The BMP Ligand Gdf6 Prevents Differentiation of Coronal Suture Mesenchyme in Early Cranial Development

Growth Differentiation Factor-6 (Gdf6) is a member of the Bone Morphogenetic Protein (BMP) family of secreted signaling molecules. Previous studies have shown that Gdf6 plays a role in formation of a diverse subset of skeletal joints. In mice, loss of Gdf6 results in fusion of the coronal suture, the intramembranous joint that separates the frontal and parietal bones. Although the role of GDFs in the development of cartilaginous limb joints has been studied, limb joints are developmentally quite distinct from cranial sutures and how Gdf6 controls suture formation has remained unclear. In this study we show that coronal suture fusion in the Gdf6−/− mouse is due to accelerated differentiation of suture mesenchyme, prior to the onset of calvarial ossification. Gdf6 is expressed in the mouse frontal bone primordia from embryonic day (E) 10.5 through 12.5. In the Gdf6−/− embryo, the coronal suture fuses prematurely and concurrently with the initiation of osteogenesis in the cranial bones. Alkaline phosphatase (ALP) activity and Runx2 expression assays both showed that the suture width is reduced in Gdf6+/− embryos and is completely absent in Gdf6−/− embryos by E12.5. ALP activity is also increased in the suture mesenchyme of Gdf6+/− embryos compared to wild-type. This suggests Gdf6 delays differentiation of the mesenchyme occupying the suture, prior to the onset of ossification. Therefore, although BMPs are known to promote bone formation, Gdf6 plays an inhibitory role to prevent the osteogenic differentiation of the coronal suture mesenchyme

SNPs in Multi-Species Conserved Sequences (MCS) as useful markers in association studies: a practical approach

<p>Abstract</p> <p>Background</p> <p>Although genes play a key role in many complex diseases, the specific genes involved in most complex diseases remain largely unidentified. Their discovery will hinge on the identification of key sequence variants that are conclusively associated with disease. While much attention has been focused on variants in protein-coding DNA, variants in noncoding regions may also play many important roles in complex disease by altering gene regulation. Since the vast majority of noncoding genomic sequence is of unknown function, this increases the challenge of identifying "functional" variants that cause disease. However, evolutionary conservation can be used as a guide to indicate regions of noncoding or coding DNA that are likely to have biological function, and thus may be more likely to harbor SNP variants with functional consequences. To help bias marker selection in favor of such variants, we devised a process that prioritizes annotated SNPs for genotyping studies based on their location within Multi-species Conserved Sequences (MCSs) and used this process to select SNPs in a region of linkage to a complex disease. This allowed us to evaluate the utility of the chosen SNPs for further association studies. Previously, a region of chromosome 1q43 was linked to Multiple Sclerosis (MS) in a genome-wide screen. We chose annotated SNPs in the region based on location within MCSs (termed MCS-SNPs). We then obtained genotypes for 478 MCS-SNPs in 989 individuals from MS families.</p> <p>Results</p> <p>Analysis of our MCS-SNP genotypes from the 1q43 region and comparison to HapMap data confirmed that annotated SNPs in MCS regions are frequently polymorphic and show subtle signatures of selective pressure, consistent with previous reports of genome-wide variation in conserved regions. We also present an online tool that allows MCS data to be directly exported to the UCSC genome browser so that MCS-SNPs can be easily identified within genomic regions of interest.</p> <p>Conclusion</p> <p>Our results showed that MCS can easily be used to prioritize markers for follow-up and candidate gene association studies. We believe that this novel approach demonstrates a paradigm for expediting the search for genes contributing to complex diseases.</p

Mapping of the neural retina leucine zipper gene, Nrl , to mouse Chromosome 14

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46998/1/335_2004_Article_BF00361397.pd

Recurrent Tissue-Specific Mtdna Mutations are Common in Humans

Mitochondrial DNA (mtDNA) variation can affect phenotypic variation; therefore, knowing its distribution within and among individuals is of importance to understanding many human diseases. Intra-individual mtDNA variation (heteroplasmy) has been generally assumed to be random. We used massively parallel sequencing to assess heteroplasmy across ten tissues and demonstrate that in unrelated individuals there are tissue-specific, recurrent mutations. Certain tissues, notably kidney, liver and skeletal muscle, displayed the identical recurrent mutations that were undetectable in other tissues in the same individuals. Using RFLP analyses we validated one of the tissue-specific mutations in the two sequenced individuals and replicated the patterns in two additional individuals. These recurrent mutations all occur within or in very close proximity to sites that regulate mtDNA replication, strongly implying that these variations alter the replication dynamics of the mutated mtDNA genome. These recurrent variants are all independent of each other and do not occur in the mtDNA coding regions. The most parsimonious explanation of the data is that these frequently repeated mutations experience tissue-specific positive selection, probably through replication advantage

Regenerative Effects of Transplanted Mesenchymal Stem Cells in Fracture Healing

Mesenchymal stem cells (MSC) have a therapeutic potential in patients with fractures to reduce the time of healing and treat non-unions. The use of MSC to treat fractures is attractive as it would be implementing a reparative process that should be in place but occurs to be defective or protracted and MSC effects would be needed only for the repairing time that is relatively brief. However, an integrated approach to define the multiple regenerative contributions of MSC to the fracture repair process is necessary before clinical trials are initiated. In this study, using a stabilized tibia fracture mouse model, we determined the dynamic migration of transplanted MSC to the fracture site, their contributions to the repair process initiation and their role in modulating the injury-related inflammatory responses. Using MSC expressing luciferase, we determined by bioluminescence imaging that the MSC migration at the fracture site is time- and dose-dependent and, it is exclusively CXCR4-dependent. MSC improved the fracture healing affecting the callus biomechanical properties and such improvement correlated with an increase in cartilage and bone content, and changes in callus morphology as determined by micro-computed-tomography and histological studies. Transplanting CMV-Cre-R26R-LacZ-MSC, we found that MSC engrafted within the callus endosteal niche. Using MSC from BMP-2-Lac-Z mice genetically modified using a bacterial artificial chromosome system to be β-gal reporters for BMP-2 expression, we found that MSC contributed to the callus initiation by expressing BMP-2. The knowledge of the multiple MSC regenerative abilities in fracture healing will allow to design novel MSC-based therapies to treat fractures

Abrupt climatic events during the last glacial-interglacial transition in Alaska

Evidence is mounting that abrupt climatic shifts occurred during the last glacial-interglacial transition (LGIT) in the North Atlantic and other regions. However, few high-resolution climatic records of the LGIT exist from the high latitudes of the North Pacific rim. We analyzed lake sediments from southwestern Alaska for biogenic silica, organic carbon, organic nitrogen, diatom assemblages, and compound-specific hydrogen isotopes. Results reveal climatic changes coincident with the Younger Dryas, Intra-Allerod Cold Period, and Pre-Boreal Oscillation. However, major discrepancies exist in the paleoclimate patterns of the Bolling-Allerod interstadial between our data and the GISP2 18O record from Greenland, and causes are uncertain. These data suggest that the North Pacific and North Atlantic experienced similar reversals during climatic warming of the LGIT but that the Bolling-Allerod cooling trend in the GISP2 18O record is probably not a hemispheric or global pattern

‘You can never cross the same river twice’: climbers’ embodied quests for ‘original adventure’ in southern Thailand

This article presents ethnographic research into individual narratives of adventure in a small, undeveloped bay called Ton Sai in southern Thailand’s Krabi Province. Ton Sai is extremely popular with Western rock climbers and increasingly with other adventure seekers and backpackers questing for ‘authentic’ Thailand, yet is subject to almost no representation in the commercial sense. It is an example of a destination that is not on the corporate ‘radar’, yet, as will be seen, is famed, desired and produced by ‘niche’ tourists seeking very specifically valued tropical adventures. This research aims to interrogate how such a destination becomes, and remains, valued as adventurous by climbers and therefore shed some light on individual, subjective production of adventure in specific Developing World contexts. Drawing on original interview and other ethnographic data collected during winter 2012/2013, this article argues that even when third-party commercial mediators are absent, the powers of quest for authenticity and adventure are powerful enough to turn the wheels of mediation themselves. In the ‘elite circles’ in which this group manoeuvres, notions of ideal adventure space run deep and are reproduced discursively and through embodied performances in an exoticised environment that is valued for its ‘primitive timelessness’. The implications of this for locals are explored

Evolution of N-terminal sequences of the vertebrate HOXA13 protein

While the the role of the homeodomain in HOX function has been evaluated extensively, little attention has been given to the non-homeodomain portions of the HOX proteins. To investigate the evolution of the HOXA13 protein and to identify conserved residues in the N-terminal region of the protein with potential functional significance, N-terminal Hoxa13 coding sequences were PCR-amplified from fish, amphibian, reptile, chicken, and marsupial and eutherian mammal genomic DNA. Compared with fish HOXA13, the mammalian protein has increased in size by 35% primarily owing to the accumulation of alanine repeats and flanking segments rich in proline, glycine, or serine within the first 215 amino acids. Certain residues and amino acid motifs were strongly conserved, and several HOXA13 N-terminal domains were also shared in the paralogous HOXB13 and HOXD13 genes; however, other conserved regions appear to be unique to HOXA13. Two domains highly conserved in HOXA13 orthologs are shared with Drosophila AbdB and other vertebrate AbdB-like proteins. Marsupial and eutherian mammalian HOXA13 proteins have three large homopolymeric alanine repeats of 14, 12, and 17–18 residues that are absent in reptiles, birds, and fish. Thus, the repeats arose after the divergence of reptiles from the lineage that would give rise to the mammals. In contrast, other short homopolymeric alanine repeats in mammalian HOXA13 have remained virtually the same length, suggesting that forces driving or limiting repeat expansion are context dependent. Consecutive stretches of identical third-base usage in alanine codons within the large repeats were found, supporting replication slippage as a mechanism for their generation. However, numerous species-specific base substitutions affecting third-base alanine repeat codon positions were observed, particularly in the largest repeat. Therefore, if the large alanine repeats were present prior to eutherian mammal development as is suggested by the opossum data, then a dynamic process of recurring replication slippage and point mutation within alanine repeat codons must be considered to reconcile these observations. This model might also explain why the alanine repeats are flanked by proline, serine, and glycine-rich sequences, and it reveals a biological mechanism that promotes increases in protein size and, potentially, acquisition of new functions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42120/1/335-11-2-151_00110151.pd

Alkaline-phosphatase (ALP) staining in the presumptive suture.

<p>Transverse sections through the E12.5 wild-type suture showed a gap in ALP activity between the frontal and parietal bones (A). The width of the gap was reduced in the <i>Gdf6</i>+/−, with an increase in ALP activity within the suture (B). In the <i>Gdf6</i>−/−, there is a continuous line of ALP activity through the region, with a reduction in ALP activity in the parietal portion of the fused bone (C). Similar changes in ALP staining were observed in the E13.5 and E14.5 rostral (G–I, M–O) and caudal (J–L, P–R) coronal sutures and through <i>in situ</i> hybridization for <i>Runx2</i> (D–F).</p