Selective ablation of differentiated cells permits isolation of embryonic stem cell lines from murine embryos with a non-permissive genetic background.

Embryonic stem (ES) cells enable the engineering of precise modifications to the mouse genome by gene targeting. Although there are reports of cultured cell contributions to chimaeras in golden hamster, rat and pig, definitive ES cell lines which contribute to the germline have not been demonstrated in any species but mouse. Among mouse strains, genetic background strongly affects the efficiency of ES isolation, and almost all ES lines in use are derived from strain 129 (refs 1,4,5) or, less commonly, C57BL/6 (refs 6-8). The CBA strain is refractory to ES isolation and there are no published reports of CBA-derived ES lines. Hence, CBA mice may provide a convenient model of ES isolation in other species. In ES derivation it is critical that the primary explant be cultured for a sufficient time to allow multiplication of ES cell progenitors, yet without allowing extensive differentiation. Thus, differences in ES derivation between mouse strains may reflect differences in the control of ES progenitor cells by other lineages within the embryo. Here we describe a strategy to continuously remove differentiated cells by drug selection, which generates germline competent ES lines from genotypes that are non-permissive in the absence of selection.

Ontogeny and tissue distribution of alpha-1-antitrypsin of the mouse.

alpha 1-Antitrypsin is the second most abundant proteinase inhibitor in plasma. The fact that it is a globular glycoprotein of relatively small size (Mr 53 500) allows it access to a wide variety of fluids and tissue sites. alpha 1-Antitrypsin has been purified from mouse plasma by affinity chromatography and ion exchange. The purified protein exhibits homogeneity on polyacrylamide electrophoresis, but electrophoretic heterogeneity on crossed immunoelectrophoresis. Mouse and rat alpha 1-antitrypsin show strong crossreactivity and the half-life for mouse alpha 1-antitrypsin is 15.5 h. Fetal levels are 15% of adult and it requires 25--30 days before adult levels are reached in the neonate. Maternal levels remain unchanged throughout pregnancy and at parturition. The inhibitory is presented in a number of body fluids including serum, breast milk, gastrointestinal washing, lung washings and bile. The source of alpha 1-antitrypsin for all of these fluids appears to be the liver.

Development of calretinin immunoreactivity in the mouse inner ear.

Calretinin is a calcium-binding protein of the EF-hand family. It has been previously identified in particular cell types of adult guinea pig, rat, and chinchilla inner ear. Development of calretinin immunoreactivity in the mouse inner ear was investigated from embryonic day 13 (E13) to the adult stage. In the adult mouse vestibule, calretinin immunoreactivity was present in the same structures as described for the rat and guinea pig: the population of afferent fibers forming calyx units and a small number of ganglion neurons. The earliest immunoreactivity was found at E17 in vestibular hair cells (VHCs), then, at E19, in afferent fibers entering the sensory epithelia and in rare ganglion neurons. At postnatal day 4 (P4), a few vestibular nerve fibers and ganglion neurons were reactive. From this stage until P14, immunoreactivity developed in the calyx units and disappeared from VHCs. At P14, immunostaining was adult-like. In the adult mouse cochlea, immunoreactivity was present in the same cell populations as described in the rat: the inner hair cells (IHCs) and most of Corti's ganglion neurons. Calretinin immunoreactivity appeared at E19-P0 in IHCs and ganglion neurons of the basal turn. At P1, outer hair cells (OHCs) of the basal turn were positive. Calretinin immunoreactivity then appeared in IHCs, OHCs, and ganglion neurons of the medial turn, then of the apical turn. At P4, all IHCs and OHCs and most of the ganglion neurons were immunostained. Immunoreactivity gradually disappeared from the OHCs starting at P10 and, at P22, only IHCs and ganglion neurons were positive. The sequences of appearance of calretinin were specific to each cell type of the inner ear and paralleled their respective maturation. Calretinin was transiently expressed in VHCs and OHCs.

Fetal-maternal immune interaction: blocking antibody and survival of the fetus.

In the late 1940s it became clear that the homograft reaction was essentially the result of an immune response. Subsequently, Medawar commented on the apparent paradox of the survival of the mammalian fetus in the face of such a potential (cell-mediated) immune response. In an outbred population the fetal-placental unit will be antigenically different to the mother by virtue of its complement of paternal genes and additionally there may be developmental or stage-specific gene products that are immunogenic. Many mechanisms have been proposed to account for the survival of the fetus in the face of a potential immune attack and, while many of these have been investigated in considerable detail, there has been no clear-cut indication that any one plays a predominant role. Either control of immune rejection of the fetus is exercised by an as yet undiscovered mechanism or, more probably, by a combination of some or all of the mechanisms that have been proposed by many workers over the last three decades. Potential controlling processes, which will be reviewed briefly, include: systemic and local modification of maternal responsiveness; altered expression of MHC antigens on extra-embryonic tissues; the placenta as a barrier; and blocking antibody responses. We discuss some of our recent studies in which we have started to look for potential blocking antibodies in a mouse model system. Cells secreting immunoglobulins M and G, characterized in hemolytic plaque assays, have been mapped to areas close to the midgestation mouse embryo, using an immunocryohistological technique. A scaled-down version of hybridoma technology has been used as an analytical probe of the specificity and isotype of immunoglobulin secreted by cells originating either from close to the embryo/fetus or from the para-aortic lymph nodes (PALN). So far monoclonal (IgG1) antibodies with specificity for embryonic cells have been derived together with some monoclonal immunoglobulins with as yet uncharacterized antibody specificity.

Embryogenesis of the mammalian inner ear. III. Formation of the tectorial membrane of the CBA/CBA mouse in vivo and in vitro.

The development of the tectorial membrane in the basal coil of the cochlea has started already in the 15th gestational day inner ear and has reached a considerable thickness and maturation at birth. The development of the tectorial membrane occurs synchronously in in vivo labyrinths and the in vitro material cultured to an age corresponding to birth. At least during this part of the development the formation of the tectorial membrane is independent of the specific composition of endolymph. In the in vivo material a secretory maximum was reached on the 18th gestational day, whereafter the secretory activity was low, especially after birth. In the in vitro specimens, however, a rather constant secretion of material occurred also post partum, which indicates a lack of control mechanisms during in vitro conditions. A complete maturation of the tectorial membrane did not occur in vitro. When passing the point of time corresponding to birth, in the in vitro inner ear explants the gross structure of the tectorial membrane is only slightly changed. In vivo a mature configuration of the tectorial membrane is observed on the 14th DAB (day after birth).

Regional differences in morphogenesis of the neuroepithelium suggest multiple mechanisms of spinal neurulation in the mouse.

A study of neuroepithelial morphogenesis in the mouse embryo has identified three modes of neural tube formation that occur consecutively as neurulation progresses along the spinal region. The three modes of neurulation differ in the extent to which the neuroepithelium exhibits formation of "hinge points', i.e. localised bending owing to reduction in apical surface area. In Mode 1, bending occurs only in the neuroepithelium overlying the notochord, creating a median hinge point. The neural folds remain straight along both apical and basal surfaces, resulting in a neural tube with a slitshaped lumen. In Mode 2, the neuroepithelium forms paired dorsolateral hinge points, as well as a median hinge point, whereas the remaining portions of the neuroepithelium do not bend. This produces a neural tube with a diamond-shaped lumen. In Mode 3 neurulation, the entire neuroepithelium exhibits bending, so that the cells specific hinge points are not discernible; the resulting neural tube has a circular lumen. The three modes of neurulation are present in all three strains of mice studied: C57BL/6, CBA/Ca and curly tail, a mutant predisposed to neural tube defects. However, curly tail embryos exhibit a delay in transition from Mode 2 to Mode 3, preceding faulty closure of the posterior neuropore. This heterogeneity of neurulation morphogenesis in the mouse embryo indicates that the underlying mechanisms may vary along the body axis. Specifically, we suggest that Mode 1 neurulation is driven largely by forces generated extrinsic to the neuroepithelium, in adjacent tissues, whereas Mode 3 neurulation is dependent primarily on forces generated intrinsic to the neuroepithelium. Down the body axis, there is a gradual decrease in the area of ectoderm involved in neural induction and, as neurulation reaches lower spinal levels, the newly induced neural plate exhibits marked indentation from the time of its first appearance. The transition from primary neurulation (neural folding of Mode 3 type) to secondary neurulation (neural tube formation by cavitation) appears to be a smooth continuation of this trend, with loss of contact between the newly induced neuroepithelium and the outside of the embryo.

Early development of the stato-acoustic and facial ganglia.

The early embryonic development of the stato-acoustic and geniculate ganglia was documented in CBA/CBA mice by light and transmission electron microscopy. The geniculate ganglion was identified at the 12-15 somite stage with its origin from the epibranchial placode as well as the neural crest. The VIII ganglion develops later at the 25-27 somite stage, having its origin both in the otic vesicle and in the neural crest cells.

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.

Development of the spinous process of the second thoracic vertebra of the mouse in the late fetal and early postnatal period.

The bony spinous process of T2 in certain inbred strains of the mouse is variable in size or in some cases absent. The development of this process has been investigated in histological sections of CBA, C57BL and tk/tk mice between birth and 14 days. The spinous process is shown to be modified in shape and size late in development.

Variability of embryonic development among three inbred strains of mice.

We examined the relationships between litter size, embryonic growth, days of gestation, onset and duration of morphological stages and development of the first arch skeleton in three inbred strains of mice--C57BL/6, CBA/J and C3H/He. Detailed embryonic staging was based on craniofacial development between 11 and 18 days of gestation. Considerable intra- and interlitter variation of morphological stages of embryonic development exists in all three inbred strains. The relationship of morphological stages to days of gestation reveals that each stage has a different duration, being shortest at Theiler's stage 18 and longest at stage 21 in all three inbred strains. Embryos of CBA/J mice tend to reach each stage later than do embryos of the other two strains, i.e., morphological development is slowest in CBA/J. The greatest length, a measurement of embryonic growth, increases at a constant rate during gestation in all three strains. In C57BL/6 and CBA/J, more embryos tend to be implanted in the right horn of the uterus than in the left, whereas in C3H/He an even number of embryos tends to be implanted in both horns. Timing of the development of Meckel's cartilage differs between the three inbred strains: both condensation and onset of matrix deposition begin one stage earlier in C57BL/6 than in CBA/J and C3H/He. On the other hand, alkaline phosphatase, one of the earliest markers for bone development, is expressed at the same time in all three inbred strains. Differences in timing of skeletal development between the strains may be attributed in part to the genealogical closeness OF CBA/J and C3H/He mice.

[An electron microscopic study of centriole and centrosome morphogenesis in the early development of the mouse]. / Elektronno-mikroskopicheskoe issledovanie morfogeneza tsentriolei i tsentrosomy v rannem razvitii myshi.

Using ultrathin section electron microscopy, the precise time of the appearance of centrioles and centrosomes and their morphogenesis in early mouse development was followed. It turns out that in murine morulae and early blastocyst stage centrosomes, centrioles or other microtubule organizing centres are absent; the network of microtubules in interphase cells is diffuse. In 4-day blastocyst four nascent centrioles were found in two cells of trophectoderm in addition to one nascent centriole in the only cell of the inner cell mass. The nascent centrioles were not connected with microtubules. In the late blastocyst (more than 4.5-days of development) among the cells of inner cell mass, cells without centrioles were found, with nascent centrioles but not connected with microtubules; sometimes cells with true centrosomes were seen, having double centrioles and microtubule organizing centres associated with them. Thus, the centrosomes appear asynchronously in different cell types of blastocyst at the preimplantation stage. The centrosome development begins from a self-assembly de novo of the nascent centrioles without any association with microtubules.

[The development of mouse embryos in vitro in a protein-free medium depending on the numbers of embryos in a microvolume of the medium]. / Razvitie myshinykh émbrionov in virto v srede bez belka v zavisimosti ot kolichestva zarodyshei v mikroob''eme sredy.

We studied the development of mouse embryos in vitro depending on the number of embryos in a given microvolume of the Ham's F-10 medium without protein or with the addition of serum. The absence of serum from the culture medium did not affect the development of two-cell embryos cultivate in groups of 5-6 (about 90% embryos developed until the stage of blastocyst and over 50% left zona pellucida), but the development of single embryos in the protein-free medium proceeded significantly worse. Single two-cell embryos cultivated in the serum-containing medium developed similar to embryos cultivated in groups. At the same time, no significant differences in development was found between eight-cell embryos cultivated individually or in groups of up to 10 embryos in the Ham's F-10 medium, either in the presence of serum or without it (about 95% embryos developed until the stage of blastocyst and over 70% left zona pellucida). The increase in the number of cultivated embryos over 10 had the adverse effect on development of either two-cell or eight-cell embryos. The attachment of blastocysts to the substrate after leaving zona pellucida and growth of trophectoderm was observed only in the presence of serum. These results suggest that interaction between preimplantation embryos in culture can probably be mediated by factor(s) released by embryos into the medium. Serum appears to contain such factor(s). Sensitivity of embryos to the factor(s) apparently depends on the stage of development.

[The development of diploid parthenogenetic mouse embryos of the inbred C57BL and CBA strains]. / Izuchenie razvitiia diploidnykh partenogeneticheskikh zarodyshei myshei inbrednykh linii C57BL i CBA.

We studied preimplantation development in vitro and postimplantation development in vivo of diploid parthenogenetic mouse embryos of C57BL/6 and CBA strains, as well as of (CBA x C57BL/6)F1 hybrids. Development to blastocyst stage of diploid eggs obtained from C57BL/6, CBA, and hybrid mice was observed in 90, 15, and 73% cases, respectively. After implantation, C57BL/6 embryos did not develop to somite stages, while CBA and hybrid embryos reached various stages of somite formation in 45 and 30% cases, respectively. Cultivation of embryos beginning from one-cell stage in the medium containing 2% newborn calf serum increased the yield of blastocysts from 15 to 59% in CBA embryos and from 73 to 90% in hybrids; However, such effect was not observed with C57BL/6 embryos. The latest stages of development observed in CBA and hybrid diploid parthenogenetic embryos were 33-35 somites and 25-30 somites, respectively. Imprinting patterns in chromosomes of CBA and C57BL/6 gametes are discussed.

[Cytological research on the argentophilic nucleolus-organizer regions of the metaphase chromosomes in parthenogenetic mouse embryos]. / Tsitologicheskoe issledovanie argentofil'nykh iadryshkoobrazuiushchikh raionov metafaznykh khromosom u partenogeneticheskikh zarodyshei myshei.

Silver staining technique visualizing argentophilic nucleolus organizer regions (Ag-NORs) was used for studying parthenogenetic mouse embryos produced by artificial activation of oocytes in Ca(2+)-Mg(2+)-free medium. Ag-NOR-containing chromosomes were detected in metaphases of parthenogenetic embryos during six successive cleavage divisions starting with the two-cell stage. The frequency of metaphases with varying AG-NOR number in diploid parthenogenones was similar to that in the control (fertilized) embryos. Average number of metaphase Ag-NOR chromosomes (calculated per diploid chromosome set) in haploid parthenogenones exceeded that in the control; in some cases all NORs were stained by silver. This is evidence that latent ribosomal cistrons in some chromosomes can be activated.

[Cytological study of the nucleolus organizer regions in the chromosomes of CBA and C57BL mice and their hybrids]. / Tsitologicheskoe issledovanie iadryshkoorganizuiushchikh raionov khromosom u myshei linii CBA, C57BL i ikh gibridov.

Transcriptionally active NORs of chromosomes visualized by AgNO3 staining were studied in bone marrow and embryos (day 10 of gestation) of CBA and C57BL mice, as well as of (CBA x C57BL)F1 hybrids. These mouse strains were shown to differ by the average number of Ag-positive NORs in marrow cells; in hybrids, the number of NORs is greater than in the parent strains. During embryogenesis, the number of chromosomes carrying silver-stained NORs increases; however, no significant differences by this parameter was detected between hybrid and C57BL embryos. The average number of silver-stained NORs was the smallest in embryos of CBA mice.

[Effect of monosomy of the autosomes on the preimplantation stages of embryogenesis in laboratory mice]. / Vliianie monosomii nekotorykh autosom na doimplantatsionnye stadii émbriogeneza laboratornykh myshei.

The effects of monosomy for the autosomes 1, 2, 3, 5, 6, 16 and 19 were studied in mice with single or double Robertsonian translocations. The monosomy for different autosomes affects the preimplantation development of the mouse embryos in different ways. The monosomy for the autosomes 1, 3, 6, 16 or 19 does not affect cleavage, compactization or blastulation and is, in some cases, even compatible with the implantation. The most these embryos are eliminated at the blastocyst stage (monosomy for the autosomes 3, 6 or 19) or, sometimes, at the postimplantation stages (1 or 16). The monosomy for the autosomes 2 or 5 is realized during cleavage causing the developmental delay, pathological changes in the nuclei of blastomeres and elimination at the morula stage. The results obtained suggest differential activity of chromosomes at the preimplantation stages of embryogenesis. Possible reasons for the early death of the embryos with particular types of monosomy are discussed. A hypothesis of mutual activation of the homologous autosomes at the early developmental stages is put forward. According to this hypothesis, the loci of a single unpaired autosome, especially of paternal origin, remain inactive during the early embryogenesis.

[Effect of maternal genotype on the rate of preimplantation development in mice]. / Vliiani genotipa materi na skorost' predimplantasionnogo razvitiia myshi.

The genetic control of the rate of preimplantation development was studied in the mouse embryos. The number of cells in the embryo and the percentage of embryos at the blastocyst stage were determined on the 3.5 day of pregnancy. The experiments were carried out with CBA, A/He, C57Bl/Mib mice and mice homozygous by the mutant genes white (Miwh), fidget (fi) and ocular retardation (or), congenic with the inbred C57Bl/Mib mice. Contrasting differences were found between C57Bl/Mib and fi/fi mice. The rate of development of the morphologically normal C57Bl/Mib and fi/fi and F1 embryo was shown to depend on the maternal genotype, rather than on the paternal one. The effect of maternal genotype of the rate of preimplantation development was related to differences in the time of beginning of the cleavage. The rate of cleavage is similar for the C57Bl/Mib, fi/fi and F1 embryos.

[Immunofluorescence study of H-2 antigens in early mouse embryogenesis]. / Immunofluorestsentnoe issledovanie H-2 antigenov v rannem émbriogeneze myshei.

The time of appearance and degree of expression were studied for antigens of the main locus of histocompatibility of the mouse embryos by indirect immunofluorescence. H-2 antigens appeared on the 5.5 day of embryogenesis on the cells of still undifferentiated rudiments of embryonic endoderm and ectoderm. By the 8th day of embryogenesis, rather intensice fluorescence of the cells of amnion, yolk sac and embryonic ectoderm was observed suggesting a marked expression of H-2 antigens during this period. The cells of trophoblast gave practically no positive reaction with anti-H-2-serum.

[Development and survival of mouse embryos in intra- and interstrain transplants]. / Razvitie i vyzhivaemost' zarodyshei myshei pri vnutri- i mezhlineinykh transplantatsiiakh.

Survival rate and weight of embryos were studied in the Balb/c, CBA//ac and C57BL/6j mice, developed from several combinations of transplantation. The weight of allogenic fetus as well as syngenic fetus which developed together with allogenic ones was detected to increase. This increased weight does not result from survival of certain genotype embryos, but seems to be due to genetical differences in the mother-embryo system.

There are no in vivo pulsations of mouse blastocysts.

An important event at the onset of the implantation process in mammals is the hatching of the blastocyst from the zona pellucida. Microcinematographic studies of in vitro preimplantation development in mice revealed the pulsatile activity of blastocysts before and during the hatching period. It is generally accepted--up to now--that the in vitro hatching is at least partially the result of repeated contractions and reexpansions of the blastocyst. The presence of pulsatile activity in vivo may confirm this hypothesis. Mouse blastocysts were obtained by a rapid flushing (5-10') from uterine horns on day 4 of pregnancy and the presence or absence of contracted blastocysts was noted. From 410 examined blastocysts only 3% were contracted as compared with the very frequent in vitro pulsations. This result suggests that in mice the in vivo pulsatile activity of blastocysts, practically, does not exist. The in vivo contractions are probably determined by the suboptimal culture conditions.