Early Mouse Embryo Development Research Articles

Biological effects and dose-response assessment of diesel exhaust particles on in vitro early embryo development in mice

OBJECTIVE: We observed recently an increased risk of early pregnancy loss in patients exposed to high levels of ambient PM10 during the preconceptional period. Yet, effects of this exposure on early embryo development are unknown. The aim of this study was to assess the dose-response and biological effects of diesel exhaust particles (DEP) on embryo development using the IVF mouse model.

A kinase-dead knock-in mutation in mTOR leads to early embryonic lethality and is dispensable for the immune system in heterozygous mice

BackgroundThe mammalian target of rapamycin protein (mTOR) is an evolutionarily conserved kinase that regulates protein synthesis, cell cycle progression and proliferation in response to various environmental cues. As a critical downstream mediator of PI3K signaling, mTOR is important for lymphocyte development and function of mature T and B-cells. Most studies of mTOR in immune responses have relied on the use of pharmacological inhibitors, such as rapamycin. Rapamycin-FKBP12 complex exerts its immunosuppressive and anti-proliferative effect by binding outside the kinase domain of mTOR, and subsequently inhibiting downstream mTOR signaling.ResultsTo determine the requirement for mTOR kinase activity in the immune system function, we generated knock-in mice carrying a mutation (D2338) in the catalytic domain of mTOR. While homozygous mTOR kd/kd embryos died before embryonic day 6.5, heterozygous mTOR+/kd mice appeared entirely normal and are fertile. mTOR +/kd mice exhibited normal T and B cell development and unaltered proliferative responses of splenocytes to IL-2 and TCR/CD28. In addition, heterozygousity for the mTOR kinase-dead allele did not sensitize T cells to rapamycin in a CD3-mediated proliferation assay. Unexpectedly, mTOR kinase activity towards its substrate 4E-BP1 was not decreased in hearts and livers from heterozygous animals.ConclusionAltogether, our findings indicate that mTOR kinase activity is indispensable for the early development of mouse embryos. Moreover, a single wild type mTOR allele is sufficient to maintain normal postnatal growth and lymphocyte development and proliferation.

A link between the interleukin‐6/Stat3 anti‐apoptotic pathway and microRNA‐21 in preimplantation mouse embryos

Signal transducers and activators of transcription-3 (Stat3) plays a central role in interleukin-6 (IL-6)-mediated cell proliferation by inhibiting apoptosis in a variety of cell types. MicroRNA-21 (miRNA-21), a ubiquitous miRNA, acts as an anti-apoptotic factor that seems to be indirectly but strictly linked to Stat3. In order to determine whether the IL-6 induced Stat3 anti-apoptosis pathway is linked with miRNA-21, we first determined the effects of recombinant mouse IL-6 on Stat3 expression, mouse embryo viability, and the mRNA levels of apoptosis related genes and miRNA-21 during mouse embryo development in vitro. Addition of 10 or 100 ng/ml of recombinant IL-6 to the culture medium did not affect the developmental ability of 2-cell stage embryos into blastocysts. However, total cell number was significantly increased and apoptosis was reduced in blastocyst stage embryos cultured in the presence of 100 ng/ml of recombinant IL-6. Furthermore, addition of recombinant IL-6 to the culture medium significantly increased the copy numbers of anti-apoptotic miRNA-21, up-regulated Bcl2l1, and down-regulated casp3. Similarly, the injection of mature miRNA-21 into cells up-regulated Bcl2l1 and down-regulated casp3. These results suggest that the induction of the Stat3 anti-apoptotic pathway by IL-6 is linked to miRNA-21 expression, which possibly results in the regulation of cell apoptosis in early mouse embryo development.

122 GLYCOSAMINOGLYCANS IMPROVE THE NUMBER OF BLASTOMERES IN ZONA-FREE RAT AND MOUSE EMBRYOS

In general, early mammalian embryos are enclosed by zona pellucida until the blastocyst stage. The possible role of zona pellucida could be to maintain the three-dimensional structure of cleaving embryos, as well as preventing blastomeres from attack by immune cells and miscellaneous germs. However, beneficial roles of the zona on early development of blastomeres are still unknown. The present study was undertaken to examine if the absence of zona pellucida of rat and mouse embryos at the 8-cell stage affected the early development to the blastocyst stage. Furthermore, we examined whether supplementation of culture medium with glycosaminoglycans, such as hyaluronic acid and heparan sulfate, improved the early development or not. In the first experiment, embryos at the 8-cell stage were collected from mated Wistar rats or ICR mice and then, directly or after removing zona pellucida by using acidity Tyrode’s solution, cultured in modified R1ECM or kSOM medium for 24 h in an atmosphere of 5% CO2 in air. Following culture, the incidence of blastocyst formation and the cell number of blastocysts were examined. In the second experiment, intact or zona-free 8-cell embryos were cultured in various concentration of hyaluronic acid (0, 65, 125, 250 μg mL–1) or heparin sulfate (0 or 15 μg mL–1) for 24 h. After culture, blastocyst formation and cell number of blastocyst were similarly examined. Statistical analyses was performed by one-way ANOVA with Bonferroni/Dunn’s post hoc test (significance, P < 0.05). All percentage data were subjected to arc-sine transformation before statistical analysis. Percentage of blastocyst formation and the mean cell number of the blastocyst were less when zona-free 8-cell rat and mouse embryos were cultured (72.1 ± 2.9% and 22.8 ± 0.7), as compared with control intact embryos (94.3 ± 3.5% and 30.6 ± 1.1). Supplementation with hyaluronic acid (250 μg mL–1) improved the blastocyst formation rate of rat embryos (86.4 ± 5.0%) and the cell number of blastocysts (28.8 ± 0.5) to the same level of zona-intact embryos. In mouse embryos, the same concentration of hyaluronic acid improved only the cell number of blastocysts (from 21.7 ± 0.7 to 28.9 ± 0.7) to the same level of zona-intact embryos (30.9 ± 0.7). When heparin sulfate was supplemented, the incidence of blastocyst formation of rat embryos did not improve, but the cell number of the blastocyst (31.1 ± 0.7) did improve to the similar level with zona-intact embryos (30.6 ± 1.1). These results indicate that the zona pellucida has a beneficial effect on the early development of rat and mouse embryos and suggest that glycosaminoglycans, such as hyaluronic acid and heparan sulfate, contribute to the beneficial effect.

Shh and ROCK1 modulate the dynamic epithelial morphogenesis in circumvallate papilla development

In rodents, a circumvallate papilla (CVP) develops with dynamic changes in epithelial morphogenesis during early tongue development. Molecular and cellular studies of CVP development revealed that there would be two different mechanisms in the apex and the trench wall forming regions with specific expression patterns of Wnt11 and Shh. Molecular interactions were examined using in vitro organ culture with over-expression of Shh, important signalling molecules and various inhibitors revealed that there are two significant different mechanisms in CVP formation by Wnt11 and Shh expressions. Wnt, a well known key molecule to initiate taste papillae, would govern Rho activation and cytoskeleton formation in the apex epithelium of CVP. In contrast, Shh regulates the cell proliferation to differentiate taste buds and to invaginate the epithelium for development of von Ebner's gland (VEG). Based on these results, we suggest that these different molecular signalling cascades of Wnt11 and Shh would play crucial roles in specific morphogenesis and pattern formation of CVP during early mouse embryo development.

Involvement of the p110α isoform of PI3K in early development of mouse embryos

Class I of phosphoinositide 3-kinases (PI3Ks) is characterized as a group of intracellular signal proteins possessing both protein and lipid kinase activities. Recent studies implicate class I of PI3Ks acts as indispensable mediators in early development of mouse embryos, but the molecular mechanisms are poorly defined. In this paper, mouse one-cell embryos were used to investigate a possible contribution of the catalytic subunit of PI3K, p110 alpha, to cell cycle progression. The expression level of p110 alpha was determined in four phases of one-cell embryos. Silencing of p110 alpha by microinjection of p110 alpha shRNA into one-cell embryos resulted in a G2/M arrest and prevented the activation of Akt and M-phase promoting factor (MPF). Further, microinjection of the synthesized mRNA coding for a constitutively active p110 alpha into one-cell embryos induced cell cleavage more effectively than microinjection of wild-type p110 alpha mRNA, whereas microinjection of mRNA of kinase-deficient p110 alpha delayed the first mitotic cleavage. Taken together, this study demonstrates that p110 alpha is significant for G2/M transition of mouse one-cell embryos and further emphasizes the importance of Akt in PI3K pathway.

Dynamic Changes of Nuclear Halo Structure of In Vivo-Fertilized and Cloned Preimplantation Mouse Embryos.

Eukaryotic chromosomes are organized into domains through matrix attachment regions playing important roles in transcription, recombination and replication. The non-matrix-associated domains form loops of DNA which appear as a nuclear halo surrounding the nuclear matrix. Little is known regarding DNA looping in mouse preimplantation embryos. In this study we monitored the changes in nuclear halo structures of in vivo fertilized embryos as well as those derived from somatic cell nuclear transfer (SCNT). Nuclear halos were prepared by extraction with 2 M NaCl then visualized with propidium iodide. The halo diameters were measured by confocal microscopy. Embryos fertilized in vivo and cultured in vitro were collected at the pronuclear, 2-, 4- and 8-cell stages. The average diameter of the nuclear halo decreased significantly from the pronuclear to the 8-cell stage (male pronuclei: 29.53± 0.86 μm (n=5); female pronuclei: 20.04±1.00 μm (n=5); 2-cell: 22.88± 0.49 μm (n=10); 4-cell: 16.16±0.31 μm (n=6); 8-cell: 12.67± 0.18 μm (n=56)) showing dynamic changes in halo size occur during mouse early embryo development. Because TSA treatment increases mouse SCNT efficiency, we then examined SCNT embryos at 1 h post donor injection, as well as 6 h and 10 h post chemical activation with and without Trichostatin A (TSA) treatment. Premature Chromosome Condensation was observed at 1 h post cell injection. Nuclear halo extraction procedure significantly increased the size of nuclear halo at 6 and 10 h post-activation. Interestingly, we also found significant decreases in nuclear halo areas after TSA treatment at 6 h post-activation (738.4± 78 μm2 (n=7) vs. 483.4± 42.5μm2 (n=7)). Taken together, DNA looping analysis can be used to access nuclear organization during preimplantation embryo development and to begin to investigate the mechanism of nuclear reprogramming by SCNT.

Requirement ofN-Myristoyltransferase 1 in the Development of Monocytic Lineage

N-myristoyltransferase (NMT) exists in two isoforms, NMT1 and NMT2, that catalyze myristoylation of various proteins crucial in signal transduction, cellular transformation, and oncogenesis. We have recently demonstrated that NMT1 is essential for the early development of mouse embryo. In this report, we have demonstrated that an invariant consequence of NMT1 knock out is defective myelopoesis. Suppressed macrophage colony forming units were observed in M-CSF-stimulated bone marrow cells from heterozygous (+/-) Nmt1-deficient mice. Homozygous (-/-) Nmt1-deficient mouse embryonic stem cells resulted in drastic reduction of macrophages when stimulated to differentiate by M-CSF. Furthermore, to understand the requirement of NMT1 in the monocytic differentiation we investigated the role of NMT, pp60c-Src (NMT substrate) and heat shock cognate protein 70 (inhibitor of NMT), during PMA-induced differentiation of U937 cells. Src kinase activity and protein expression increased during the differentiation process along with regulation of NMT activity by hsc70. NMT1 knock down in PMA treated U937 cells showed defective monocytic differentiation. We report in this study novel observation that regulated total NMT activity and NMT1 is essential for proper monocytic differentiation of the mouse bone marrow cells.

Partners in Crime

See related article, pages 12–15 Vertebrates have evolved 2 types of blood vessels—arteries and veins—that function to supply oxygen and nutrients and remove cellular waste. To efficiently accomplish these specialized functions, arteries and veins have developed distinct morphological and molecular differences. As a result, the endothelial cells that compose arteries and veins are vastly different in their biochemical and cellular properties. During development, the nascent endothelial cells generated from hemangioblasts or angioblasts1,2 undergo tightly regulated specification and differentiation processes to adopt either the arterial or venous endothelial fate. Differentiated endothelial cells migrate and aggregate to form either arteries or veins according to their adopted fate.3 Dysregulation of these regulated events often results in devastating consequences. For instance, failure to segregate arterial and venous endothelial cells causes potentially fatal clinical conditions such as arteriovenous malformation, hereditary hemorrhagic telangiectasia, and cerebral cavernous malformation.4 Significant progress has been made in understanding how nascent endothelial cells adopt the arterial fate, resulting in identification of several key signaling molecules and their intracellular transducers involved in the specification of arterial endothelial cells.5 However, transcription factors that function …

EMB-003 Approach for microdeletion screening in congenital heart defects

s Aspire: ART Paving the way for new frontiers mouse embryonic development. Materials/Methods: Whole-mount in-situ hybridization was carried out as described by Correia and Conlon. Using the cDNA of mouse p19 cells as the template, DNA was amplified with the primers for mouse borealin: forward, 5′-TGCCTTCCATCCAAGAAGAG-3′ and reverse, 5′AGGTGTTGGCAGTGAAGGAC-3′. The amplified DNA was cloned into pGEM-T Easy vector (Promega) and sequenced. Hybridization was performed with a digoxygenin-labelling and detection kit (Roche). Protein extracts were separated by SDS-PAGE and detected by western blotting using the western blot kit (KPL). Human anti-borealin antibody (gift from Dr Earnshaw and Dr Chang) and anti-rabbit IgG (KPL) were used at 1:500 and 1:10,000 dilutions, respectively. Anti-GAPDH antibody, anti-mouse IgG, anti-Oct-4 antibody (Santa) and anti-survivin antibody (Chemicon) were used according to the manufacturer’s protocol. After injection with the anti-borealin antibody or rabbit IgG (Santa), zygotes were brought back into the medium for culture. Indirect immunofluorescence was performed as described previously. After injection, the zygotes in the first mitotic division were immediately fixed in 4% formaldehyde at 4°C for 30 min, followed by incubation with the anti-β-tubulin antibody (Sigma) and fluoresceinconjugated anti-mouse IgG (Sigma). DNA was stained with 0.5 μg/ml DAPI (Chemicon) in 1× PBS. Results: Borealin was highly expressed in the undifferentiated ES cells, the mouse preimplantation embryos and the embryonic brain of 8.5 dpc–9.5 dpc mouse embryo. The mouse embryos were arrested at the 2-cell or 4-cell stage with the Borealin depleted. Conclusion: The authors have indicated for the first time that borealin plays crucial roles in the early development of mouse embryos and in the self-renewal and proliferation of undifferentiated HES cells.

Early Embryonic Lethality Caused by Disruption of the Gene for Choline Kinase α, the First Enzyme in Phosphatidylcholine Biosynthesis

Choline kinase alpha (CK-alpha) is one of two mammalian enzymes that catalyze the phosphorylation of choline to phosphocholine in the biosynthesis of the major membrane phospholipid, phosphatidylcholine. We created mice lacking CK-alpha with an embryonic stem cell line containing an insertional mutation in the gene for CK-alpha (Chka). Embryos homozygous for the mutant Chka allele were recovered at the blastocyst stage, but not at embryonic day 7.5, indicating that CK-alpha is crucial for the early development of mouse embryos. Heterozygous mutant mice (Chka(+/-)) appeared entirely normal in their embryonic development and gross anatomy, and they were fertile. Although choline kinase activity was decreased by approximately 30%, the amount of phosphatidylcholine in cells and the levels of other enzymes involved in phosphatidylcholine biosynthesis were unaffected. Phosphatidylcholine biosynthesis measured by choline incorporation into hepatocytes was also not compromised in Chka(+/-) mice. Enhanced levels of choline and attenuated levels of phosphocholine were observed in both the livers and testes of Chka(+/-) mice. Triacylglycerol and cholesterol ester were elevated approximately 2-fold in the livers, whereas neutral lipid profiles in plasma were similar in Chka(+/-) and wild-type (Chka(+/+)) mice. Thus, Chka is an essential gene for early embryonic development, but adult mice do not require full expression of the gene for normal levels of phosphatidylcholine.

EMB-004 Effect of oxytocin on folliculogenesis and ovulation

s Aspire: ART Paving the way for new frontiers mouse embryonic development. Materials/Methods: Whole-mount in-situ hybridization was carried out as described by Correia and Conlon. Using the cDNA of mouse p19 cells as the template, DNA was amplified with the primers for mouse borealin: forward, 5′-TGCCTTCCATCCAAGAAGAG-3′ and reverse, 5′AGGTGTTGGCAGTGAAGGAC-3′. The amplified DNA was cloned into pGEM-T Easy vector (Promega) and sequenced. Hybridization was performed with a digoxygenin-labelling and detection kit (Roche). Protein extracts were separated by SDS-PAGE and detected by western blotting using the western blot kit (KPL). Human anti-borealin antibody (gift from Dr Earnshaw and Dr Chang) and anti-rabbit IgG (KPL) were used at 1:500 and 1:10,000 dilutions, respectively. Anti-GAPDH antibody, anti-mouse IgG, anti-Oct-4 antibody (Santa) and anti-survivin antibody (Chemicon) were used according to the manufacturer’s protocol. After injection with the anti-borealin antibody or rabbit IgG (Santa), zygotes were brought back into the medium for culture. Indirect immunofluorescence was performed as described previously. After injection, the zygotes in the first mitotic division were immediately fixed in 4% formaldehyde at 4°C for 30 min, followed by incubation with the anti-β-tubulin antibody (Sigma) and fluoresceinconjugated anti-mouse IgG (Sigma). DNA was stained with 0.5 μg/ml DAPI (Chemicon) in 1× PBS. Results: Borealin was highly expressed in the undifferentiated ES cells, the mouse preimplantation embryos and the embryonic brain of 8.5 dpc–9.5 dpc mouse embryo. The mouse embryos were arrested at the 2-cell or 4-cell stage with the Borealin depleted. Conclusion: The authors have indicated for the first time that borealin plays crucial roles in the early development of mouse embryos and in the self-renewal and proliferation of undifferentiated HES cells.

Photobiomodulation of the development of early mouse embryos by luminescent irradiation with λmax = 626 nm

Photobiomodulation of the development of early mouse embryos by luminescent irradiation with λmax = 626 nm

In vitro effects of phthalic acid mono-n-butyl ester and di-n-butyl ester on early mouse embryo development

The diesters of 1,2-benzenedicarboxylic acid, commonly known as phthalates, are synthetic chemicals often used as plasticizers in the manufacture of flexible vinyl. Recently, phthalate esters have been shown to disrupt implantation and pregnancy maintenance in mated rats.

Redundant roles of Sox17 and Sox18 in early cardiovascular development of mouse embryos

Sox7, -17 and -18 constitute the Sox subgroup F ( SoxF) of HMG box transcription factor genes, which all are co-expressed in developing vascular endothelial cells in mice. Here we characterized cardiovascular phenotypes of Sox17/ Sox18-double and Sox17-single null embryos during early-somite stages. Whole-mount PECAM staining demonstrated the aberrant heart looping, enlarged cardinal vein and mild defects in anterior dorsal aorta formation in Sox17 single-null embryos. The Sox17/ Sox18 double-null embryos showed more severe defects in formation of anterior dorsal aorta and head/cervical microvasculature, and in some cases, aberrant differentiation of endocardial cells and defective fusion of the endocardial tube. However, the posterior dorsal aorta and allantoic microvasculature was properly formed in all of the Sox17/ Sox18 double-null embryos. The anomalies in both anterior dorsal aorta and head/cervical vasculature corresponded with the weak Sox7 expression sites. This suggests the region-specific redundant activities of three SoxF members along the anteroposterior axis of embryonic vascular network.

Involvement of Protein Kinase B/AKT in Early Development of Mouse Fertilized Eggs1

The activation of AKT (also called protein kinase B) is thought to be a critical step in the phosphoinositide 3-kinase pathway that regulates cell growth and differentiation. In this report, we investigated the role of AKT in the regulation of mouse early embryo development. Injection of mRNA coding for a constitutively active myristoylated AKT (myr-Akt1) into one-cell stage fertilized eggs induced cell division more effectively than injection of wild-type AKT (Akt1-WT) mRNA, whereas microinjection of mRNA of kinase-deficient AKT (Akt1-KD) delayed the first mitotic division. Meanwhile, microinjection of different kinds of mRNA of AKT affected the phosphorylation status of CDC2A-Tyr15 and the activation of M-phase promoting factor (MPF). To investigate the intermediate factor between AKT and MPF, we then injected one-cell stage eggs first with Akt1-WT mRNA or myr-Akt1 mRNA and then with mRNA encoding either wild-type CDC25B (Cdc25b-WT) or a AKT-nonphosphorylatable Ser351 to Ala CDC25B mutant (Cdc25b-S351A). Cdc25b-S351A strongly inhibited the effect of AKT. Therefore, AKT causes the activation of MPF and strongly promotes the development of one-cell stage mouse fertilized eggs by inducing AKT-dependent phosphorylation of CDC25B, a member of the CDC25 phosphatase family. Our finding that CDC25B acts as a potential target of AKT provides new insight into the effect of AKT in the regulation of early development of mouse embryos.

Early mouse embryo development: could epigenetics influence cell fate determination?

It is generally assumed that the developmental program of embryogenesis relies on epigenetic mechanisms. However, a mechanistic link between epigenetic marks and cell fate decisions had not been established so far. In a recent article, Torres-Padilla and colleagues show that epigenetic information and, more precisely, histone arginine methylation mediated by CARM1 could contribute to cell fate decisions in the mouse 4-cell-stage embryo. It provides the first indications that global epigenetic information influences allocation of pluripotent cells toward the first cell lineages.

Histone Phosphorylation and Pericentromeric Histone Modifications in Oocyte Meiosis

Epigenetic regulation of pericentromeric heterochromatin is crucial for proper interactions between kinetochores and spindle microtubules governing accurate chromosome segregation. Here, we first examined the dynamic distribution of phosphorylated serine 10 and 28 on H3 during mouse oocyte maturation and early embryo development using immunofluorescent staining and confocal microscopy. Our results revealed strong signals of phosphorylated H3/ser10 and 28 in the pericentromeric heterochromatin area and continuous persistent staining of the chromosome periphery, respectively. A panel of specific antibodies against various acetylated lysine, dimethylated lysine or phosphorylated serine residues on histone H3 or H4 were used to investigate the effects of Trichostatin A (TSA), a general inhibitor of histone deacetylases (HDACs), on histone modifications of pericentromeric heterochromatin. Unexpectedly, TSA treatment was unable to alter the acetylation and methylation status of pericentromeric heterochromatin, however, it resulted in significant dephosphorylation of H3/ser10 at this site during mouse oocyte meiosis, which is likely to play a role in the TSA-induced defective chromosome segregation. Furthermore, by using ZM447439, an inhibitor of Aurora kinases, we revealed that Aurora kinases may participate in the regulation of histone phosphorylation during mouse oocyte maturation.

The Effect of Vero Cell Coculture on the Development of Mouse Embryos Exposed to Monoclonal Antibodies Specific for Mammalian Heat Shock Protein 60

Heat shock proteins (HSP) have been identified as an important factor of a very complex and highly conserved cellular defense mechanism to preserve cell survival under adverse environmental conditions. HSP 60 are immunodominant antigens of microbe such as Chlamydia trachomatis and have a potentiality to become a target antigen due to antigenic similarity between chlamydial and human HSP. This study was conducted to investigate the effects of Vero cell coculture to anti-HSP 60 on the early mouse embryo development in vitro. The 2-cell mouse embryos (ICR) were cultured and mouse embryo development was observed every 24 hr for 3 days. 45% and 22.1% of the embryos cultured in Ham's F-10 plus anti HSP 60 with Vero cells developed to the 4- to 8-cell stage (day 1) and morular stage (day 2) as compared with 29.2% and 2.7% of those cultured without Vero cells respectively. But at day 3, the beneficial effect of Vero cells was not noted. These findings suggest that Vero cells have some roles to overcome the detrimental effect of anti-HSP 60 to some degree. These results suggest that Vero cells coculture will promote reproductive outcome in patient previously sensitized to microbial (e.g. Chlamydia trachomatis) HSP 60.

Huntingtin is localized in the nucleus during preimplanatation embryo development in mice

Huntington's disease (HD) is a dominant neurodegenerative disorder caused by the expansion of a CAG repeat in the gene encoding huntingtin. Moreover, the nuclear targeting of mutant huntingtin increases cellular toxicity, whereas normal huntingtin resides mainly in the cytoplasm, and is associated with membranes or microtubules. Huntingtin is enriched in neurons and its expression is increased during neural development. The inactivation of the HD gene results in embryonic lethality before nervous system development. Thus, huntingtin is critical during early embryonic development. Nevertheless, the function of huntingtin at this stage is unknown, even the distribution of the protein has not been described.The present study was undertaken to elucidate the distribution of huntingtin during the early developmental period in the mouse embryo. At the preimplantation stage, huntingtin was detected in nuclei up to 2.5 days post coitum (dpc), but disappeared from nuclei during the blastocyst stage (3.5dpc). Following this stage, huntingtin was mainly localized in the cytoplasm and co-localized with mitotic spindles. These data suggest that the nuclear targeting of normal huntingtin is required during early embryo development in mice.