Connexins and E-cadherin are differentially expressed during trophoblast invasion and placenta differentiation in the rat

We have characterized the spatial and temporal expression pattern of six different connexin genes and E-cadherin during trophectoderm development in the rat. During the initial phase of trophoblast invasion at 6 days postcoitum (dpc), the trophoblast expressed E-cadherin but no connexin expression could be observed. With progressing invasion of the polar trophoblast into the maternal decidua, from 7 dpc onwards E-cadherin expression in the ectoplacental cone cells was lost and was now restricted to the extraembryonic ectoderm. In the ectoplacental cone and extraembryonic ectoderm instead connexin31 mRNA and protein could be found. This pattern was maintained up to day 10 postcoitum. The start of labyrinthine trophoblast differentiation from day 11 postcoitum onwards was characterized by persisting expression of E-cadherin in the extraembryonic ectoderm and its derivative, the chorionic plate. In addition to E-cadherin, from 10 dpc onwards, connexin26 started to be expressed in the chorionic plate, and both molecules remained coexpressed in the labyrinthine trophoblast of the mature placenta. During this differentiation process connexin31 remained expressed mainly in the proliferating spongiotrophoblast. From day 14 postcoitum onwards, the expression of connexin31 in the spongiotrophoblastic cells decreased, and in parallel they started to express connexin43. The trophoblastic giant cells, first characterized by connexin31, lost all of the investigated connexins during midgestation on day 12 postcoitum but started to express connexin43 from day 18 postcoitum onwards. Our studies suggest that loss of E-cadherin and induction of connexin31 expression is correlated with the proliferative and invasive stages of the ectoplacental cone, whereas appearance of connexin26, E-cadherin and connexin43 reflects the switch to the differentiated phenotypes of the mature placenta.     

Spatiotemporal expression of C-CAM in the rat placenta

We investigated the expression of the immunoglobulin superfamily cell adhesion molecule, C-CAM, in developing and mature rat placenta. By immunohistochemical staining at the light microscopic level, no C-CAM-expression was seen before Day 9 of gestation, when it appeared in the trophoblasts of ectoplacental cones. On Day 10.5, spongiotrophoblasts and invasive trophoblasts around the maternal vessels of the decidua basalis were stained positively. On Day 12.5, C-CAM was detected in the spongiotrophoblasts of the junctional layer, but labyrinth trophoblasts and secondary giant trophoblasts were not stained. On Day 17.5, C-CAM was found only in the labyrinth and lacunae of the junctional layer. At this stage, both the labyrinth cytotrophoblasts of the maternal blood vessels and the endothelial cells of the embryonic capillaries were strongly stained. Placental tissues from gestational Days 12.5 and 17.5 were analyzed by immunoelectron microscopy to determine the location of C-CAM at the subcellular level. On Day 12.5, positive staining of the spongiotrophoblasts was observed, mainly on surface membranes and microvilli between loosely associated cells. On Day 17.5, staining was found primarily on the microvilli of the maternal luminal surfaces of the labyrinth cytotrophoblasts, and both on the luminal surface and in the cytoplasm of endothelial cells of the embryonic vessels. RT-PCR analysis and Southern blotting of the PCR products revealed expression of mRNA species for both of the major isoforms, C-CAM1 and C-CAM2. Immunoblotting analysis of C-CAM isolated from 12.5-day and 14.5-day placentae showed that it appeared as a broad band with an apparent molecular mass of 110-170 kD. In summary, C-CAM was strongly expressed in a specific spatiotemporal pattern in trophoblasts actively involved in formation of the placental tissue, suggesting an important role in placental development. In the mature placenta, C-CAM expression was confined to the trophoblastic and endothelial cells lining the maternal and embryonic vessels, respectively, suggesting important functions in placental physiology.     

Two cases of male infertility with pathological dilatation of ejaculatory duct

Culturing of rabbit pre-implantation embryos was performed in Ham's F10 medium supplemented with polyvinylpyrrolidone. Under these culture conditions, day 6 post coitum blastocysts increased their diameter within 24 h to 80% of that of day 7 blastocysts grown in vivo. Despite this substained growth, the embryonic disc remained undifferentiated with clear signs of degeneration after 24 h of culture. Basic fibroblast growth factor (bFGF) was able to overcome this developmental block. After 12 h of culture, day 6 blastocysts showed pear-shaped embryonic discs, and after 24 h, the primitive streak with Hensen's node was visible. The bFGF had no comparable effects on day 5 and day 7 blastocysts. The embryonic discs of day 5 blastocysts degenerated, even in the presence of bFGF, whereas day 7 blastocysts were able to form their primitive streak, also in the absence of bFGF. TGF beta 1 did not promote embryonic development in vitro. The data indicate that the onset of mesoderm formation in the rabbit is controlled by a growth factor of the FGF-family.     

Abnormal functional connectivity in Alzheimer''s disease: intrahemispheric EEG coherence during rest and photic stimulation

Tolbutamide is a sulfonylurea oral hypoglycaemic agent with suspected teratogenicity in humans and demonstrated teratogenicity in laboratory animals, but the underlying mechanism is unknown. This study examined maternal-to-conceptus tolbutamide transfer on gestational days 9.5 and 10.5 and drug concentration in embryonic head, heart, and trunk regions on gestational day 10.5 after maternal dosing in mouse. Embryos exposed to tolbutamide in vitro on gestational day 8.5 were assayed for glucose uptake, glycolysis, and protein content after 6, 12, and 24 hr. Dose-dependent tolbutamide transfer from maternal serum to extraembryonic fluid occurred on gestational day 9.5 and 10.5, with highest tolbutamide levels in embryonic heart on gestational day 10.5. In vitro tolbutamide exposure on gestational day 8.5 decreased glycolysis at 6 hr, increased glycolysis at 24 hr, and had no effect on glucose uptake at 6, 12, or 24 hr. Embryonic protein content reflected growth retardation after 24 hr tolbutamide exposure. Thus, mouse embryos are directly exposed to tolbutamide after maternal dosing on gestational day 9.5 and 10.5, with concentration of drug within embryonic heart. Tolbutamide-induced changes in glucose metabolism are less apparent in whole embryos than reported in adult tissues.     

Z-321, a prolyl endopeptidase inhibitor, augments the potentiation of synaptic transmission in rat hippocampal slices

In toto mouse embryos were cultivated at embryonic day 8.5 for 26 h with 105, 310 or 620 microM caffeine; 105-310 microM correspond to concentrations transferred by the placenta of heavy caffeine consumers. Failure of neural tube closure, excessive proliferation of neuroepithelial cells and premature evagination of telencephalic vesicles were present in 50% of treated embryos. When reaching the embryonic neural tube before neuronal migration, caffeine regionally modifies the schedule and/or rate of neural cell proliferation.     

Immunohistochemical analysis of insulin-like growth factor-binding proteins -1, -2, and -3 in implantation sites of the mouse

PURPOSE: Our purpose was to analyze potential interactions between the embryo and the maternal endometrial interface in vivo by analyzing immunolocalization of insulin-like growth factor-binding proteins (IGFBPs) -1, -2, and -3 in implantation sites of the mouse. METHODS: Six-week-old B6D2F1 female mice underwent superovulation followed by mating and sacrifice at timed intervals. Formalin-fixed paraffin-embedded tissue was used for avidin-biotin immunocytochemical localization of IGFBPs utilizing standard methodology. RESULTS: Immunostaining at 1.5 days post coitum revealed light staining in the epithelial glandular cells and faint staining in decidual stroma for both IGFBP-1 and IGFBP-2. At 7.5-10.5 days post coitum, there was moderate-dense immunostaining in the decidualized stromal cells at the implantation site for all three IGFBPs, whereas light immunostaining was seen in nonimplantation site decidua. CONCLUSIONS: Compartmentalization of immunostaining for IGFBP-1, -2, and -3 within decidualized stroma suggests that these proteins may be regulated by trophoblastic and/or embryonic signals.     

Abnormal eye development associated with Cat4a, a dominant mouse cataract mutation on chromosome 8

PURPOSE: Cat4a, one of four mutant alleles at the mouse Cat4 locus, causes central corneal opacity and anterior polar cataract in heterozygotes and microphthalmia in homozygotes. The Cat4 locus has been mapped to chromosome 8, 31 cM from the centromere. In this study ocular development of Cat4a mutant mice was investigated to characterize the defects in eye morphogenesis. METHODS: Serial sections from eyes of wild-type, heterozygous, and homozygous littermates were examined by means of light microscopy at selected intervals from embryonic day 11 to postnatal day 1. Eyes of adult heterozygous and homozygous mice also were evaluated histologically. RESULTS: Failure of separation of the lens vesicle from the surface ectoderm was the earliest structural defect observed. In heterozygous embryos, the abnormality was limited to persistent connection of the anterior pole of the lens to the cornea. Adult heterozygotes had defects in the central corneal stroma and endothelium and anterior polar cataracts with or without keratolenticular adhesion. In homozygous embryos, the persistent connection of lens to surface ectoderm was associated with aborted lens development, failure of closure of the optic fissure, and impairment of growth of the eyecup. Microphthalmic eyes of adult homozygous mice had a poorly developed cornea, and the anterior chamber and vitreous compartment were absent. An extensively folded retina and remnants of a degenerated lens filled the interior of the globe. CONCLUSIONS: A developmental defect inhibits separation of the lens vesicle from surface ectoderm in mice heterozygous or homozygous for the Cat4a mutation. In homozygotes subsequent lens and eye morphogenesis are also severely affected. Cat4a shows phenotypical similarity to several other independent mouse mutations including Small eye, a mutation of the Pax6 gene. Cat4 may be one of several genes involved in a common developmental path and may be part of the Pax6-regulated gene cascade governing eye morphogenesis.     

Phenotypic analysis and molecular cloning of discolored-1 (dsc1), a maize gene required for early kernel development

Recessive mutations in the maize dsc1 locus prevent normal kernel development. Solidification of the endosperm in homozygous dsc1- mutant kernels was undetectable 12 days after pollination, at which time the tissue was apparently completely solidified in wild-type kernels. At later times endosperm did solidify in homozygous dsc1- mutant kernels, but there was a marked reduction in the volume of the tissue. Embryo growth in homozygous dsc1- kernels was delayed compared to wild-type kernels, but proceeded to an apparently normal stage 1 in which the scutellum, coleoptile, and shoot apex were clearly defined. Embryo growth then ceased and the embryonic tissues degraded. Late in kernel development no tissue distinctions were obvious in dsc1- mutant embryos. Immature mutant embryos germinated when transplanted from kernels to tissue culture medium prior to embryonic degeneration, but only coleoptile proliferation was observed. The dsc1 gene was isolated by transposon tagging. Analysis of the two different dsc1- mutations confirmed that transposon insertion into the cloned genomic locus was responsible for the observed phenotype. Dsc1 mRNA was detected specifically in kernels 5-7 days after pollination. These data indicate Dsc1 function is required for progression of embryo development beyond a specific stage, and also is required for endosperm development.     

Histotypic in vitro reorganization of dissociated cells from mouse fetal gonads

Cell suspensions obtained from the testes of 13.5-14.5 day post coitum (dpc) mouse embryos reaggregate in cord-like structures following in vitro culture for 24 h on a reconstituted basement membrane (Matrigel). Ovarian cells of the same fetal age or cell suspensions from sex indifferent gonadal ridges (11.5 dpc embryos) form an organized cell network but not cord-like structures. Antibodies directed against laminin or against the alpha 6 subunit of its integrin receptor prevent such morphogenetic processes. The addition of 5 micrograms/ml cycloheximide, cytochalasin B or tunicamycin also inhibits the phenomenon. Interestingly, compounds that increase intracellular cyclic AMP (cAMP) (dbcAMP, forskolin and isobutyl-1-methylxanthine) induce embryonic testicular cells to organize into structures similar to those assembled by ovarian or sex indifferent cell suspensions. These findings offer a simple in vitro model for studying some aspects of early gonad development and provide novel experimental evidence that cell motility and cell-cell adhesion, possibly regulated by cAMP dependent mechanisms, are likely to play an important role in gonad morphogenesis.     

A null c-myc mutation causes lethality before 10.5 days of gestation in homozygotes and reduced fertility in heterozygous female mice

To directly assess c-myc function in cellular proliferation, differentiation, and embryogenesis, we have used homologous recombination in embryonic stem cells to generate both heterozygous and homozygous c-myc mutant ES cell lines. The mutation is a null allele at the protein level. Mouse chimeras from seven heterozygous cell lines transmitted the mutant allele to their offspring. The analysis of embryos from two clones has shown that the mutation is lethal in homozygotes between 9.5 and 10.5 days of gestation. The embryos are generally smaller and retarded in development compared with their littermates. Pathologic abnormalities include the heart, pericardium, neural tube, and delay or failure in turning of the embryo. Heterozygous females have reduced fertility owing to embryonic resorption before 9.5 days of gestation in 14% of implanted embryos. c-Myc protein is necessary for embryonic survival beyond 10.5 days of gestation; however, it appears to be dispensable for cell division both in ES cell lines and in the embryo before that time.     

A deletion within the murine Werner syndrome helicase induces sensitivity to inhibitors of topoisomerase and loss of cellular proliferative capacity

Werner syndrome (WS) is an autosomal recessive disorder characterized by genomic instability and the premature onset of a number of age-related diseases. The gene responsible for WS encodes a member of the RecQ-like subfamily of DNA helicases. Here we show that its murine homologue maps to murine chromosome 8 in a region syntenic with the human WRN gene. We have deleted a segment of this gene and created Wrn-deficient embryonic stem (ES) cells and WS mice. While displaying reduced embryonic survival, live-born WS mice otherwise appear normal during their first year of life. Nonetheless, although several DNA repair systems are apparently intact in homozygous WS ES cells, such cells display a higher mutation rate and are significantly more sensitive to topoisomerase inhibitors (especially camptothecin) than are wild-type ES cells. Furthermore, mouse embryo fibroblasts derived from homozygous WS embryos show premature loss of proliferative capacity. At the molecular level, wild-type, but not mutant, WS protein copurifies through a series of centrifugation and chromatography steps with a multiprotein DNA replication complex.     

Biased suppression of hematopoiesis and multiple developmental defects in chimeric mice containing Shp-2 mutant cells

Shp-2 is a cytoplasmic tyrosine phosphatase that contains two Src homology 2 (SH2) domains at the N terminus. Biochemical data suggests that Shp-2 acts downstream of a variety of receptor and cytoplasmic tyrosine kinases. A targeted deletion mutation in the N-terminal SH2 (SH2-N) domain results in embryonic lethality of homozygous mutant mice at midgestation. In vitro embryonic stem (ES) cell differentiation assays suggest that Shp-2 might play an important role in hematopoiesis. By aggregating homozygous mutant (Shp-2(-/-)) ES cells and wild-type (WT) embryos, we created Shp-2(-/-)-WT chimeric animals. We report here an essential role of Shp-2 in the control of blood cell development. Despite the widespread contribution of mutant cells to various tissues, no Shp-2(-/-) progenitors for erythroid or myeloid cells were detected in the fetal liver and bone marrow of chimeric animals by using the in vitro CFU assay. Furthermore, hematopoiesis was defective in Shp-2(-/-) yolk sacs. In addition, the Shp-2 mutation caused multiple developmental defects in chimeric mice, characterized by short hind legs, aberrant limb features, split lumbar vertebrae, abnormal rib patterning, and pathological changes in the lungs, intestines, and skin. These results demonstrate a functional involvement of Shp-2 in the differentiation of multiple tissue-specific cells and in body organization. More importantly, the requirement for Shp-2 is more stringent in hematopoiesis than in other systems.     

A Y-chromosomal effect on blastocyst cell number in mice

Karyotopic and cell number analysis of 3.5 day post coitum preimplantation mouse embryos was used to determine whether XY embryos had more cells than XX embryos at the late morula/early blastocyst stage. This proved to be the case for the CD1 strain (for which it had previously been shown that XY embryos form a blastocoel earlier than XX embryos) and for the MF1 strain. However, this increased cell number was not seen in MF1 embryos carrying an RIII strain Y in place of the MF1 Y. Furthermore, interstrain crosses between CD1 and the MF1,YRIII strain showed that the cell number increase segregated with the CD1 Y but not with the RIII Y. It is concluded that the CD1 and MF1 Y chromosomes carry a factor that accelerates the rate of preimplantation development.     

Abnormalities in the cerebellum and brainstem in homozygous lurcher mice

The lurcher mutation induces Purkinje cell degeneration in heterozygous mice, and neonatal death in homozygous animals. Using the D6Mit16 Simple Sequence Length Polymorphic marker in F2 hybrids between AKR +/+ mice and B6+/Lc mice, homozygous lurcher fetuses and newborns as well as heterozygous and normal littermates were identified, and their brain morphology was analysed. In homozygous lurcher embryos at embryonic day 18 and neonates the cerebellum was hypotrophic, particularly in the posterior half. Purkinje cells were smaller in the whole cerebellum and showed a maturational delay. Calretinin-positive cells were less frequently observed in the depth of the vermis than in normal mice. Both Purkinje cells and the vermal calretinin-positive cells were more abnormal in fetuses at day 19 and newborn mutants than one day earlier. An abnormal number of pycnotic cells were observed in the cerebellum, especially in newborn mutants. Brainstem abnormalities were characterized by abnormal curvature, caudal displacement of the pontine gray nuclei which were located caudally along the ventral border of the superior olivary complex, a drastic decrease in Purkinje cell axons in all the vestibular nuclei and the presence of dystrophic processes in at least two calbindin-positive cell groups of the dorsal pontine region. These results show that the mutation, which is semidominant in Purkinje cells, is recessive in other cell groups of the cerebellum and brainstem. They reveal that the sequence leading to Purkinje cell death appears to be similar in homozygous and heterozygous mice, although occurring earlier and worsening more quickly in the former. Lastly, they confirm the absence of effect of the mutation on the neurons of the inferior olivary complex.     

Expression of gap junctions in cultured rat L6 cells during myogenesis

Although gap junctions are absent from adult skeletal muscle, they have been described in embryonic and neonatal rat skeletal muscle and in cultured rat myoblasts. In order to determine the precise developmental expression and molecular composition of gap junctions during myogenesis, RNA was isolated from cultures of rat L6 myoblasts and examined using Northern blot analysis with cDNA probes specific for connexin32 and connexin43. Connexin32 mRNA could not be detected in rat myoblast and myotube samples. However, connexin43 mRNA was expressed at high levels in cycling L6 myoblasts and this expression decreased by approximately 60% in L6 myotubes following fusion. Immunofluorescent localization with an antibody specific for connexin43 confirmed the accumulation of connexin43 protein in membranes shared between adjacent myoblasts at 12 hr of culture. By 24 hr of culture, connexin43 disappeared from most cells, only to reappear at 36 hr at a low level that was maintained through 72 hr in culture. Although most myoblasts in these cultures expressed connexin43, myotubes expressed little or no membrane-associated connexin43. Dye transfer experiments established that, at 12 hr of culture, the majority of myoblasts were dye coupled suggesting that connexin43 protein is assembled into functional gap junctions. At 24 hr, the number of coupled cells decreased slightly, while at 48 hr, most of the myoblasts were not dye coupled. These results demonstrate that the expression of connexin43 is temporally correlated with myoblast fusion and may play a role in this process.     

Gene transfer to the rodent placenta in situ. A new strategy for delivering gene products to the fetus

The delivery of biologically active factors to the developing mammalian embryo by in utero gene transfer has generated considerable interest but limited success. The chorioallantoic placenta is a potential alternative target for providing therapeutic transgenes to the fetus during gestation. We demonstrate that somatic gene transfer to the midgestation rat placenta may be efficiently accomplished in situ through the implantation of a variety of genetically modified cells with different antigenic and growth properties. Ex vivo-modified cells survived and retained transgene expression until term. Proteins secreted from the transplanted cells were detectable within the fetal trunk blood. These studies suggest that gene transfer to the placenta may be a useful tool for answering questions of both embryonic and placental development and providing therapeutic proteins during gestation for amelioration of diseases with onset during embryonic life.     

Differential expression of gap junctions in neurons and astrocytes derived from P19 embryonal carcinoma cells

The P19 embryonal carcinoma cell line represents a pluripotential stem cell that can differentiate along the neural or muscle cell lineage when exposed to different environments. Exposure to retinoic acid induces P19 cells to differentiate into neurons and astrocytes that express similar developmental markers as their embryonic counterparts. We examined the expression of gap junction genes during differentiation of these stem cells into neurons and astrocytes. Untreated P19 cells express at least two gap junction proteins, connexins 26 and 43. Connexin32 could not be detected in these cells. Treatment for 96 hr with 0.3 mM retinoic acid induced the P19 cells to differentiate first into neurons followed by astrocytes. Retinoic acid produced a decrease in connexin43 mRNA, protein, and functional gap junctions. Connexin26 message was not affected by retinoic acid treatment. The neurons that developed consisted of small round cell bodies extending two to three neurites and expressed MAP2. Connexin26 was detected at sites of cell-cell and cell-neurite contact within 3 days following differentiation with retinoic acid. The astrocytes were examined for production of their intermediate filament marker, glial fibrillary acidic protein (GFAP). GFAP was first detected at 8 days by Western blotting. In culture, astrocytes co-expressed GFAP and connexin43 similar to primary cultures of mouse brain astrocytes. These results suggest that differentiation of neurons and glial cells involves specific connexin expression in each cell type. The P19 cell line will provide a valuable model with which to examine the role gap junctions play during differentiation events of developing neurons and astrocytes.     

Abrogation of the Cripto gene in mouse leads to failure of postgastrulation morphogenesis and lack of differentiation of cardiomyocytes

Cripto-1(Cr1) protein encoded by the tdgf1 gene, is a secreted growth factor that is expressed early in embryonic development and is re-expressed in some tumors of the breast and colon. During embryonic development, Cr1 is expressed in inner cell mass cells and the primitive streak, and later is restricted to the developing heart. To investigate the role of Cr1 during mouse development, mice were generated that contain a null mutation of both Cr1 genes, derived from homologous recombination in embryonic stem cells. No homozygous Cr1-/- mice were born, indicating that Cr1 is necessary for embryonic development. Embryos initiated gastrulation and some embryos produced mesoderm up to day E7.5. Increasingly aberrant morphogenesis gave rise to disordered neuroepithelium that failed to produce a recognizable neural tube, or head-fold. Although some biochemical markers of differentiating ectoderm, mesoderm and endoderm were expressed, all the cardiac-specific markers were absent from day E8.7 embryos: (&agr;)MHC, betaMHC, MLC2A, MLC2V and ANF, whereas they were expressed in wild-type embryos. The yolk sac and placental tissues continued development in the absence of the embryo until day E9.5 but lacked large yolk sac blood vessels. Chimeric mice were constructed by microinjection of double targeted Cr1(-/- )embryonic stem cells into normal C57BL/6 blastocysts. The Cr1 produced by the normal C57BL/6 cells fully rescued the phenotype of Cr1(-/-) cells, indicating that Cr1 protein acted in a paracrine manner. Cells derived from the embryo proliferated and migrated poorly and had different adhesion properties compared to wild type. Therefore, lethality in the absence of Cr1, likely resulted largely from defective precardiac mesoderm that was unable to differentiate into functional cardiomyocytes.