No abstract

Cre transgenic mice can be used to delete gene sequences flanked by loxP sites in specific somatic tissues. We have generated vavCre transgenic mice, which can be used to inactivate genes specifically in adult hematopoietic and endothelial cells. In these animals, a Cre transgene is expressed under control of murine vav gene regulatory elements. To assess their usefulness, vavCre transgenic mice were bred with R26R mice, which express a lacZ reporter gene only in cells where Cre-mediated recombination has occurred. VavCre/R26R double-heterozygous offspring were analyzed by beta-galactosidase histochemistry and flow cytometry. VavCre-mediated recombination occurred in most hematopoietic cells of all hematopoietic organs, including the hematopoietic progenitor-rich bone marrow. Recombination also occurred in most endothelial and germ cells, but only rarely in other cell types. The recombination in both hematopoietic and endothelial lineages may partly reflect their putative shared ontogeny and provides a unique tool for simultaneous pan-hematopoietic and endothelial mutagenesis.

The distal portion of mouse chromosome 12 is imprinted. To date, however, Gtl2 is the only imprinted gene identified on chromosome 12. Gtl2 encodes multiple alternatively spliced transcripts with no apparent open reading frame. Using conceptuses with maternal or paternal uniparental disomy for chromosome 12 (UPD12), we found that Gtl2 is expressed from the maternal allele and methylated at the 5' end of the silent paternal allele. A reciprocally imprinted gene, Delta-like (Dlk), with homology to genes involved in the Notch signalling pathway was identified 80kb upstream of Gtl2. Dlk was expressed exclusively from the paternal allele in both the embryo and placenta, but the CpG-island promoter of Dlk was completely unmethylated on both parental alleles. Rather, a paternally methylated region was identified in the last exon of the active Dlk allele. The proximity, reciprocal imprinting and methylation in this domain are reminiscent of the co-ordinately regulated Igf2-H19 imprinted domain on mouse chromosome 7. Like H19 and Igf2, Gtl2 and Dlk were found to be co-expressed in the same tissues throughout development, though not after birth. These results have implications for the regulation, function and evolution of imprinted domains.

ABSTRACT:The rheological characteristics of gastric and duodenal mucin solutions, the building blocks of the mucus layer that covers the epithelia of the two organs, were investigated using particle tracking microrheology. We used biochemically well characterized purified porcine mucins for MUC5AC and h $ c 5:160:8 for MUC2. The dynamics of the self-assembled comb polymers is examined in terms of a scaling model for flexible polyelectrolyte combs. Both duodenum and gastric mucin are found to be pH switchable gels, gelation occurring at low pHs. There is a hundred-fold increase in the elastic shear modulus once the pH is decreased. The addition of DTT, guanidinium chloride and urea disassembles both the semi-dilute and gel structures causing a large increase in the compliance (decrease in their shear moduli). Addition of the polyphenol EGCG has a reverse effect on mucin viscoelasticity, that is, it triggers a sol-gel transition in semi-dilute mucin solutions at neutral pH.

Although the trophoblast is necessary for the growth, viability and patterning of the mammalian embryo, understanding of its patterning role is still rudimentary. Expression of the transcription factor Ets2 is restricted to the trophoblast in early postimplantation stages and Ets2 mutants have been previously shown to have defects in trophoblast development. We show here that Ets2 is necessary in the trophoblast for fundamental aspects of anteroposterior (AP) epiblast axis initiation, including mesoderm initiation at the primitive streak, establishment of posterior character in the epiblast and appropriate spatial restriction of the anterior visceral endoderm (AVE). Most homozygous Ets2 mutants also show highly reduced development of the trophoblast with an absence of extraembryonic ectoderm (EXE) markers. Embryos in which the EXE has been physically removed before culture in vitro phenocopy the patterning defects of Ets2 mutants. These defects cannot be rescued by providing Ets2 mutants with wildtype epiblast in tetraploid aggregations. Thus, EXE-derived signals are necessary for normal embryonic patterning. Ets2 is likely to be required in the EXE downstream of epiblast signals, such as Fgf, and, in turn, helps to regulate signals from the EXE that signal back to the epiblast to promote proper primitive streak and AVE development. This study provides new insights about the genetic and cellular basis of the patterning role and development of the early trophoblast.

The inner ear develops from the otic placode, one of the cranial placodes that arise from a region of ectoderm adjacent to the anterior neural plate called the pre-placodal domain. We have identified a Forkhead family transcription factor, Foxi3, that is expressed in the pre-placodal domain and down-regulated when the otic placode is induced. We now show that Foxi3 mutant mice do not form otic placodes as evidenced by expression changes in early molecular markers and the lack of thickened placodal ectoderm, an otic cup or otocyst. Some preplacodal genes downstream of Foxi3 - Gata3, Six1 and Eya1 - are not expressed in the ectoderm of Foxi3 mutant mice, and the ectoderm exhibits signs of increased apoptosis. We also show that Fgf signals from the hindbrain and cranial mesoderm, which are necessary for otic placode induction, are received by pre-placodal ectoderm in Foxi3 mutants, but do not initiate otic induction. Finally, we show that the epibranchial placodes that develop in close proximity to the otic placode and the mandibular division of the trigeminal ganglion are missing in Foxi3 mutants. Our data suggest that Foxi3 is necessary to prime pre-placodal ectoderm for the correct interpretation of inductive signals for the otic and epibranchial placodes.

The placenta contains several types of feto-maternal interfaces where zygote-derived cells interact with maternal cells or maternal blood for the promotion of fetal growth and viability. The genetic factors regulating the interactions between different cell types within fetomaternal interfaces and the relative contributions of the maternal and zygotic genomes are poorly understood. Genomic imprinting, the epigenetic process responsible for parental origin-dependent functional differences between homologous chromosomes, has been proposed to contribute to these events. Previous studies showed that mouse conceptuses with an absence of imprinted differences between the two copies of chromosome 12 (upon paternal inheritance of both copies) die late in gestation and have a variety of defects, including placentomegaly. Here we examined the role of chromosome 12 imprinting in these placentae in more detail. We show that the spatial interactions between different cell types within fetomaternal interfaces are defective and identify abnormal behaviors in both zygote-derived and maternal cells that are attributed to the genome of the zygote but not the mother. These include compromised invasion of the maternal decidualized endometrium and the central maternal artery situated within it by zygote-derived trophoblast, abnormalities in the wall of the central maternal artery, and defects within the zygote-derived cellular layer of the labyrinth, which is in direct contact with maternal blood. These findings demonstrate multiple roles for chromosome 12 imprinting in the placenta that have not previously been associated with imprinting effects. They provide insights into the function of imprinting in placental development and have evolutionary and clinical implications.

Super-resolution fluorescence microscopy, specifically stochastic reconstruction microscopy (STORM), and atomic force microscopy (AFM) were used to image the self-assembly processes of the peptide surfactant IK. The peptide surfactants self-assembled into giant helical fibrils with diameters between 5 and 10 nm with significant helical twisting. The resolution of the STORM images was 30 nm, calculated using the Fourier ring correlation method. STORM compares favorably with AFM for the calculation of contour lengths (∼6 μm) and persistence lengths (10.1 ± 1.2 μm) due to its increased field of view (50 μm), and its ability to image bulk morphologies away from surfaces under ambient solution conditions. Two-color STORM experiments were performed to investigate the dynamic process of self-assembly after mixing of two separately labeled samples, and the results revealed the formation of long nanofibers via end-to-end connections of short ones. No evidence was found for significant monomer exchange between the samples, and the self-assembled structures were very stable and long-lived.