Intra-Organ Variation in Age-Related Mutation Accumulation in the Mouse

Using a transgenic mouse model harboring chromosomally integrated lacZ mutational target genes, we previously demonstrated that mutations accumulate with age much more rapidly in the small intestine than in the brain. Here it is shown that in the small intestine point mutations preferentially accumulate in epithelial cells of the mucosa scraped off the underlying serosa. The mucosal cells are the differentiated villus cells that have undergone multiple cell divisions. A smaller age-related increase, also involving genome rearrangements, was observed in the serosa, which consists mainly of the remaining crypts and non-dividing smooth muscle cells. In the brain we observed an accumulation of only point mutations in no other areas than hypothalamus and hippocampus. To directly test for cell division as the determining factor in the generation of point mutations we compared mutation induction between mitotically active and quiescent embryonic fibroblasts from the same lacZ mice, treated with either UV (a point mutagen) or hydrogen peroxide (a clastogen). The results indicate that while point mutations are highly replication-dependent, genome rearrangements are as easily induced in non-dividing cells as in mitotically active ones. This strongly suggests that the point mutations found to have accumulated in the mucosal part of the small intestine are the consequence of replication errors. The same is likely true for point mutations accumulating in hippocampus and hypothalamus of the brain since neurogenesis in these two areas continues throughout life. The observed intra-organ variation in mutation susceptibility as well as the variation in replication dependency of different types of mutations indicates the need to not only extend observations made on whole organs to their sub-structures but also take the type of mutations and mitotic activity of the cells into consideration. This should help elucidating the impact of genome instability and its consequences on aging and disease

Mice with targeted disruptions in the paralogous genes hoxa-3 and hoxd-3 reveal synergistic interactions.

Journal ArticleThe Hox genes encode transcription factors which mediate the formation of the mammalian body plan along the anteroposterior and appendicular axes. Paralogous Hox genes within the separate linkage groups are closely related with respect to DNA sequence and expression, suggesting that they could have at least partially redundant functions. We showed previously that mice homozygous for independent targeted disruptions in the paralogous genes hoxa-3 and hoxd-3 had no defects in common. But our current analysis of double mutants has revealed strong, dosage-dependent interactions between these genes. We report here that in hoxd-3- homozygotes the first cervical vertebra, the atlas, is homeotically transformed to the adjacent anterior structure. Unexpectedly, in double mutants, rather than observing a more extensive homeotic transformation, the entire atlas is deleted. These observations are interpreted in terms of a model in which these Hox genes differentially regulate the proliferation rates of the appropriate sets of precursor cells

Age- and Temperature-Dependent Somatic Mutation Accumulation in Drosophila melanogaster

Using a transgenic mouse model harboring a mutation reporter gene that can be efficiently recovered from genomic DNA, we previously demonstrated that mutations accumulate in aging mice in a tissue-specific manner. Applying a recently developed, similar reporter-based assay in Drosophila melanogaster, we now show that the mutation frequency at the lacZ locus in somatic tissue of flies is about three times as high as in mouse tissues, with a much higher fraction of large genome rearrangements. Similar to mice, somatic mutations in the fly also accumulate as a function of age, but they do so much more quickly at higher temperature, a condition which in invertebrates is associated with decreased life span. Most mutations were found to accumulate in the thorax and less in abdomen, suggesting the highly oxidative flight muscles as a possible source of genotoxic stress. These results show that somatic mutation loads in short-lived flies are much more severe than in the much longer-lived mice, with the mutation rate in flies proportional to biological rather than chronological aging

Molecular evolution of HoxA13 and the multiple origins of limbless morphologies in amphibians and reptiles

Developmental processes and their results, morphological characters, are inherited through transmission of genes regulating development. While there is ample evidence that cis-regulatory elements tend to be modular, with sequence segments dedicated to different roles, the situation for proteins is less clear, being particularly complex for transcription factors with multiple functions. Some motifs mediating protein-protein interactions may be exclusive to particular developmental roles, but it is also possible that motifs are mostly shared among different processes. Here we focus on HoxA13, a protein essential for limb development. We asked whether the HoxA13 amino acid sequence evolved similarly in three limbless clades: Gymnophiona, Amphisbaenia and Serpentes. We explored variation in ω (dN/dS) using a maximum-likelihood framework and HoxA13sequences from 47 species. Comparisons of evolutionary models provided low ω global values and no evidence that HoxA13 experienced relaxed selection in limbless clades. Branch-site models failed to detect evidence for positive selection acting on any site along branches of Amphisbaena and Gymnophiona, while three sites were identified in Serpentes. Examination of alignments did not reveal consistent sequence differences between limbed and limbless species. We conclude that HoxA13 has no modules exclusive to limb development, which may be explained by its involvement in multiple developmental processes

LIFE BEEF CARBON: a common framework for quantifying grass and corn based beef farms’ carbon footprints

peer-reviewedEurope’s roadmap to a low-carbon economy aims to cut greenhouse gas (GHG) emissions 80% below 1990 levels by 2050. Beef production is an important source of GHG emissions and is expected to increase as the world population grows. LIFE BEEF CARBON is a voluntary European initiative that aims to reduce GHG emissions per unit of beef (carbon footprint) by 15% over a 10-year period on 2172 farms in four large beef-producing countries. Changes in farms beef carbon footprint are normally estimated via simulation modelling, but the methods current models apply differ. Thus, our initial goal was to develop a common modelling framework to estimate beef farms carbon footprint. The framework was developed for a diverse set of Western Europe farms located in Ireland, Spain, Italy and France. Whole farm and life cycle assessment (LCA) models were selected to quantify emissions for the different production contexts and harmonized. Carbon Audit was chosen for Ireland, Bovid-CO2 for Spain and CAP’2ER for France and Italy. All models were tested using 20 case study farms, that is, 5 per country and quantified GHG emissions associated with on-farm live weight gain. The comparison showed the ranking of beef systems gross carbon footprint was consistent across the three models. Suckler to weaning or store systems generally had the highest carbon footprint followed by suckler to beef systems and fattening beef systems. When applied to the same farm, Carbon Audit’s footprint estimates were slightly lower than CAP’2ER, but marginally higher than Bovid-CO2. These differences occurred because the models were adapted to a specific region’s production circumstances, which meant their emission factors for key sources; that is, methane from enteric fermentation and GHG emissions from concentrates were less accurate when used outside their target region. Thus, for the common modelling framework, regionspecific LCA models were chosen to estimate beef carbon footprints instead of a single generic model. Additionally, the Carbon Audit and Bovid-CO2 models were updated to include carbon removal by soil and other environmental metrics included in CAP’2ER, for example, acidification. This allows all models to assess the effect carbon mitigation strategies have on other potential pollutants. Several options were identified to reduce beef farms carbon footprint, for example, improving genetic merit. These options were assessed for beef systems, and a mitigation plan was created by each nation. The cumulative mitigation effect of the LIFE BEEF CARBON plan was estimated to exceed the projects reduction target (−15%)

Molars and incisors: show your microarray IDs.

BACKGROUND: One of the key questions in developmental biology is how, from a relatively small number of conserved signaling pathways, is it possible to generate organs displaying a wide range of shapes, tissue organization, and function. The dentition and its distinct specific tooth types represent a valuable system to address the issues of differential molecular signatures. To identify such signatures, we performed a comparative transcriptomic analysis of developing murine lower incisors, mandibular molars and maxillary molars at the developmental cap stage (E14.5). RESULTS: 231 genes were identified as being differentially expressed between mandibular incisors and molars, with a fold change higher than 2 and a false discovery rate lower than 0.1, whereas only 96 genes were discovered as being differentially expressed between mandibular and maxillary molars. Numerous genes belonging to specific signaling pathways (the Hedgehog, Notch, Wnt, FGF, TGFβ/BMP, and retinoic acid pathways), and/or to the homeobox gene superfamily, were also uncovered when a less stringent fold change threshold was used. Differential expressions for 10 out of 12 (mandibular incisors versus molars) and 9 out of 10 selected genes were confirmed by quantitative reverse transcription-PCR (qRT-PCR). A bioinformatics tool (Ingenuity Pathway Analysis) used to analyze biological functions and pathways on the group of incisor versus molar differentially expressed genes revealed that 143 genes belonged to 9 networks with intermolecular connections. Networks with the highest significance scores were centered on the TNF/NFκB complex and the ERK1/2 kinases. Two networks ERK1/2 kinases and tretinoin were involved in differential molar morphogenesis. CONCLUSION: These data allowed us to build several regulatory networks that may distinguish incisor versus molar identity, and may be useful for further investigations of these tooth-specific ontogenetic programs. These programs may be dysregulated in transgenic animal models and related human diseases leading to dental anomalies.journal articleresearch support, non-u.s. gov't2013 Mar 262013 03 26importe

EpCAM and the biology of hepatic stem/progenitor cells

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein, which is frequently and highly expressed on carcinomas, tumor-initiating cells, selected tissue progenitors, and embryonic and adult stem cells. During liver development, EpCAM demonstrates a dynamic expression, since it can be detected in fetal liver, including cells of the parenchyma, whereas mature hepatocytes are devoid of EpCAM. Liver regeneration is associated with a population of EpCAM-positive cells within ductular reactions, which gradually lose the expression of EpCAM along with maturation into hepatocytes. EpCAM can be switched on and off through a wide panel of strategies to fine-tune EpCAM-dependent functional and differentiative traits. EpCAM-associated functions relate to cell–cell adhesion, proliferation, maintenance of a pluripotent state, regulation of differentiation, migration, and invasion. These functions can be conferred by the full-length protein and/or EpCAM-derived fragments, which are generated upon regulated intramembrane proteolysis. Control by EpCAM therefore not only depends on the presence of full-length EpCAM at cellular membranes but also on varying rates of the formation of EpCAM-derived fragments that have their own regulatory properties and on changes in the association of EpCAM with interaction partners. Thus spatiotemporal localization of EpCAM in immature liver progenitors, transit-amplifying cells, and mature liver cells will decisively impact the regulation of EpCAM functions and might be one of the triggers that contributes to the adaptive processes in stem/progenitor cell lineages. This review will summarize EpCAM-related molecular events and how they relate to hepatobiliary differentiation and regeneration

Nutritional Factors Modulating Alu Methylation inan Italian Sample from The Mark-Age StudyIncluding Offspring of Healthy Nonagenarians

Alu hypomethylation promotes genomic instability and is associated with aging and age-related diseases. Dietary factors affect global DNA methylation, leading to changes in genomic stability and gene expression with an impact on longevity and the risk of disease. This preliminary study aims to investigate the relationship between nutritional factors, such as circulating trace elements, lipids and antioxidants, and Alu methylation in elderly subjects and offspring of healthy nonagenarians. Alu DNA methylation was analyzed in sixty RASIG (randomly recruited age-stratified individuals from the general population) and thirty-two GO (GeHA offspring) enrolled in Italy in the framework of the MARK-AGE project. Factor analysis revealed a different clustering between Alu CpG1 and the other CpG sites. RASIG over 65 years showed lower Alu CpG1 methylation than those of GO subjects in the same age class. Moreover, Alu CpG1 methylation was associated with fruit and whole-grain bread consumption, LDL2-Cholesterol and plasma copper. The preserved Alu methylation status in GO, suggests Alu epigenetic changes as a potential marker of aging. Our preliminary investigation shows that Alu methylation may be affected by food rich in fibers and antioxidants, or circulating LDL subfractions and plasma copper

Developmental expression of retinoic acid receptors (RARs)

Here, I review the developmental expression features of genes encoding the retinoic acid receptors (RARs) and the 'retinoid X' or rexinoid receptors (RXRs). The first detailed expression studies were performed in the mouse over two decades ago, following the cloning of the murine Rar genes. These studies revealed complex expression features at all stages of post-implantation development, one receptor gene (Rara) showing widespread expression, the two others (Rarb and Rarg) with highly regionalized and/or cell type-specific expression in both neural and non-neural tissues. Rxr genes also have either widespread (Rxra, Rxrb), or highly-restricted (Rxrg) expression patterns. Studies performed in zebrafish and Xenopus demonstrated expression of Rar and Rxr genes (both maternal and zygotic), at early pre-gastrulation stages. The eventual characterization of specific enzymes involved in the synthesis of retinoic acid (retinol/retinaldehyde dehydrogenases), or the triggering of its catabolism (CYP26 cytochrome P450s), all of them showing differential expression patterns, led to a clearer understanding of the phenomenons regulated by retinoic acid signaling during development. Functional studies involving targeted gene disruptions in the mouse, and additional approaches such as dominant negative receptor expression in other models, have pinpointed the specific, versus partly redundant, roles of the RARs and RXRs in many developing organ systems. These pleiotropic roles are summarized hereafter in relationship to the receptors’ expression patterns