Hazard assessment of air pollutants: The transforming ability of complex pollutant mixtures in the Bhas 42 cell model.

The use of in vitro alternative methods is a promising approach to characterize the hazardous properties of environmental chemical mixtures, including urban airborne particulate matter (PM). The aim of this study was to examine seasonal differences in the toxic and transforming potential of PM samples, by using the in vitro cell transformation assay in Bhas 42 cells for the prediction of potential carcinogenic effects. Bhas 42 cells are already initiated, and the v-Ha-ras transfection, together with genetic modification following the immortalization process, makes them a valuable model to study the late steps of cellular transformation leading to the acquisition of the malignant phenotype. Exposure to organic extracts of PM1 and PM2.5 induced dose-related effects. The transforming and cytotoxic properties are related to the amount of PM collected during the sampling campaign and associated with the concentrations of polycyclic aromatic hydrocarbons (PAHs) in the samples. All the samples induced cell transformation following prolonged exposure of 2 weeks. Our results support the utility of the in vitro top-down approach to characterise the toxicity of real mixtures, thereby supporting regulators in the decision-making process. The results also identify the need for appropriate assay selection within the in vitro testing strategy to address the complexity of the final adverse outcomes.

Expression of delta-like 1 homologue and insulin-like growth factor 2 through epigenetic regulation of the genes during development of mouse molar.

Delta-like 1 homolog (Dlk1) and insulin-like growth factor 2 (Igf2) are two of six well-studied mouse imprinted gene clusters that are paternally expressed. Their expression is also linked to their maternally expressed non-coding RNAs, encoded by Gene trap locus 2 (Gtl2) and Imprinted maternally expressed transcript (H19), co-located as imprinted gene clusters. Using deoxyoligonucleotide microarrays and real-time RT-PCR analysis we showed Dlk1 and Gtl2 to exhibit a time-course of expression during tooth development that was similar to that of Igf2 and H19. Western blot analysis of proteins encoded by Dlk1 and Igf2 suggested that the levels of these proteins reflected those of the corresponding mRNAs. Immunohistochemical studies of DLK1 in murine molars detected the protein in both epithelial and mesenchymal regions, in developing cusp mesenchyme, and in newly synthesized enamel and dentin tubules. IGF2 protein was detected primarily at prenatal stages, suggesting that it may be active before birth. Analysis of methylation of cytosine-phosphate-guanine (CpG) islands in both Dlk1 and Igf2 suggested the presence of an increasing fraction of hypermethylated bases with increasing time of development. The increased levels of hypermethylation coincided both with the diminished levels of expression of Dlk1 and Igf2 and with decreased levels of DLK1 and IGF2 proteins in the tooth germ, suggesting that their expression is regulated via methylation of CpG islands present in these genes.

Albendazole sulphoxide administered prior to mating andits relation with fertilization and mouse embryo development / Albendazol sulfóxido administrado previo al apareamiento y su relación con la fertilización y el desarrollo embrionario de ratón

The aim of this work was to evaluate the effect of albendazole sulphoxide (ABZSO) administered to Balb C mice prior to mating on fertilization rate and preimplantational embryo development. Twenty four female mice 5-8 weeks of age were superovulated by intraperitoneal injection of 7.5 UI of equine chorionic gonadotropin (eCG, Novormon®, Laboratorios Syntex S.A., Argentina); 48 h later they received 10 IU of human chorionic gonadotropin (hCG, Profasi®, Laboratorios Serono, Méjico) and were paired with males of proven fertility. Females received 100 mg/kg or 200 mg/kg of ABZSO orally at the time of hCG administration, prior to mating. The control group received carboxymethylcellulose, vehicle used to prepare the drug suspension. Pregnant females were killed by cervical dislocation at day 4 of pregnancy and non fertilized oocyte and embryos were flushed from uteri. The possible effects of ABZSO were evaluated considering the fertilization rate, the total number of collected embryos per female; the percentage of embryos morphologically normal; the differentiation rate (determined by the relation between the number of blastocyst and the total of morphologically normal embryos) and the cleavage rate determined by counting the nuclei. The variables were analyzed on a per litter basis using the Kruskal-Wallis test. The fertilization rate was lower in females administered ABZSO at a dose of 200 mg/kg (P<0.05). However, no statistically significant differences were found in the embryonic parameters after the administration of 100 mg/kg or 200 mg/kg of ABZSO compared to the untreated control group (P>0.05). In conclusion, a single acute exposure to ABZSO prior to mating at around the time of fertilization at a dose higher than the one usually administered in human and veterinary medicine affects the fertilization rate but it has no adverse effects on early embryo development.

El objetivo de este trabajo fue evaluar el efecto de albendazol sulfóxido (ABZSO) administrado a ratonas Balb C previo al apareamiento, sobre la tasa de fertilización y el desarrollo embrionario preimplantacional. Se utilizaron 24 hembras de 5 a 8 semanas de edad las que fueron inducidas a superovular por inyección intraperitoneal de 7,5 UI de gonadotrofina coriónica equina (eCG, Novormon®, Laboratorios Syntex S.A. Argentina) seguidas, 48 h más tarde por 10 UI de gonadotrofina coriónica humana (hCG, Profasi®, Laboratorios Serono, México). Al momento de recibir la dosis de hCG, fueron apareadas con machos de fertilidad probada. Las hembras fueron dosificadas oralmente con ABZSO disuelto en carboximetilcelulosa en dosis de 100 mg/kg (Grupo 100) y 200 mg/kg (Grupo 200) previo al apareamiento. El grupo control recibió carboximetilcelulosa. Las hembras preñadas fueron sacrificadas por dislocación cervical en el día 4 de preñez y se recolectaron ovocitos sin fertilizar y embriones preimplantacionales mediante el lavado de cuernos uterinos. Se determinó la tasa de fertilización, el número promedio de embriones recolectados por hembra, el porcentaje de embriones morfológicamente normales, el porcentaje de diferenciación y la velocidad de clivaje estimada por recuento de núcleos. Las variables fueron analizadas sobre la base de la camada utilizando el test de Kruskal-Wallis. La tasa de fertilización resultó menor para hembras que recibieron albendazol sulfóxido a razón de 200 mg/kg de peso (P<0,05); no obstante, no se observaron diferencias significativas en los parámetros embrionarios luego de la administración de 100 mg/kg ó 200 mg/kg de ABZSO comparado con el grupo control (P>0,05). En conclusión, la exposición aguda de ABZSO realizada previo al apareamiento a una dosis mayor de aquella utilizada en medicina humana y veterinaria afecta la tasa de fertilización pero no muestra efectos adversos sobre el desarrollo embrionario temprano.

Study of embryotoxicity of mentha piperita L. during organogenesis in balb/c mice / Estudio de la embriotoxicidad de mentha piperita L. durante la organogénesis en ratones balb/c

Mentha piperita (Labiatae), commonly known as peppermint is a native Iranian herb which is used in folk medicine for various purposes. This study was carried out to reveal the teratogenic effect of Mentha piperita on mice fetuses. In this experimental study, pregnant Balb/c mice divided to four groups. Case group received 600 (treatment I) and 1200 (treatment II) mg/kg/day the hydroalcoholic extract of Mentha piperita during 6-15 of gestational days and one control group received normal saline during GD6-GD15 by gavages and other control group did not receive any matter during 6-15 of gestational days. Mice sacrificed at GD18 and embryos were collected. Macroscopic observation was done by stereomicroscope. 20 fetuses of each group were stained by Alizarin red-S and Alcian blue staining method. The Mean weight of fetuses decreased in treatment groups rather than control (P<0.05) but CRL there was no significant difference between treatments and controls groups. In the treatment I (600 mg/kg/day) and treatment II (1200 mg/kg/day), normal saline and control group, no gross congenital malformations were observed in fetuses. Treated fetuses also had no delayed bone ossification as determined by Alizarin red-S and Alcian blue staining method. This study showed that the hydroalcoholic extract of Mentha piperita (600 and 1200 mg/kg/day) has no teratogenic effect in mice fetuses if used continuously during embryonic period.

Mentha piperita (Labiatae), comúnmente conocida como menta, es una hierba nativa de Irán, que se utiliza en la medicina tradicional para diversos fines. Este estudio fue realizado para descubrir el efecto teratogénico de la Mentha piperita en fetos de ratones. Los ratones Balb/c preñadas fueron divididas en cuatro grupos. Los grupos recibieron 600 (tratamiento I) y 1200 (tratamiento II) mg/kg/día del extracto hidroalcohólico de Mentha piperita durante los días 6-15 de gestación (DG), mientras que un grupo control recibió solución salina normal durante los DG 6-15 vía oral y otro grupo control sano no recibió substancia durante los DG 6-15. Los ratones fueron sacrificados el DG 18, recolectando los fetos. Se realizó la observación macroscópica mediante un estereomicroscopio. 20 fetos de cada grupo se tiñeron por el método de rojo de alizarina-S y azul de Alcián. La media de peso de los fetos disminuyó más en los grupos de tratamientos que los controles (p <0,05), pero CRL no presentó diferencias significativas entre los tratamientos y los grupos control. En los fetos del grupos tratamiento I (600 mg/kg/día), tratamiento II (1200 mg/kg/día), solución salina normal y control no se observó ninguna malformación congénita grave. Los fetos tratados tampoco tuvieron osificación ósea retrasada según lo determinado por el método de rojo de alizarina-S y azul de Alcián. Este estudio mostró que el extracto hidroalcohólico de Mentha piperita (600 y 1200 mg/kg/día) no tiene efectos teratogénicos en fetos de ratones al ser utilizado continuamente durante el período embrionario.

Morphological study of the transition of haematopoietic sites in the developing mouse during the peri-natal period.

The transition of fetal haematopoietic sites in mice was examined histologically from 12.5-day embryos (E12.5) until 10 days of age (D10), and the expression of the adhesive molecules VCAM-1 and fibronectin was examined immunohistologically. Erythropoiesis occurred in the liver (E12.5-18.5), spleen (E18.5-D4) and bone marrow (D6-10), in that sequence. Even at D10, some erythropoiesis occurred in the liver, and more so in the spleen, although the active haematopoietic site was the bone marrow. Similarly, granulopoiesis of neutrophils occurred in the liver, spleen and bone marrow in turn. Granulopoiesis still occurred in the spleen at D10, but no neutrophils were found in the liver after D4. VCAM-1 appeared in the liver, spleen and bone marrow in parallel with active erythropoiesis and granulopoiesis. The co-expression of VCAM-1 and fibronectin was recognized in the endothelial cells of the sinus at the onset of haematopoiesis. This study showed that haematopoiesis in the liver, spleen and bone marrow overlapped peri-natally, although it shifted sequentially. VCAM-1 appears to be closely associated with erythropoiesis and granulopoiesis, and the co-expression of VCAM-1 and fibronectin plays a role in inducing haematopoietic stem cells to move to the tissues.

Tra2betal regulates P19 neuronal differentiation and the splicing of FGF-2R and GluR-B minigenes.

The present study demonstrates that the expression of Tra2beta1 (Transformer 2-beta1) proteins, an SR (serine/arginine rich) protein, is developmentally up-regulated in a neural-specific pattern. The up-regulation is also observed in RA (retinoic acid) induced neural differentiation of P19 cells. Tra2betal proteins are located in the nuclei of P19 cells, which are consistent with its functional site as an SR protein. The over-expression of Tra2betal proteins promotes RA induced neuronal differentiation of P19 cells. In P19 cells, the splicing of FGF-2R (fibroblast growth factor receptor 2) minigene produces the BEK form, while the alternative splicing of GluR-B (glutamate receptor subunit B) minigene generates two products, the Flop and the Truncated isoforms. Tra2betal inhibits the BEK splicing, but it promotes the Flop splicing. The results therefore suggest that Tra2betal is involved in the regulation of alternative splicing processes during neural development, peculiarly the splicing of FGF-2R and GluR-B genes. Both FGF-2R and GluR-B genes are known to play important roles in neural differentiation.

Inheritance patterns of maternal alleles in imprinted regions of the mouse genome at different stages of development.

Deviations from Mendelian 1:1 transmission ratio have been observed in mice and humans. With few exceptions, the mechanism leading to transmission-ratio distortion (TRD) remains obscure. We proposed that a genomic imprinting mechanism plays a key role in the genesis of grandparental origin-dependent TRD (Naumova et al. 2001). To further test this hypothesis, we analyzed the transmission of grandparental alleles at three imprinted regions of the mouse genome known to contain genes required for embryo development. We found and replicated moderate (58%: 42%) TRD in favor of grandmaternal alleles in the imprinted region of maternal distal Chromosome (Chr) 12 among female offspring. Comparison of transmission ratios at the distorted region of Chr 12 among 3-week-old mice with those in embryos suggests that the distortion in favor of grandmaternal alleles is owing to postimplantation embryo loss. The absence of grandparental origin-dependent TRD for maternal Chr 6 and 7 implies that the relationship between TRD and imprinting is complex. Most likely, multiple conditions are required for TRD to occur.

Expression of specific glycoconjugates in both primary and secondary olfactory pathways in BALB/C mice.

Binding of cell surface carbohydrates to their receptors specifically promotes axon growth and synaptogenesis in select regions of the developing nervous system. In some cases these interactions depend upon cell-cell adhesion mediated by the same glycoconjugates present on the surface of apposing cells or their processes. We have previously shown that the plant lectin Dolichos biflorus agglutinin (DBA) binds to a subpopulation of mouse primary olfactory neurons whose axons selectively fasciculate prior to terminating in the olfactory bulb. In the present study, we investigated whether these glycoconjugates were also expressed by postsynaptic olfactory neurons specifically within the olfactory pathway. We show here for the first time that DBA ligands were expressed both by a subset of primary olfactory neurons as well as by the postsynaptic mitral/tufted cells in BALB/C mice. These glycoconjugates were first detected on mitral/tufted cell axons during the early postnatal period, at a time when there is considerable synaptogenesis and synaptic remodelling in the primary olfactory cortex. This is one of the few examples of the selective expression of molecules in contiguous axon tracts in the mammalian nervous system. These results suggest that glycoconjugates recognized by DBA may have a specific role in the formation and maintenance of neural connections within a select functional pathway in the brain.

Axonal secretion of Reelin by Cajal-Retzius cells: evidence from comparison of normal and Reln(Orl) mutant mice.

A novel secretory pathway has been identified in the study of mice homozygous for the Reln(Orl) mutation, a line characterised by the defective secretion of the large extracellular matrix glycoprotein Reelin. By using both light and electron microscopy, immunohistochemical studies for Reelin in these mutants identified morphological changes in their Cajal-Retzius cells (CR cells). The CR cells of the mutant displayed the characteristic features of bipolar, tangentially elongated neurons with a dendritic proximal pole and an axonal cone at the opposite end of the soma. At either pole, cisterns of prominent rough endoplasmic reticulum (RER) were found to be rich in Reelin. However, the Reelin-positive RER cisterns of the axonal cones were hugely dilated in homozygous Reln(Orl) mice as compared with their wild type counterparts. CR cell axons displayed beads throughout their length, each contained a smooth spheroidal cistern filled with Reelin-immunoreactive fibrillar material, and were increased in number and size in Reln(Orl) mice. RER phenotype was rescued in the Reln(Alb2) mice, a mutation in which no Reelin protein is produced. We propose that the RER dilations viewed in the Reln(Orl) mutation are due to the accumulation of the defective Reelin protein, and the large axonal beads in Reln(Orl) mice reflect the accumulation of truncated Reelin as the result of defects in its secretion. These observations point to an original, hitherto unrecognised, mechanism of secretion by bulk transport in smooth cisterns from the axonal cone into the axon, followed by secretion in the cortical marginal zone from the axonal cisterns that we have named axonal reelin reservoirs.

BRCA2-null embryonic survival is prolonged on the BALB/c genetic background.

Women who inherit mutations in the BRCA2 cancer susceptibility gene have an 85% chance of developing breast cancer. The function of the BRCA2 gene remains elusive, but there is evidence to support its role in transcriptional transactivation, tumor suppression, and the maintenance of genomic integrity. Individuals with identical BRCA2 mutations display a different distribution of cancers, suggesting that there are low-penetrance genes that can modify disease outcome. We hypothesized that genetic background could influence embryonic survival of a Brca2 mutation in mice. Brca2-null embryos with a 129/SvEv genetic background (129(B2-/-)) died before embryonic day 8. 5. Transfer of this Brca2 mutation onto the BALB/cJ genetic background (BALB/c(B2-/-)) extended survival to embryonic day 10.5. These results indicate that the BALB/c background harbors genetic modifiers that can prolong Brca2-null embryonic survival. The extended survival of BALB/c(B2-/-) embryos enabled us to ask whether transcriptional regulation of the Brca1 and Brca2 genes is interdependent. The interdependence of Brca1 and Brca2 was evaluated by studying Brca2 gene expression in BALB/c(B1-/-) embryos and Brca1 gene expression in BALB/c(B2-/-) embryos. Nonisotopic in situ hybridization demonstrated that Brca2 transcript levels were comparable in BALB/c(B1-/-) embryos and wild-type littermates. Likewise, reverse transcriptase-polymerase chain reactions confirmed Brca1 mRNA expression in embryonic day 8.5 BALB/c(B2-/-) embryos that was comparable to Brca2-heterozygous littermates. Thus, the Brca1 and Brca2 transcripts are expressed independently of one another in Brca1- and Brca2-null embryos. Mol. Carcinog. 28:174-183, 2000.

Regional patterning of reticulospinal and vestibulospinal neurons in the hindbrain of mouse and rat embryos.

The dispositions and axonal trajectories of bulbospinal neurons in the pons and medulla of mouse and rat embryos is described from the earliest times these projections can be labelled retrogradely from the cervical spinal cord. Reticulospinal and vestibulospinal neurons are clustered into identifiable groups, each with a characteristic combination of spatial domain and axon trajectory. The various groups can be labelled retrogradely in a specific developmental sequence. The position of some groups shifts from medial to lateral with development, apparently through cell migration. These observations show that the basic regional organization of the reticulospinal and vestibulospinal projections is similar in mouse and rat and is already established during early stages of axon outgrowth.

Ultrastructural changes of the filtration barrier during mouse nephron development.

The development of mouse renal corpuscles localized in the juxtamedullary zone of renal cortex was assessed with the aid of light and electron microscopy. The observations have confirmed the existence of four developmental phases of the nephron. The analysis of filtration barrier components has shown that during the development of the kidney the height of maturing podocytes becomes markedly reduced, and that adherence and occluding junctions formed between them shift from their apices towards their bases. Ladder-like junctions have also been observed between podocytes. The number of their foot processes steadily grows, they become narrower and filtration slit diaphragms begin appear between them. Fusion of podocytes basement membranes and endotheliocytes leads to formation of a doubly thick filtration membrane. As the tuft of the capillary glomerulus grows, newly formed fragments of the basement membrane in the form of loops and pouches start to be evident in its close neighborhood. Large endotheliocytes become greatly flattened and numerous fenestrae appear in their cytoplasm.

[Changes in the cavitation start time in chimeric CB6F1 <--> BALB/c embryos]. / Izmenenie vremeni zapuska kavitatsii u khimernykh zarodyshei CB6F1 <--> BALB/c.

It is found out that the acceleration of preimplantation morphogenesis is observed in mouse chimeras between single two-cell-stage blastomeres of BALB/c and [BALB/c x C57B1/6] x C57B1/6 (F2) embryos. The paired aggregation of blastomeres of two-cell stage made F2-BALB/c chimeras to start cavitation 8 +/- 2 hours earlier than F2<-->F2 and BALB/c-BALB/c control embryos. We have demonstrated for the first time the 'chimeric' heterosis on preimplantation stages. Possible approaches to the future studies of this phenomenon are discussed.

Light and electron microscopic examination of various doses of given vitamin A on the development of cornea in mice. Partly presented in ICEM 13-Paris.

Vitamin A (0.2 micrograms, 0.6 micrograms, 1.2 micrograms) was administered orally to the mice on days 8-11 of gestation. Fetuses were removed on day 17 of gestation. No external malformation of the fetuses was seen on the stereomicroscope investigation. Corneal degeneration was seen on the light and electron microscopic examination. As a result it was accepted that vitamin A taken during the critical periods of gestation affected the development of cornea.

Identification of Elf-1 and B61 as high affinity ligands for the receptor tyrosine kinase MDK1.

Mouse Developmental Kinase 1 (MDK1) is a receptor tyrosine kinase of the eck/eph subfamily expressed in a variety of tissues during early mouse embryogenesis. To obtain further insight into the function of MDK1, we determined identity and localisation of its physiological ligand(s). Staining whole embryos with fusion proteins between the extracellular domain of MDK1 and human secreted alkaline phosphatase revealed areas of high receptor binding in the caudal mesencephalon, the frontal neocortex and the limb buds. This staining was sensitive to treatment with phosphatidylinositol-specific phospholipase C. Using Scatchard analysis, high affinity binding of Elf-1 (1.7 x 10(-10) M) and B61 (2.2 x 10(-10) M) towards MDK1 could be demonstrated. However, the transmembrane ligand Lerk2 displayed no measurable affinity for MDK1. Elf-1 and B61 bind to the three full-length MDK1 isoforms with similar dissociation constants. Slightly lower affinities were observed for the two truncated receptors MDK1-Tl and MDK1-T2. The activation of MDK1 with Elf-1 or B61 leads to the rapid autophosphorylation of MDK1 as well as tyrosine phosphorylation of an unknown 62 kDa phosphoprotein in Rat1 cells. These findings implicate MDK1 in patterning processes during early mouse embryogenesis and suggest MDK1 involvement in early organogenesis and midbrain development.

["2-cell block" and the viability of BALB/c embryos when explanted at the second cleavage cell cycle]. / "Dvukletochnyi blok" i zhizhnesposobnost' zarodyshei myshei linii BALB/c pri éksplantatsii na vtorom kletochnom tsikle drobleniia.

The embryos of BALB/c mice were explanted from hormonally stimulated females in a standard culture during the second cleavage division corresponding to the stage of the embryonic genome activation. Observations over the cultivated embryos have shown that explantation of the embryos both at the two-cell stage and during the first half of the second cleavage division brings most embryos (more than 80%) to the state of "two-cell block." Thereafter, the number of blocked embryos progressively decreases to less than 20% after explantation in the end of the cleavage division, which corresponds to the proportion of the embryos in the reproductive tract which were delayed at the 2-cell stage by the beginning of the third cleavage division. After explantation at the time close to the middle of the cleavage division, the survival of the blocked embryos is reduced and in the end of the two-cell stage again increases. The BALB/c embryos should stay in the maternal reproductive tract during the entire second cleavage division before explantation in order to provide for viability and developmental potencies in vitro close to those in vivo. The results obtained are discussed with reference to the controlling mechanisms of early embryogenesis and cell cycle.

Lack of transforming activity of fumonisin B1 in BALB/3T3 A31-1-1 mouse embryo cells.

The capacity of fumonisin B1 (FB1) to induce morphological transformation of cultured mammalian cells was assessed using BALB/3T3 A31-1-1 mouse embryo cells. FB1 with 90% purity was prepared from Fusarium proliferatum grown on whole corn. Cell growth was not inhibited by 48 hr of exposure at concentrations up to 1000 micrograms/ml. Moderate inhibition was induced by 6 days of exposure. In transformation assays with a 48-hr exposure, increases in transformed foci were observed at some concentrations; however, the responses were not reproducible. Prolonged exposure for up to 4 wk at 10, 100 and 500 micrograms/ml failed to induce increases in transformed foci. Analysis of combined results showed that only the increase induced by a 48-hr exposure at 500 micrograms/ml was significant. A trend test indicated the lack of a dose response for concentrations of 10-1000 micrograms/ml. FB1 seems to lack in vitro transforming activity.

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).

Developmental pattern of expression of the mouse alpha 1 (XI) collagen gene (Col11a1).

Fibrillar networks are intimately involved in several morphogenetic processes which underlie the harmonious development of the vertebrate embryo. Recent genetic evidence has demonstrated that the minor types V and XI collagen are key regulators of types I and II fibrillogenesis in non-cartilaginous and cartilaginous matrices, respectively. A comprehensive understanding of the expression and regulation of the genes coding for the chains of the minor collagen types is therefore relevant to animal morphogenesis and development. The present study was undertaken to elucidate the embryonic pattern of expression of the gene coding for the mouse alpha 1 chain of type XI colagen (Col11a1) using the technique of in situ hybridization. Transcripts of the Col11a1 gene were detected as early as 11 days of gestation. The alpha 1(XI) transcripts were found to accumulate mostly in cartilaginous tissues, such as the chondrocranium and the developing limbs. Like the major cartilage-specific collagen (type II), Col11a1 expression was also noted in the neuro-epithelium of the brain. However, alpha 1(XI) transcripts accumulated in several other non-cartilaginous sites. They include odontoblasts, trabecular bones, atrioventricular valve of the heart, the tongue, the intestine, and the otic vesicle. Altogether, the data confirm that Col11a1 has a broader spectrum of expression than previously thought. This finding raises the possibility that the alpha 1(XI) chain may participate in the formation of stage- and tissue-specific trimers with distinct functional properties.

Development of muscarinic receptor subtypes in the forebrain of the mouse.

Cholinergic mechanisms are involved in the regulation of developmental events in the nervous system. Muscarinic cholinergic receptors are thought to be the predominant mediator of cholinergic neurotransmission in the forebrain; however, their developmental role is less well understood. The present study takes advantage of subtype-specific antibodies to muscarinic receptor proteins to investigate the cellular localization of the subtypes in developing mouse forebrain. Receptor protein expression was assessed between postnatal day (PND) 5 and adulthood by immunocytochemical methods with antibodies to m1, m2, and m4 receptors, the most abundant subtypes in rodent brain. We have found dramatic developmental changes in the distribution of all three receptors. In the adult mouse, m1 and m2 receptor immunoreactivity displayed complementary staining patterns in most forebrain areas with m4 sharing similarities in pattern with both m1 and m2. Furthermore, each receptor was expressed transiently in gray matter areas or fiber bundles at various developmental stages. The m4 receptor was also expressed in developing blood vessels. Such transient immunoreactivity was usually associated with times and areas of dynamic morphogenesis, thus suggesting distinct roles for the receptor subtypes in ontogenetic events.