation of erythroblasts, the fraction of those cells that were engaged in Stat5 signaling, and the level of phosphorylated Stat5 within signaling cells. They found that Stat5 signaling was highest in precursor cells, and declined with maturation. Furthermore, precursors exhibited an ‘‘analog,’’ graded increase in signaling in response to rising Epo levels. This graded increase was a combination of an increase in the number of signaling cells, and an increase in the signaling intensity within each cell. In contrast, older cells, which overall had much lower Stat5 signaling, responded to an increase in Epo by increasing the number of signaling cells, but within each cell, the signal intensity was binary, either ‘‘on’’ or ‘‘off,’’ with little change in intensity in the ‘‘on’’ position. What regulates this transition from analog to binary response? In large part, they found, the ability to mount a highintensity and graded response was due to larger amounts of Stat5 protein in earlier cells, rather than differences in Epo receptor level. When the authors added exogenous Stat5 to older cells, they too were able to signal at the higher level and in a graded way. Conversely, in cells with a mutant Epo receptor unable to bind Stat5, only basal-level, binary signaling was present, similar to the older cells. A binary signal, the authors argue, provides an ideal way to filter out the noise at low incoming Epo signal strength, while the analog response at higher Epo concentrations provides the cell with a more dynamic ability to respond to varying levels of stress. Additionally, they found, part of the stress response was an increased production of transferrin receptor (CD71), probably to help bring in extra iron to make more hemoglobin. Understanding this system is important not only for a better theoretical understanding of the dynamics of cell signaling. Targeting the high-level Stat5 signal, while maintaining the low-level one, may be a viable strategy for treating myeloproliferative disorders, including some types of leukemia, in which Stat5 is constitutively active.