The proline-arginine repeat protein linked to C9-ALS/FTD causes neuronal toxicity by inhibiting the DEAD-box RNA helicase-mediated ribosome biogenesis.

A GGGGCC repeat expansion in the C9ORF72 gene has been identified as the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeat expansion undergoes unconventional translation to produce dipeptide repeat (DPR) proteins. Although it has been reported that DPR proteins cause neurotoxicity, the underlying mechanism has not been fully elucidated. In this study, we have first confirmed that proline-arginine repeat protein (poly-PR) reduces levels of ribosomal RNA and causes neurotoxicity and found that the poly-PR-induced neurotoxicity is repressed by the acceleration of ribosomal RNA synthesis. These results suggest that the poly-PR-induced inhibition of ribosome biogenesis contributes to the poly-PR-induced neurotoxicity. We have further identified DEAD-box RNA helicases as poly-PR-binding proteins, the functions of which are inhibited by poly-PR. The enforced reduction in the expression of DEAD-box RNA helicases causes impairment of ribosome biogenesis and neuronal cell death. These results together suggest that poly-PR causes neurotoxicity by inhibiting the DEAD-box RNA helicase-mediated ribosome biogenesis.

Components of RNA granules affect their localization and dynamics in neuronal dendrites.

In neurons, RNA transport is important for local protein synthesis. mRNAs are transported along dendrites as large RNA granules. The localization and dynamics of Puralpha and Staufen1 (Stau1), major components of RNA transport granules, were investigated in cultured hippocampal neurons. Puralpha-positive granules were localized in both the shafts and spines of dendrites. In contrast, Stau1-positive granules tended to be localized mainly in dendritic shafts. More than 90% of Puralpha-positive granules were positive for Stau1 in immature dendrites, while only half were positive in mature dendrites. Stau1-negative Puralpha granules tended to be stationary with fewer anterograde and retrograde movements than Stau1-positive Puralpha granules. After metabotropic glutamate receptor 5 activation, Stau1-positive granules remained in the dendritic shafts, while Puralpha granules translocated from the shaft to the spine. The translocation of Puralpha granules was dependent on myosin Va, an actin-based molecular motor protein. Collectively our findings suggest the possibility that the loss of Stau1 in Puralpha-positive RNA granules might promote their activity-dependent translocation into dendritic spines, which could underlie the regulation of protein synthesis in synapses.

Up-regulation of HP1γ expression during neuronal maturation promotes axonal and dendritic development in mouse embryonic neocortex.

Immature neurons undergo morphological and physiological changes including axonal and dendritic development to establish neuronal networks. As the transcriptional status changes at a large number of genes during neuronal maturation, global changes in chromatin modifiers may take place in this process. We now show that the amount of heterochromatin protein 1γ (HP1γ) increases during neuronal maturation in the mouse neocortex. Knockdown of HP1γ suppressed axonal and dendritic development in mouse embryonic neocortical neurons in culture, and either knockdown or knockout of HP1γ impaired the projection of callosal axons of superficial layer neurons to the contralateral hemisphere in the developing neocortex. Conversely, forced expression of HP1γ facilitated axonal and dendritic development, suggesting that the increase of HP1γ is a rate limiting step in neuronal maturation. These results together show an important role for HP1γ in promoting axonal and dendritic development in maturing neurons.

Littermate influence on infant growth in mice: comparison of SJL/J and ICR as cotransferred carrier embryos.

In mice, a minimum number of healthy embryos is required to trigger and maintain pregnancy. Therefore, when recovering mouse embryos from a limited litter, one useful technique is to transfer carrier ICR embryos along with the embryos of interest, a technique referred to as cotransfer. In this study, we examined suitable mouse strains for cotransfer with C57BL/6J (B6) embryos in regards to the maintenance of pregnancy, number of pups born, intrauterine growth, and postnatal growth. Because the coat color of B6 is black, we compared two white coat-colored strains, SJL/J and ICR. Cotransfer of SJL/J and ICR embryos had similar effects on maintenance of pregnancy, number of pups born, and intrauterine growth. However, the postnatal growth of B6 mouse pups cotransferred and grown with SJL/J pups was better than for B6 mouse pups cotransferred and grown with ICR pups, suggesting competition among littermates. These results demonstrate that cotransfer of SJL/J embryos will be useful not only as carrier embryos with B6-background embryos but also as a model system to examine littermate competition.

Arrhythmogenic calmodulin mutations disrupt intracellular cardiomyocyte Ca2+ regulation by distinct mechanisms.

BACKGROUND: Calmodulin (CaM) mutations have been identified recently in subjects with congenital long QT syndrome (LQTS) or catecholaminergic polymorphic ventricular tachycardia (CPVT), but the mechanisms responsible for these divergent arrhythmia-susceptibility syndromes in this context are unknown. We tested the hypothesis that LQTS-associated CaM mutants disrupt Ca2+ homeostasis in developing cardiomyocytes possibly by affecting either late Na current or Ca2+-dependent inactivation of L-type Ca2+ current. METHODS AND RESULTS: We coexpressed CaM mutants with the human cardiac Na channel (NaV1.5) in tsA201 cells, and we used mammalian fetal ventricular cardiomyocytes to investigate LQTS- and CPVT-associated CaM mutations (LQTS- and CPVT-CaM). LQTS-CaM mutants do not consistently affect L-type Na current in heterologous cells or native cardiomyocytes, suggesting that the Na channel does not contribute to LQTS pathogenesis in the context of CaM mutations. LQTS-CaM mutants (D96V, D130G, F142L) impaired Ca2+-dependent inactivation, whereas the CPVT-CaM mutant N54I had no effect on Ca2+-dependent inactivation. LQTS-CaM mutants led to loss of Ca2+-transient entrainment with the rank order from greatest to least effect: CaM-D130G~CaM-D96V>>CaM-F142L. This rank order follows measured Ca2+-CaM affinities for wild-type and mutant CaM. Acute isoproterenol restored entrainment for CaM-130G and CaM-D96V but caused irreversible cytosolic Ca2+ overload for cells expressing a CPVT-CaM mutant. CONCLUSIONS: CaM mutations associated with LQTS may not affect L-type Na+ current but may evoke defective Ca2+-dependent inactivation of L-type Ca2+ current.

Gene expression and development of mouse zygotes following droplet vitrification.

The concept of ultra-rapid vitrification has emerged in recent years; the accelerated cooling rate reduced injury attributed to cryopreservation and improved post-freezing developmental competence of vitrified oocytes and embryos. The objectives of the present study were to develop a simple and effective ultra-rapid vitrification method (droplet vitrification) and evaluate its effects on post-thaw development and apoptosis-related gene expression in mouse zygotes. Presumptive zygotes were equilibrated for 3 min in equilibration medium and washed 3 times in vitrification solution. A drop (5 microL) of vitrification solution containing 10-12 embryos was placed directly onto surface of liquid nitrogen, with additional liquid nitrogen poured over the drop. For thawing and cryoprotectant removal, vitrified drops were put into dilution medium for 3 min, followed by M2 medium for 5 min. Although cleavage rate did not differ significantly among the control (90.8+/-2.8%; mean+/-S.E.M.), toxicity control (83.5+/-3.2%), and vitrified (86.2+/-3.1%) zygotes, rates of blastocyst and hatched blastocyst formation were lower (P<0.01) in vitrified zygotes (49.7+/-4.7% and 36.0+/-4.7%) and toxicity controls (47.3+/-4.6% and 40.3+/-4.6%) compared with controls (65.5+/-4.1% and 54.2+/-4.3%). Exposure of zygotes to vitrification solution, as well as the vitrification process, down-regulated the expression of Bax, Bcl2, and p53 genes in blastocysts. Although droplet vitrification was efficient and easy, it altered the transcriptional activities of Bax, Bcl2, and p53 genes in vitrified embryos, indicating a strong relationship between reduced developmental competence and the altered transcriptional activities of these genes.

Chronological appearance of spontaneous and induced apoptosis during preimplantation development of rabbit and mouse embryos.

This study was undertaken to obtain specific information on the characteristics of spontaneous and induced apoptosis during preimplantation development of rabbit in vivo and in vitro developed embryos and mouse in vitro embryos. After reaching appropriate developmental stages, embryos were transferred into culture media with or without apoptotic inductor (actinomycin D 500 ng/mL) and cultured for 10 h. The identification of apoptotic cells was based on morphological assessment of nuclei and on detection of specific DNA degradation, phosphatidylserine redistribution and active caspase-3 under fluorescence microscope. Our experiments proved that apoptosis is a frequent physiological event occurring during normal preimplantation development. A high number of untreated rabbit and mouse blastocysts contained at least one apoptotic cell. Rabbit embryos showed a lower incidence of spontaneous apoptosis. Treated blastocysts of both species responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and significant increase in the incidence of apoptotic cell death. The occurrence of spontaneous apoptosis during earlier preimplantation development was sporadic and its presence was observed only at stages following embryonic genome activation (at 4-cell stage and later in mouse, at 16-cell and morula stage in rabbit). The susceptibility of embryos at early stages to the apoptotic inductor was much lower. The presence of actinomycin D did not increase the incidence of apoptotic embryos or apoptotic cells. Nevertheless, it slowed down embryo growth and triggered earlier appearance of some apoptotic features (at the 6-cell stage in rabbit). The results show that the occurrence of both spontaneous and induced apoptosis in preimplantation embryos is stage- and species-specific.

Mouse tooth development time sequence determination for the ICR/Jcl strain.

To establish the normal dental development pattern of the ICR/Jcl strain of mouse, we analyzed a significant number of observations of the different developmental stages of the first mandibular molar, accurately recording the chronology of their daily embryonic development. Proliferation of the dental sheet began at day 12.5 in utero (E-12.5), the bud stage appeared at days E-13.5 and E-14.5, the cap stage was observed at days E-14.5, E-15.5 and E-16.5 and the early bell stage at day E-17.5. The presence of predentin was observed at day E-18.5 and dentin was observed 1 and 2 days after birth (D-1 and D-2). The late bell stage with presence of enamel was detected more than 3 days after birth. Embryonic and dental development in the ICR/Jcl strain of mouse is faster than in other well-known strains. The establishment of this developmental pattern will be useful for future investigations of transgenic mice.

Opposing regulation of cell proliferation by retinoic acid and the serotonin2B receptor in the mouse frontonasal mass.

Development of the frontonasal mass (FNM), branchial arches, heart, and limbs depends on neural crest-mediated epithelial-mesenchymal (E-M) interactions. Teratogenesis by retinoic acid (RA) or blockade of serotonergic (5-HT) signaling by the pan-5-HT(2) receptor antagonist, ritanserin, perturbs development of these embryonic structures. In both cases, resulting phenotypes include forebrain and olfactory placode anomalies, malformations of the face, eye and lens, as well as posterior neural tube and cardiac defects. Similar sites of malformations, together with the presence of RA response elements in the 5-HT(2B) receptor promoter, have led to the suggestion that a negative regulatory relationship may exist between RA and 5-HT(2)-mediated 5-HT signaling at sites of E-M interaction (Choi et al. 1997); however, another possibility is that RA and 5-HT act independently as opposing signals to regulate development of common embryonic targets. Together with recent evidence for opposite effects on chondrogenic differentiation in hindlimb micromass cultures (Bhasin et al. 2003a), results of the present study raise the possibility that these pathways may act as opposing signals for common targets in the mouse embryo. The RA receptors, co-factors and metabolic enzymes, and 5-HT(2B) receptors were found to be are coordinately expressed at sites of E-M interaction, including the FNM, in the embryonic day (E)10.5 mouse. Cell proliferation experiments using [(3)H]thymidine incorporation demonstrated that RA or activation of 5-HT(2B) receptors caused opposite effects in FNM explants, namely stimulation or inhibition of cell proliferation, respectively, 5-HT(2B) receptor activation did not appreciably alter patterning in FNM explants. While RA has been shown to regulate lateral patterning in the FNM (LaMantia et al. 2000), 5-HT(2B) receptor activation did not alter patterning in FNM explants. Quantification of 5-HT(2B) receptor transcripts by real-time PCR provided no evidence of negative regulation of 5-HT(2B) receptor expression by RA in FNM explants, although preliminary studies using in situ hybridization had suggested that this was a possibility in both explants and RA teratogenized embryos. Future studies using quantitative PCR may still show this to be the case in teratogenized embryos. Together with the finding of coordinate expression of 5-HT(2B )receptors and RA signaling molecules, results of the present study suggest that RA, and 5-HT mediated by 5-HT(2B )receptors, may act as opposing signals to regulate cell proliferation during craniofacial development in the mouse embryo.

Myosin heavy chain isoforms of the murine masseter muscle during pre- and post-natal development.

Masticatory muscles that are derived from the branchial arches express different compositions of myosin heavy chain (MHC) isoforms during the transitional phase from suckling to mastication. To clarify the developmental changes of murine masseter muscle, the composition of MHC isoforms was examined using immunohistochemical staining and competitive reverse transcription PCR. We found that MHC1 was expressed transiently in the pre and post-natal stages. In the compositional change of isoforms, the embryonic type MHCp was expressed consistently, whereas the adult isoforms increased with the developmental process. In particular, a significant change was observed between embryonic days 14 and 16, a stage when murine facial development is conspicuous. This suggests that the development of murine masseter muscle is closely associated with facial development.

Development of pharyngeal arch arteries in early mouse embryo.

The formation and transformation of the pharyngeal arch arteries in the mouse embryo, from 8.5 to 13 days of gestation (DG), was observed using scanning electron microscopy of vascular casts and graphic reconstruction of 1-microm serial epoxy-resin sections. Late in 8.5-9DG (12 somites), the paired ventral aortae were connected to the dorsal aortae via a loop anterior to the foregut which we call the 'primitive aortic arch', as in the chick embryo. The primitive aortic arch extended cranio-caudally to be transformed into the primitive internal carotid artery, which in turn gave rise to the primitive maxillary artery and the arteries supplying the brain. The second pharyngeal arch artery (PAA) appeared late in 9-9.5DG (16-17 somites), and the ventral aorta bent dorsolaterally to form the first PAA anterior to the first pharyngeal pouch by early in 9.5-10DG (21-23 somites). The third PAA appeared early in 9.5-10DG (21-23 somites), the fourth late in 9.5-10DG (27-29 somites), and the sixth at 10DG (31-34 somites). By 10.5DG (35-39 somites), the first and second PAAs had been transformed into other arteries, and the third, fourth and sixth PAAs had developed well, though the PAA system still exhibited bilateral symmetry. By 13DG, the right sixth PAA had disappeared, and the remaining PAAs formed an aortic-arch system that was almost of the adult type.

Characteristics of the cell membrane fluidity, actin fibers, and mitochondrial dysfunctions of frozen-thawed two-cell mouse embryos.

Physical and chemical alterations caused by the freezing and thawing and their effects on survivals/developments in vitro were investigated. Of a total of 452 two-cell mouse embryos, the overall survival rate of the frozen-thawed embryos was 76.1% (344/452). The blastocyst formation of the frozen-thawed embryos was 32.6% (44/136) compared to 74.5% (117/157) in the fresh embryos (P<0.05). The total number of cells in a blastocyst also decreased from 96.0 +/- 19.0 (n=26) in the fresh embryos to 42.0 +/- 11 .34 (n=30) in the frozen-thawed embryos (P<0.05). Fluorescence recovery after photobleaching (FRAP) measurement revealed about 5-fold decrease in the cell membrane fluidity with a characteristic time constant (tau) of 1.46 +/- 0.13 sec (n=5) in the frozen-thawed embryos as opposed to 0.28 +/- 0.04 sec (n=5) in the fresh embryos (P<0.05). The relative amount of H(2)O(2) in an embryo as quantified by the fluorescence intensity of 2',7'-dichlorofluorescein (DCF) showed 62.8 +/- 23.5 (n=24) and 34.2 +/- 14.5 (n=20) in the frozen-thawed embryos and in the fresh embryos, respectively (P<0.05). The distribution of actin filaments in the frozen-thawed embryos revealed an uneven distribution, particularly discontinuities at the "actin band," which contrasted to an even distribution shown in the fresh embryos. Mitochondrial staining by Rhodamine 123 showed that there was no significant difference between the two treatments in the number and in the distribution of viable mitochondria, but a marked aggregation was seen in the arrested embryos. No Annexin V binding was detected in two-cell or four-cell embryos while the binding was positive in the arrested embryos. The mitochondrial membrane potential measured by a membrane potential-sensitive fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol- carbocyanine iodide (JC-1) revealed a marked depolarization in the frozen-thawed embryos. Finally, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) was employed to quantify the DNA fragmentation. In 75.0% cells of blastocysts (n=24) in the frozen-thawed embryos, the DNA fragmentation was detected as opposed to 37.0% in the fresh embryos (n=20) (P<0.05). Taken together, it is proposed that during the cryopreservation, two-cell mouse embryos are subjected to physical and chemical alterations, including destruction of the cell membrane integrity, redistribution of actin fibers, mitochondrial depolarizations, and increased reactive oxygen species (ROS) productions, which then may trigger the apoptotic cascade leading to a decrease in the survival rate and in the developmental rate of the embryos.

Developmental ability of mouse late 2-cell stage blastomeres fused to chemically enucleated oocytes in vitro.

The effects of different concentrations of etoposide and cycloheximide (ETO-CHXM), used for chemical enucleation of mouse oocytes, on polar body extrusion and chromatin expulsion were tested. The developmental ability of blastomeres of late 2-cell stage embryos fused to chemically enucleated oocytes of different ages or cytoplasts from different sources was also examined in vitro. Metaphase I oocytes cultured in different concentrations of ETO-CHXM (10-50 micrograms/ml/each) extruded polar bodies at rates similar to those cultured without ETO-CHXM (58.5-65.9% and 64.6%, respectively). However, low percent of the oocytes (1.7-6.2%) expressed signs of meiotic perturbation, which was manifested by blebbing of the cytoplasmic membrane and extrusion of two or more polar body-like fragments. Twenty-three percent of the chemically enucleated oocytes cultured in ETO-CHXM-free medium spontaneously fused to their polar bodies. The rates of total chromatin expulsion were similar when ETO-CHXM concentrations were 36 and 50 micrograms/ml (93.5 and 98%, respectively). The results also showed that the cleavage rates of reconstituted embryos were significantly (P < 0.001) affected by the age of the chemically enucleated oocytes. Cytoplasts of bisected oocytes that matured in vivo supported the development of 31.7% of the reconstituted embryos to the blastocyst stage. However, both cytoplasts of chemically enucleated oocytes and in vitro matured oocytes did not support the development to the blastocyst stage. A high percentage (85.5%) of the reconstituted embryos with chemically enucleated recipients displayed abnormality of the metaphase plate. These results suggest that concentrations of etoposide between 36 and 50 micrograms/ml are optimum for enucleation of mouse oocytes. Furthermore, increasing the age or reducing the cytoplasmic volume of the chemically enucleated oocytes did not improve the development of the reconstituted embryos to the blastocyst stage.

Mouse embryonic cardiac metabolism under euglycemic and hypoglycemic conditions.

Children of mothers with insulin-dependent diabetic mothers (IDDM) have a 2-4 times higher incidence of congenital birth defects as compared to the general population, including cardiac abnormalities, of unknown etiology. Using rodent embryos to explore potential teratogenic factors of the altered IDDM metabolism, it has been shown that exposure to hypoglycemia in vitro results in a variety of defects, including cardiac malformations. Since pregnant diabetics experience frequent episodes of low blood glucose, it was hypothesized that hypoglycemia may play a role in the generation of heart abnormalities seen in children born to IDDM mothers. Several studies have indicated that during embryogenesis the heart is dependent on glucose for energy production such that under hypoglycemic conditions, insufficient amounts of ATP may be produced resulting in abnormalities. To test this hypothesis, cardiac ATP content was monitored in D10-D12 (plug day = D1) hearts. In addition, the contribution of glycolysis and the Krebs cycle to ATP production was monitored. D10 hearts exposed to euglycemic control conditions were found to be primarily dependent on glycolysis for ATP production from glucose before switching to the Krebs cycle and oxidative phosphorylation for energy production from this substrate on D11. Exposure to hypoglycemia did not alter the timing of this maturation process or deplete cardiac ATP content. However, cardiac lactate levels increased approximately twofold in the presence of hypoglycemia on d10. Since increased concentrations of lactate are harmful to many tissues and have been shown to be detrimental to the adult rat heart, lactic acidosis may explain the origin of cardiac defects produced by hypoglycemia, and not a deficiency of ATP.

Developing allantois is a primary site of 2'-deoxycoformycin toxicity.

In this study, an assessment of normal mouse allantoic development and its sensitivity to 2'-(R)-deoxycoformycin (dCF; Pentostatin) exposure were examined. Both dissecting microscopy and scanning electron microscopy were used to describe the normal growth and morphogenesis of the mouse allantois over gestational days 7-10 as a preliminary step in evaluating potential abnormal allantoic ontogeny and its effect on umbilical cord and placental development. Two abnormal allantoic/umbilical cord phenotypes were observed subsequent to injecting pregnant mice with 5 mg dCF/kg, i.p., on gestational day 7 (GD 7) and evaluating litters on GD 10, 11, and 12. Abnormal phenotypes included: (1) an allantois which extended approximately halfway across the exocoelom but failed to establish a functional contact with the chorion; and (2) a phenotype characterized by reduced expansion of the allantois across the chorionic surface, a very thin umbilical cord, and aberrant vascularization throughout the structure. Both abnormal phenotypes exhibited either an agenesis or hypogenesis of the umbilical cord and chorioallantoic plate, respectively. Neither abnormal phenotype, however, exhibited errors in the directionality of allantoic growth toward the chorion nor in the formation of aberrant contacts between allantois and adjacent yolk sac or amnionic mesenchyme. Statistical interpretation of the experimental data strongly suggested that abnormalities in allantoic/umbilical cord development were directly associated with embryolethality as evidenced by a decline in the frequency of abnormal allantoic/umbilical cord phenotypes over GD 10-12 (73, 36, and 4%; respectively) and a concomitant increase in the frequency of implantation site resorptions over the same time period (7, 47, and 78%). These results strongly suggest that the developing allantois is very sensitive to the effects of dCF exposure, and that interference with its development leads to embryolethality by GD 12.

Localization of bone morphogenetic protein-4 messenger RNA in developing mouse cochlea.

Bone morphogenetic protein-4 (BMP-4) is a cytokine that belongs to the TGF-beta superfamily. It has been implicated that it plays an important role during embryogenesis including epithelial-mesenchymal interactions and mesenchymal cells condensation. To investigate whether BMP-4 is involved in the embryogenesis of the inner ear, we performed in situ hybridization at various stages of the developing inner ear of mice. BMP-4 mRNA was detected only in the developmental stage. Its initial expression was detected in part of the otic vesicle at 9 days post-coitum (PC). As development proceeded, the hair cells of the cochlear duct were morphologically distinguishable. and BMP-4 expressing cells were found in Claudius cell region at 16 days PC. At this stage, the signal was most intense during development and the structure notably changed under the expressing cells. Mesenchymal cells were assembled and condensed underneath the BMP-4-expressing cells. The basilar membrane, with a rich extracellular matrix and elasticity, would be produced in the area between the Claudius' cells and condensed mesenchymal cells. Previous reports support the notion that BMP-4 expressed in the epithelium causes mesenchymal cell condensation and induces a structure with elasticity. In the developing cochlea. BMP-4 may be involved in the condensation and differentiation of the mesenchymal cells as well as basilar membrane formation. BMP-4 might be an essential factor for the normal development of the basilar membrane.

Magnetic resonance imaging of the internal structure of the mouse fetus.

Magnetic resonance imaging (MRI) has potential as an imaging technique in fetal anatomy. In this article are presented images obtained from mouse fetuses at 16 days of pregnancy by means of the ultraconductive MRI system (JEOL AIM270). When fetuses removed from the uterus were fixed with 10% neutral formalin, various organs including heart, liver, lungs and bones were clearly seen, and a clear outline was obtained of the fetal skin and subcutaneous tissue by varying the setting. The same specimens were able to be used for histological evaluation. This MRI system will allow wider application in fetal anatomy and provide additional information about the developing mouse fetus.

Expression of the recombinase-activating gene (RAG-1) in murine early embryogenesis.

The recombinase activation genes, RAG-1 and RAG-2, are expressed together in immature T or B lymphocytes and possess activity to induce V(D)J rearrangement in T cell receptor (TCR) and Ig genes. In vertebrates, only Ig and TCR molecules are reported to have recombination in their development using multiple V, D, J component gene segments. Thus, expression of RAG genes are localized only in lymphoid organs and sites of extrathymic T cell differentiation. In this study, we have used RAG-1 and RAG-2 genes as markers of possible genetic recombination in developing murine preimplantation embryos, using the highly sensitive reverse transcriptase polymerase chain reaction (RT-PCR) technique and in situ hybridization. From 40 preimplantation embryos of various developmental stages we extracted RNA, reverse-transcribed it into cDNA and used it in RT-PCR studies. A PCR of 35 cycles disclosed expression of RAG-1 but not RAG-2 in morulae and blastocysts. Southern blot hybridization using a specific synthetic oligonucleotide probe for RAG-1 and RT-PCR with another primer pair identified RAG-1 expression in developing embryos. In situ hybridization using a cooled CCD camera also revealed localization of RAG-1 mRNA in blastocysts. We propose possible genetic recombination during late preimplantation murine embryogenesis which may contribute to the loss of totipotency and differentiation of inner cell mass and trophoectoderm.

Study by scanning electron microscopy of the morphogenesis of three types of lingual papilla in the mouse.

Tongues were removed from fetuses of mice on the 15th day of gestation (E15), from newborns (P0), from juveniles on the 7th day (P7) and on the 14th day (P14) after birth for examination by scanning electron microscopy. In the fetuses at E15, rudiments of fungiform papillae with a relatively regular, lattice-like pattern were visible on the anterior half of the dorsal surface of the tongue. The outline of the rudiment of a circumvallate papilla could be recognized on the median line between the lingual body and the lingual radix. No rudiments of filiform papillae could be seen. At P0, rudiments of filiform papillae were compactly distributed over the dorsal surface, as are the filiform papillae in the adult, their width was approximately one-third of that of fungiform papillae, and their tips were rounded than those of the filiform papillae in the adult. The fungiform papillae then increased in size and became somewhat irregular in shape. In juveniles at P7, the filiform papillae were long and slender, being relatively large on the intermolar eminence. A taste pore was visible in the center of each fungiform papilla at this stage. The shape of the circumvallate papilla was similar to that in the adult. In juveniles at P14, the shapes of all three types of papilla were almost the same as those in the adult. The rudiments of each of the three kinds of lingual papilla appeared at a different stage of development of mice; rudiments of the fungiform and circumvallate papillae, which are related to the sense of taste, were formed earlier than those of the filiform papillae, which are not involved in taste.

Electrofusion of zona-free mouse embryonic cells in electrolytes and their development in vitro.

The influence of increasing the physical electrofusion parameters, direct current (DC) pulse strength, pulse duration, pulse number, alternating current (AC) voltage and alignment time, in electrolytes on the rates of fusion, degeneration and development of zona-free mouse 2-cell embryos were examined. Furthermore, the effects of physiological saline and mannitol as fusion media and various mouse strains were also evaluated. Dulbecco's phosphate-buffered saline (PBS) supplemented with 10% fetal calf serum was used as the main fusion solution. A significant increase in the rate of fusion (P < 0.05) was obtained by increasing pulse strength from 30 to 300 V/mm. The embryos fused at the pulse strengths of 30 to 70 V/mm had significantly higher development rates to blastocysts compared with those fused at 100 to 300 V/mm (P < 0.05). There were no significant differences in the rates of fusion, degeneration and development to blastocysts when the pulse duration was increased from 30 to 90 microseconds. Although fusion rates were increased (P < 0.05) by increasing the pulse number up to 4, a significant decrease (P < 0.05) in development to blastocysts was observed when the pulse number was 5. Application of AC voltage prior to the DC pulse tended to increase the fusion rate (89.2-93.8%), compared with fusion with the DC pulse only (75.0%). Prolongation of alignment time from 5 to 15 sec had no effect on the fusion rate. Under the optimum conditions (2 pulses of DC of 70 V/mm, 70 microseconds pulse duration and AC of 5 V/mm for 5 sec), no significant difference was obtained in the fusion and development rates in different mouse strains, nor were fusion and development rates significantly different among PBS, physiological saline and mannitol solutions (P > 0.05).