Histochemical analysis of extracellular matrix material in embryonic trisomy 1 mouse eye

Trisomic animals produced from mice doubly heterozygous for Robertsonian translocation chromosomes [Rb(1.3)/Rb(1.10)]consistently show eye defects (e.g., aphakia, microphakia, and retention of lens stalk). To determine if changes in distribution or composition of extracellular matrix material may be a factor in development of these defects, eye structures of trisomy (ts) 1 embryos and normal littermates were studied histochemically using the following methods: Alcian blue 8GX, pH 2.5; periodic acid-Schiff (PAS), Alcian blue/PAS combined; high-iron diamine (HID), and HID/Alcian blue combined. Eye development was divided into stages to account for the known delay in ts 1 mouse development. Differences were found in staining patterns as early as stage 1. In later stages, the most consistent difference was an increased period of contact between lens and optic cup due to retardation of interface matrix dissolution between these rudiments in ts 1 embryos. Eyes in which this occurred had abnormally shaped lenses. Overall, the ts 1 optic cup appeared to have fewer staining abnormalities and dysmorphology than did the lens or interface matrix. Triplication of a chromosome may indirectly alter temporal and spatial organization of extracellular matrix through action on cells responsible for the production of this material. Possible mechanisms of action are discussed.     

Drapc1 expression during mouse embryonic development

We identified the mouse homolog of human DRAPC1 (APCDD1) gene, shown to be a target of Wnt/beta-catenin signaling pathway in cancer cell lines. Analysis of its spatiotemporal expression in mouse embryos from E7.5 to E14 showed that Drapc1 is expressed during development of the extraembryonic structures, nervous system, vascular system and inner ear. In addition, Drapc1 is expressed in the mesenchyme of several developing organs at sites of epithelio-mesenchymal interactions. Drapc1 expression was also found in the hair follicles of the adult mouse skin. Similarity of Drapc1 expression pattern to location of active beta-catenin in developing mouse embryo further suggests that mouse Drapc1 is a novel in vivo target gene of Wnt/beta-catenin signaling pathway.     

The expression of Visinin-like 1 during mouse embryonic development

Visinin like 1 (Vsnl1) encodes a calcium binding protein which is well conserved between species. It was originally found in the brain and its biological functions in central nervous system have been addressed in several studies. Low expression levels have also been found in some peripheral organs, but very little information is available regarding its physiological roles in non-neuronal tissues. Except for the kidney, the expression pattern of Vsnl1 mRNA and protein has not yet been addressed during embryogenesis. By in situ hybridization and immunolabeling we have extensively analyzed the expression pattern of Vsnl1 during murine development. Vsnl1 specifies the cardiac primordia and its expression becomes restricted to the atrial myocardium after heart looping. However, in the adult heart, Vsnl1 is expressed by all four cardiac chambers. It also serves as a specific marker for the cardiomyocyte-derived structures in the systemic and pulmonary circulation. Vsnl1 is dynamically expressed also by many other organs during development e.g. taste buds, cochlea, thyroid, tooth, salivary and adrenal gland. The stage specific expression pattern of Vsnl1 makes it a potentially useful marker particularly in studies of cardiac and vascular morphogenesis.     

Enzyme patterns in trisomy 19 of the mouse

Activity patterns of cytosolic and mitochondrial enzymes of carbohydrate and amino acid metabolism have been measured in murine trisomy 19. In spite of marked hypoplasia, no significant alterations of the patterns (per gram of organ weight) were observed, with the exception of glutamate oxaloacetate transaminase (GOT-1), and phosphoglycerate mutase (PGAM). Clear-cut gene dosage effects in liver, brain, heart, skeletal muscle, and erythrocytes of fetal and newborn mice, confirm the assignment of GOT-1 to chromosome 19. Data obtained for PGAM demonstrate that one of the two different subunits leading to organ-specific isozyme patterns of the dimer enzyme protein is coded on chromosome 19 (gene Pgam-1). Dosage effects are fully expressed in liver, brain, and erythrocytes (AA-type isozyme), but not in skeletal muscle (BB-type isozyme). Dosage effects on the hybrid AA-AB-BB-isozyme pattern in the course of development of the heart muscle, were demonstrated by means of quantitative activity measurement after electrophoretic separation. The comparison of enzyme patterns of eusomic and trisomic erythrocytes, produced after injection of fetal stem cells into irradiated adult carriers (transplantation chimaeras), revealed enzyme activity ratios that were similar to those produced by erythrocytes of adult euploid and trisomic mice. This is in agreement with the chromosome assignments and dosage effects mentioned above.     

Viability of trisomy 12 cells in mouse chimaeras

Summary Mouse aggregation chimaeras consisting of trisomy 12 and normal euploid cells were produced. The analysis of one trisomy 12euploid chimaera, using biochemical and cytological markers, showed that the trisomic cells were able to participate in the formation of most tissues including the ovary. On the other hand, no trisomy 12 cells were found in lymphocyte populations, which is most likely due to early selection in this particular cell lineage. The viability of two adult trisomy 12 chimaeras demonstrates that trisomy 12 cells are able to develop beyond the fetal stage which is not observed in completely trisomic fetuses.Furthermore, these chimaeras did not show any sign of a trisomy 12 syndrome, indicating that the trisomy 12 cells were functionally integrated and participated normally in the differentiation of the various tissues. Our results suggest that trisomy 12 in the mouse is not autonomously cell lethal but can be rescued and is perfectly viable in the presence of normal diploid cells.This article is dedicated to the memory of Prof. A. Gropp     

Role of Lef1 in sustaining self-renewal in mouse embryonic stem cells

Embryonic stem cells (ESCs) can self-renew indefinitely while maintaining the ability to generate all three germ-layer derivatives.Despite the importance of ESCs in developmental biology and their potential impact on regenerative medicine,the molecular mechanisms controlling ESC behavior are incompletely understood.Previously,activation of the canonical Wnt signaling pathway has been shown to contribute to mouse ESC self-renewal.Here we report that ectopic expression of Lef1,a component of the Wnt signaling pathway,has a positive effect on the self-renewal of mouse ESCs.Lef1 up-regulates Oct4 promoter activity and physically interacts with Nanog,two key components of the ESC pluripotency machinery.Moreover,siRNA for Lef1 induced mouse ESC differentiation.Our results thus suggest that in response to Wnt signaling Lef1 binds to stabilized β-catenin and helps maintain the undifferentiated status of ESCs through modulation of Oct4 and Nanog.     

Cardiac alternans in embryonic mouse ventricles

T-wave alternans, an important arrhythmogenic factor, has recently been described in human fetuses. Here we sought to determine whether alternans can be induced in the embryonic mouse hearts, despite its underdeveloped sarcoplasmic reticulum (SR) and, if so, to analyze the response to pharmacological and autonomic interventions. Immunohistochemistry confirmed minimal sarcoplasmic-endoplasmic reticulum Ca-ATPase 2a expression in embryonic mouse hearts at embryonic day (E) 10.5 to E12.5, compared with neonatal or adult mouse hearts. We optically mapped voltage and/or intracellular Ca (Ca(i)) in 99 embryonic mouse hearts (dual mapping in 64 hearts) at these ages. Under control conditions, ventricular action potential duration (APD) and Ca(i) transient alternans occurred during rapid pacing at an average cycle length of 212 +/- 34 ms in 57% (n = 15/26) of E10.5-E12.5 hearts. Maximum APD restitution slope was steeper in hearts developing alternans than those that did not (2.2 +/- 0.6 vs. 0.8 +/- 0.4; P < 0.001). Disabling SR Ca(i) cycling with thapsigargin plus ryanodine did not significantly reduce alternans incidence (44%, n = 8/18, P = 0.5), whereas isoproterenol (n = 14) increased the incidence to 100% (P < 0.05), coincident with steepening APD restitution slope. Verapamil abolished Ca(i) transients (n = 9). Thapsigargin plus ryanodine had no major effects on Ca(i)-transient amplitude or its half time of recovery in E10.5 hearts, but significantly depressed Ca(i)-transient amplitude (by 47 +/- 8%) and prolonged its half time of recovery (by 18 +/- 3%) in E11.5 and older hearts. Embryonic mouse ventricles can develop cardiac alternans, which generally is well correlated with APD restitution slope and does not depend on fully functional SR Ca(i) cycling.     

Targeted disruption of the mouse Asna1 gene results in embryonic lethality

The bacterial ArsA ATPase is the catalytic component of an oxyanion pump that is responsible for resistance to arsenicals and antimonials. Homologues of the bacterial ArsA ATPase are widespread in nature. We had earlier identified the mouse homologue (Asna1) that exhibits 27% identity to the bacterial ArsA ATPase. To identify the physiological role of the protein, heterozygous Asna1 knockout mice (Asna1+/-) were generated by homologous recombination. The Asna1+/- mice displayed similar phenotype as the wild-type mice. However, early embryonic lethality was observed in homozygous Asna1 knockout embryos, between E3.5 (E=embryonic day) and E8.5 stage. These findings indicate that Asna1 plays a crucial role during early embryonic development.     

Delayed thymocyte maturation in the trisomy 16 mouse fetus

Mouse fetuses with trisomy 16, an animal model for human trisomy 21 (Down syndrome), have severe defects in several hematopoietic stem cell populations and a marked reduction in thymocyte number. To determine whether there are other defects in the development of the trisomic thymus, the ontogeny of the cell surface antigenic determinants, Thy-1, Ly-1, CD3, CD4, CD8, and TCR v beta, was investigated. The trisomy 16 thymocytes were able to express all of determinants either during fetal life (days 14 to 19 of gestation) or in cultures of intact thymus lobes. However, in all instances (except for Thy-1, which already had a high proportion of expressing thymocytes by day 14), there was a delay in the time at which the determinants were first expressed, as manifested by reduced numbers of positively staining cells. Furthermore, there was also a delay in the rate at which the positively staining cells attained maximal Ag densities. Overall, there was an approximate 2 day lag in development of the fetal trisomic thymocytes. This lag permitted the identification of a large population of CD4-8+ cells prior to the appearance of CD4+8+ thymocytes. These findings are consistent with the identification of CD4-8+ as an intermediate stage between CD4-8- and CD4+8+ in fetal thymocyte ontogeny.     

Caveolin-1 and migration of bone-marrow derived cells in the mouse eye

Bone marrow derived cells (BMDCs) can be found in almost every tissue showing a distinct turnover and density. Since caveolin-1 regulates junction-associated proteins in endothelial and epithelial cells, its role for BMDC was investigated in the eyes of caveolin-1 knock-out mice transplanted with GFP-marked BMDC. Distribution and turnover of BMDC in connective tissues (cornea, iris, ciliary body and choroid) was not altered. The absence of caveolin-1, however, caused a significant decrease of BMDC turnover in cornea epithelium, ciliary epithelium, and in the retina. This finding emphasizes an important, hitherto unknown role of caveolin-1 in neuronal and epithelial tissues.     

Cloning and embryonic expression analysis of the mouse Gbx1 gene

We report the cloning of a cDNA encoding the complete mouse Gbx1 coding region as well as a comparative expression analysis of Gbx1 and Gbx2 during murine development. Gbx1 is expressed first during gastrulation and later in a dynamic pattern in the central nervous system, including rhombomeres 3 and 5, optic vesicles, and the medial ganglionic eminence. Gbx1 expression is not upregulated in Gbx2 null homozygotes. Therefore, the only regions of potential genetic redundancy are where Gbx1 and 2 are normally coexpressed: the primitive streak, regions of the ventricular zone of the neural tube and the medial ganglionic eminence. Finally, we demonstrate that neither Gbx1 nor Gbx2 require FGF8 for expression during gastrulation, contrary to previous published reports.     

Fibronectin in the development of embryonic chick eye.

The time course of appearance and distribution of fibronectin in the developing eye have been studied in chick embryos by indirect immunofluorescence. At the 12-somite stage, fibronectin was detected as a layer under the ectodermal cells overlying the forebrain vesicle; it was also present in the head mesenchyme. During formation of the lens placode and its invagination, a zone containing fibronectin persisted around the lens as a component of the capsule. The fibronectin-containing layer was separated from the corneal epithelial cells during the formation of the acellular stroma. The migrating corneal endothelial cells were seen posterior to the fibronectin layer. The secondary stroma was strongly positive for fibronectin. Fibronectin disappeared from the cornea starting from its posterior part along with the corneal condensation. In the newborn chicken cornea, fibronectin was present only in Descemet's membrane. In addition, the embryonic vitreous body had a network of fibronectin-containing material. The distribution of fibronectin in the developing cornea, as well as other data available on this glycoprotein, is consistent with the proposed role of fibronectin in positioning and migration of cells and in organization of the extracellular matrix.