Imaging filopodia dynamics in the mouse blastocyst

During mammalian development, the first cell lineage diversification event occurs in the blastocyst, when the trophectoderm (TE) and the inner cell mass (ICM) become established. Part of the TE (polar) remains in contact with the ICM and differs from the mural TE (mTE) which is separated from the ICM by a cavity known as the blastocoele. The presence of filopodia connecting ICM cells with the distant mural TE cells through the blastocoelic fluid was investigated in this work. We describe two types of actin-based cell projections found in freshly dissected and in vitro cultured expanding blastocysts: abundant short filopodia projecting into the blastocoelic cavity that present a continuous undulating behavior; and long, thin traversing filopodia connecting the mural TE with the ICM. Videomicroscopy analyses revealed the presence of vesicle-like structures moving along traversing filopodia and dynamic cytoskeletal rearrangements. These observations, together with immunolocalization of the FGFR2 and the ErbB3 receptors to these cell extensions, suggest that they display signal transduction activity. We propose that traversing filopodia are employed by mitotic mTE cells to receive the required signals for cell division after they become distant to the ICM.     

Intergenic enhancers with distinct activities regulate Dlx gene expression in the mesenchyme of the branchial arches

The vertebrate Dlx genes, generally organized as tail-to-tail bigene clusters, are expressed in the branchial arch epithelium and mesenchyme with nested proximodistal expression implicating a code that underlies the fates of jaws. Little is known of the regulatory architecture that is responsible for Dlx gene expression in developing arches. We have identified two distinct cis-acting regulatory sequences, I12a and I56i, in the intergenic regions of the Dlx1/2 and Dlx5/6 clusters that act as enhancers in the arch mesenchyme. LacZ transgene expression containing I12a is restricted to a subset of Dlx-expressing ectomesenchyme in the first arch. The I56i enhancer is active in a broader domain in the first arch mesenchyme. Expression of transgenes containing either the I12a or the I56i enhancers is dependent on the presence of epithelium between the onset of their expression at E9-10 until independence at E11. Both enhancers positively respond to FGF8 and FGF9; however, the responses of the reporter transgenes were limited to their normal domain of expression. BMP4 had a negative effect on expression of both transgenes and counteracted the effects of FGF8. Furthermore, bosentan, a pharmacological inhibitor of Endothelin-1 signaling caused a loss of I56i-lacZ expression in the most distal aspects of the expression domain, corresponding to the area of Dlx-6 expression previously shown to be under the control of Endothelin-1. Thus, the combinatorial branchial arch expression of Dlx genes is achieved through interactions between signaling pathways and intrinsic cellular factors. I56i drives the entire expression of Dlx5/6 in the first arch and contains necessary sequences for regulation by at least three separate pathways, whereas I12a only replicates a small domain of endogenous expression, regulated in part by BMP-4 and FGF-8.     

Detection of a new cerebral malaria susceptibility locus,using CBA mice

Human cerebral malaria (CM) during acute Plasmodium falciparum infection is a serious neurological complication that leads to coma and death. P. berghei ANKA infection of CBA mice is a useful experimental model of CM. To identify host susceptibility loci, we performed chromosomal mapping in crossbred populations of both CM-susceptible CBA and CM-resistant DBA/2 mice. One significant region for a CM-susceptible locus in CBA mice was mapped to H2 region on Chromosome 17, tentatively designated cmsc. cmsc was mapped to a different chromosomal region from that previously reported in the C57BL/6 mouse model of CM. It is possible that different loci contribute to CM in CBA and C57BL/6 mouse strains. Comparison of the function of CM susceptibility loci between CBA and C57BL/6 mice could have important implications for the study of the complex pathogenesis of CM in humans.     

Fibroblast growth factor 10 is required for proper development of the mouse whiskers

Fibroblast Growth Factor (FGF) signaling is known to play an important role during cutaneous development. To elucidate the role of FGF10 during whisker formation, we examined the expression of Fgf10 in normal developing whiskers and phenotypes of Fgf10-deficient whiskers. Fgf10 is first expressed in the maxillary process, lateral and medial nasal processes, then in the mesenchymal cells underneath the future whisker placodes, and in the surrounding mesenchyme of developing whiskers. Fgf10-null whiskers exhibit a significant decrease in number and their structure is disorganized as revealed by scanning electron microscopy. Hair follicle marker genes such as Sonic hedgehog, Patched, and Patched 2 are aberrantly expressed in the mutant whiskers. Thus, FGF10 is required for proper whisker development mediated by SHH signaling in the mouse.     

Anthracycline cardiotoxicity in transgenic mice overexpressing SR Ca2+-ATPase

Chronic anthracycline administration results in a time- and dose-dependent cardiomyopathy. The Ca-ATPase of the sarcoplasmic reticulum, SERCA2, has been implicated as a principal target for anthracycline-induced cardiotoxicity. This hypothesis predicts that improved SERCA2 function would provide protection from cardiotoxic effects of anthracycline administration. Doxorubicin was administered (1.7 mg/kg three times weekly; cumulative dose of 20 mg/kg) to 10 transgenic mice that overexpressed SERCA2 and to 10 isogenic littermates. Survival was monitored for 60 days and histologic comparisons were made of cardiac tissue. Survival in the transgenic mice was worse (1/10 60-day survivors) compared to isogenic control mice (7/10 60-day survivors). There was a greater degree of histologic damage exhibited in hearts from transgenic mice compared to isogenic controls when all available hearts were examined. These data do not support a role of SERCA2 in ameliorating anthracycline cardiotoxicity.     

Multiple single-nucleotide polymorphisms in the methylenetetrahydrofolate reductase and its truncated pseudogene of 23 inbred strains of mice

Reduced 5,10-methylenetetrahydrofolate reductase (MTHFR) results in a number of human diseases. To find a model mouse sensitive to these diseases, we analyzed single-nucleotide polymorphisms (SNPs) of the mouse Mthfr using 23 phylogenetically distant strains of mouse. We found five SNPs: two nonsynonymous and three synonymous. The CAST/Ei strain has the nonsynonymous SNP L350V and five strains (NMRI, KJR, SWN2, MSM, and JF1) have the nonsynonymous SNP S22G. The MTHFR activity of CAST/Ei and MSM showed no significant difference in activity or thermostability compared with that of C57BL/6J. We also found a pseudogene segment of the mouse Mthfr that was not present in human and was more frequently variable than the functional gene. These results suggest a possibility that the truncated pseudogene may buffer variations of the mouse Mthfr functional gene, and the mouse has evolved fewer variations of the gene than human.     

Imprinted X-chromosome inactivation: enlightenment from embryos in vivo

There are two forms of X chromosome inactivation (XCI) in the laboratory mouse, random XCI in the fetus and imprinted paternal XCI limited to the extraembryonic tissues supporting the fetal life in utero. Imprinted XCI has been studied extensively because it takes place first in embryogenesis and it may hold clues to the mechanism of control of XCI in general and to the evolution of random' XCI. Classical microscopic and biochemical studies of embryos in vivo provide a basis for interpreting the multifaceted information yielded by various inventive approaches and for planning further experiments.     

Sex-dependent antioxidant enzyme activities and lipid peroxidation in ageing mouse brain

We investigated whether oxidant status and antioxidant enzyme activities during ageing of mouse brain are regulated in sex-dependent manner. In the homogenate from the brain of 1, 4, 10 and 18 months old male and female CBA mice, lipid peroxidation (LPO), total superoxide dismutase (tSOD), catalase (CAT) and glutathione peroxidase (Gpx) were determined. LPO was age- and sex-related, favoring males over females throughout the lifespan with the peak in both sexes at 10 months of age. Throughout ageing, no difference in tSOD activity between male and female brains was observed, except in immature 1 month old mice. Gender-related difference in Gpx activity was observed, with higher level in females comparing to males, reaching statistical significance in senescent (18 months old) animals. CAT activity was drastically changed with ageing in both the male and female brain. We found different age associated trends in CAT activity in males and females: decreased with age in males and increased with age in females. Taken together, the present findings indicate that brains of female mice have lower oxidant and higher antioxidant capacity mostly related to CAT and to a lesser extent to Gpx activity.