Active cell migration drives the unilateral movements of the anterior visceral endoderm

The anterior visceral endoderm (AVE) of the mouse embryo is a specialised extra-embryonic tissue that is essential for anterior patterning of the embryo. It is characterised by the expression of anterior markers such as Hex, Cerberus-like and Lhx1. At pre-gastrula stages, cells of the AVE are initially located at the distal tip of the embryo, but they then move unilaterally to the future anterior. This movement is essential for converting the existing proximodistal axis into an anteroposterior axis. To investigate this process, we developed a culture system capable of imaging embryos in real time with single cell resolution. Our results show that AVE cells continuously change shape and project filopodial processes in their direction of motion, suggesting that they are actively migrating. Their proximal movement stops abruptly at the junction of the epiblast and extra-embryonic ectoderm, whereupon they move laterally. Confocal microscope images show that AVE cells migrate as a single layer in direct contact with the epiblast, suggesting that this tissue might provide directional cues. Together, these results show that the anteroposterior axis is correctly positioned by the active movement of cells of the AVE in response to cues from their environment, and by a 'barrier' to their movement that provides an endpoint for this migration.     

Contribution of direct electron transfer mechanisms to overall electron transfer in microbial fuel cells utilising <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> as biocatalyst

Objectives

To investigate the contribution of direct electron transfer mechanisms to electricity production in microbial fuel cells by physically retaining Shewanella oneidensis cells close to or away from the anode electrode.

Results

A maximum power output of 114 ± 6 mWm^?2 was obtained when cells were retained close to the anode using a dialysis membrane. This was 3.5 times more than when the cells were separated away from the anode. Without the membrane the maximum power output was 129 ± 6 mWm^?2. The direct mechanisms of electron transfer contributed significantly to overall electron transfer from S. oneidensis to electrodes, a result that was corroborated by another experiment where S. oneidensis cells were entrapped in alginate gels.

Conclusion

S. oneidensis transfers electrons primarily by direct electron transfer as opposed to mediated electron transfer.

     

Correct patterning of the primitive streak requires the anterior visceral endoderm

Anterior-posterior axis specification in the mouse requires signalling from a specialised extra-embryonic tissue called the anterior visceral endoderm (AVE). AVE precursors are induced at the distal tip of the embryo and move to the prospective anterior. Embryological and genetic analysis has demonstrated that the AVE is required for anterior patterning and for correctly positioning the site of primitive streak formation by inhibiting Nodal activity. We have carried out a genetic ablation of the Hex-expressing cells of the AVE (Hex-AVE) by knocking the Diphtheria toxin subunit A into the Hex locus in an inducible manner. Using this model we have identified that, in addition to its requirement in the anterior of the embryo, the Hex-AVE sub-population has a novel role between 5.5 and 6.5dpc in patterning the primitive streak. Embryos lacking the Hex-AVE display delayed initiation of primitive streak formation and miss-patterning of the anterior primitive streak. We demonstrate that in the absence of the Hex-AVE the restriction of Bmp2 expression to the proximal visceral endoderm is also defective and expression of Wnt3 and Nodal is not correctly restricted to the posterior epiblast. These results, coupled with the observation that reducing Nodal signalling in Hex-AVE ablated embryos increases the frequency of phenotypes observed, suggests that these primitive streak patterning defects are due to defective Nodal signalling. Together, our experiments demonstrate that the AVE is not only required for anterior patterning, but also that specific sub-populations of this tissue are required to pattern the posterior of the embryo.     

Quantitative trait loci mapping for canine hip dysplasia and its related traits in UK Labrador Retrievers

Background

Canine hip dysplasia (CHD) is characterised by a malformation of the hip joint, leading to osteoarthritis and lameness. Current breeding schemes against CHD have resulted in measurable but moderate responses. The application of marker-assisted selection, incorporating specific markers associated with the disease, or genomic selection, incorporating genome-wide markers, has the potential to dramatically improve results of breeding schemes. Our aims were to identify regions associated with hip dysplasia or its related traits using genome and chromosome-wide analysis, study the linkage disequilibrium (LD) in these regions and provide plausible gene candidates. This study is focused on the UK Labrador Retriever population, which has a high prevalence of the disease and participates in a recording program led by the British Veterinary Association (BVA) and The Kennel Club (KC).

Results

Two genome-wide and several chromosome-wide QTLs affecting CHD and its related traits were identified, indicating regions related to hip dysplasia.

Conclusion

Consistent with previous studies, the genetic architecture of CHD appears to be based on many genes with small or moderate effect, suggesting that genomic selection rather than marker-assisted selection may be an appropriate strategy for reducing this disease.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-833) contains supplementary material, which is available to authorized users.     

Revisiting the phylogeography and demography of European badgers (Meles meles) based on broad sampling,multiple markers and simulations

Although the phylogeography of European mammals has been extensively investigated since the 1990s, many studies were limited in terms of sampling distribution, the number of molecular markers used and the analytical techniques employed, frequently leading to incomplete postglacial recolonisation scenarios. The broad-scale genetic structure of the European badger (Meles meles) is of interest as it may result from historic restriction to glacial refugia and/or recent anthropogenic impact. However, previous studies were based mostly on samples from western Europe, making it difficult to draw robust conclusions about the location of refugia, patterns of postglacial expansion and recent demography. In the present study, continent-wide sampling and analyses with multiple markers provided evidence for two glacial refugia (Iberia and southeast Europe) that contributed to the genetic variation observed in badgers in Europe today. Approximate Bayesian computation provided support for a colonisation of Scandinavia from both Iberian and southeastern refugia. In the whole of Europe, we observed a decline in genetic diversity with increasing latitude, suggesting that the reduced diversity in the peripheral populations resulted from a postglacial expansion processes. Although MSVAR v.1.3 also provided evidence for recent genetic bottlenecks in some of these peripheral populations, the simulations performed to estimate the method''s power to correctly infer the past demography of our empirical populations suggested that the timing and severity of bottlenecks could not be established with certainty. We urge caution against trying to relate demographic declines inferred using MSVAR with particular historic or climatological events.     

Immunoquantification and enzyme kinetics of alpha-L-iduronidase in cultured fibroblasts from normal controls and mucopolysaccharidosis type I patients.

alpha-L-Iduronidase activity is deficient in mucopolysaccharidosis type I (MPS I; Hurler syndrome, Scheie syndrome) patients and results in the disruption of the sequential degradation of the glycosaminoglycans dermatan sulfate and heparan sulfate. A monoclonal antibody-based immunoquantification assay has been developed for alpha-L-iduronidase, which enables the detection of at least 16 pg alpha-L-iduronidase protein. Cultured human skin fibroblasts from 12 normal controls contained 17-54 ng alpha-L-iduronidase protein/mg extracted cell protein. Fibroblasts from 23 MPS I patients were assayed for alpha-L-iduronidase protein content. Fibroblast extracts from one MPS I patient contained at least six times the level of alpha-L-iduronidase protein for normal controls--but contained no associated enzyme activity--and is proposed to represent a mutation affecting the active site of the enzyme. Fibroblast extracts from 11 MPS I patients contained 0.05-2.03 ng alpha-L-iduronidase protein/mg extracted cell protein, whereas immunodetectable protein could not be detected in the other 11 patients. Four fibroblast extracts with no immunodetectable alpha-L-iduronidase protein had residual alpha-L-iduronidase activity, suggesting that the mutant alpha-L-iduronidase in cultured cells from these MPS I patients has been modified to mask or remove the epitopes detected by two monoclonal antibodies used in the quantification assay. Both the absence of immunoreactivity in a mild MPS I patient and high protein level in a severe MPS I patient present limitations to the use of immunoquantification analysis as a sole measure of patient phenotype. Enzyme kinetic analysis of alpha-L-iduronidase from MPS I fibroblasts revealed a number of patients with either abnormal substrate binding or catalytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the novel protein kinase activity present in the R1 subunit of herpes simplex virus ribonucleotide reductase.

We have compared the protein kinase activities of the R1 subunits from herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) ribonucleotide reductase following expression in Escherichia coli. Autophosphorylation activity was observed when kinase assays were performed with immunoprecipitated R1 or proteins purified to homogeneity, and the activity was stimulated by the basic protein protamine. Transphosphorylation of histones or calmodulin by purified or immunoprecipitated HSV-1 and HSV-2 R1 was not observed, and our results suggest that the activities of these two proteins are similar. We further characterized the protein kinase activity of HSV-1 R1 by producing insertion and deletion mutants constructed with a plasmid expressing R1 amino acids 1 to 449. C-terminal deletion analysis identified the catalytic core of the enzyme as comprising residues 1 to 292, and this polypeptide will be useful for structural determinations by X-ray crystallography. Insertion of a 4-amino-acid sequence at sites within the protein kinase domain identified regions essential for activity; insertions at residues 22 and 112 completely inactivated activity, and an insertion at residue 136 reduced activity sixfold. Similar insertions at residues 257, 262, 292, and 343 had no effect on activity. The ATP analog 5'-fluorosulfonylbenzoyladenosine, which covalently modifies conventional eukaryotic kinases at an essential lysine residue within the active site, did label HSV R1, but this labelling occurred outside the N-terminal domain. These data indicate that the HSV R1 kinase is novel and distinct from other eukaryotic protein kinases.