Clonal expansion of early to mid-life mitochondrial DNA point mutations drives mitochondrial dysfunction during human ageing

Age-related decline in the integrity of mitochondria is an important contributor to the human ageing process. In a number of ageing stem cell populations, this decline in mitochondrial function is due to clonal expansion of individual mitochondrial DNA (mtDNA) point mutations within single cells. However the dynamics of this process and when these mtDNA mutations occur initially are poorly understood. Using human colorectal epithelium as an exemplar tissue with a well-defined stem cell population, we analysed samples from 207 healthy participants aged 17-78 years using a combination of techniques (Random Mutation Capture, Next Generation Sequencing and mitochondrial enzyme histochemistry), and show that: 1) non-pathogenic mtDNA mutations are present from early embryogenesis or may be transmitted through the germline, whereas pathogenic mtDNA mutations are detected in the somatic cells, providing evidence for purifying selection in humans, 2) pathogenic mtDNA mutations are present from early adulthood (<20 years of age), at both low levels and as clonal expansions, 3) low level mtDNA mutation frequency does not change significantly with age, suggesting that mtDNA mutation rate does not increase significantly with age, and 4) clonally expanded mtDNA mutations increase dramatically with age. These data confirm that clonal expansion of mtDNA mutations, some of which are generated very early in life, is the major driving force behind the mitochondrial dysfunction associated with ageing of the human colorectal epithelium.

Highly efficient neural differentiation of human somatic stem cells, isolated by minimally invasive periodontal surgery

Neural stem cells (NSCs) are potential sources for cell therapy of neurodegenerative diseases and for drug screening. Despite their potential benefits, ethical and practical considerations limit the application of NSCs derived from human embryonic stem cells (ES) or adult brain tissue. Thus, alternative sources are required to satisfy the criteria of ready accessibility, rapid expansion in chemically defined media and reliable induction to a neuronal fate. We isolated somatic stem cells from the human periodontium that were collected during minimally invasive periodontal access flap surgery as part of guided tissue regeneration therapy. These cells could be propagated as neurospheres in serum-free medium, which underscores their cranial neural crest cell origin. Culture in the presence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) under serum-free conditions resulted in large numbers of nestin-positive/Sox-2-positive NSCs. These periodontium-derived (pd) NSCs are highly proliferative and migrate in response to chemokines that have been described as inducing NSC migration. We used immunocytochemical techniques and RT-PCR analysis to assess neural differentiation after treatment of the expanded cells with a novel induction medium. Adherence to substrate, growth factor deprivation, and retinoic acid treatment led to the acquisition of neuronal morphology and stable expression of markers of neuronal differentiation by more than 90% of the cells. Thus, our novel method might provide nearly limitless numbers of neuronal precursors from a readily accessible autologous adult human source, which could be used as a platform for further experimental studies and has potential therapeutic implications.

Pollen ultrastructure in anther cultures of Datura innoxia. III. Incomplete microspore division

During the microspore division in Datura innoxia, the mitotic spindle is oriented in planes both perpendicular (PE) and oblique (OB) to the spore wall against which the nucleus is situated. However, irrespective of polarity, the usual type of hemispherical wall is laid down at cytokinesis and isolates the generative cell from the rest of the pollen grain (type A). In PE spores the vegetative nucleus initially occupies a central position in the pollen grain, whereas in OB spores the vegetative nucleus lies at the periphery of the grain close to the generative cell. In anther cultures initiated just before the microspore division is due to take place, no marked change can be observed in either orientation or symmetry of the mitotic spindle when the spores divide. In some, however, cytokinesis is disrupted and deposition of the hemispherical wall arrested. In the absence of a complete wall, differentiation of the generative cell cannot take place and binucleate pollen grains are formed having 2 vegetative-type nuclei (type B). The 2 nuclei in the B pollens are always situated against the pollen-grain wall, suggesting that the disruption phenomenon is related to the OB spores. The incomplete wall always makes contact with the intine on the intine-side of the spindle. Wall material may be represented merely as short stubs projecting out from the intine into the cytoplasm, in which event the 2 nuclei lie close to each other and are separated by only a narrow zone of cytoplasm. In other grains the wall is partially developed between the nuclei and terminates at varying distances from the tonoplast; in these, the nuclei are separated by a wider zone of cytoplasm. The significance of these binucleate grains in pollen embryogenesis is discussed.

Pollen ultrastructure in anther cultures of Datura innoxia. II. The generative-cell wall.

In young pollen grains of Datura innoxia, a wall of the usual hemispherical type separates the 2 gametophytic cells initially and, in the electron microscope, appears as an electron-translucent matrix which is contiguous with the intine. Before detachment of the generative cell from the intine, the matrix decreases in thickness and in places is dispersed altogether leaving the plasmalemmae on either side of it in close apposition. A particularly prominent zone, triangular in profile, is left where the wall joins with the intine. After detachment of the cell, remnants of the matrix can be seen distributed irregularly around the cell and it is supposed that these are partly derived from material in the triangular zone as the cell is drawn away from the intine. The wall residues persist throughout the maturation phase of the pollen and are considered to be either callose resulting from incomplete digestion of the initial wall, or some other polysaccharide material which is unevenly laid down along the wall and concentrated at the junction with the intine. In pollen induced into embryogenesis by anther culture, wall material is also distributed irregularly around the detached cell in a series of discrete zones, but these are more extensive than in vivo, closer together and in many instances highly dilated. The wall profiles thus have a beaded appearance, the 'beads' being connected together by short links of the 2 apposed plasmalemmae. The contents of the swollen zones have a similar electron density to that of the matrix in vivo but also show traces of a fibrillar component. It is postulated that this unusual swelling is a prelude to dispersal of the wall by disruption of the plasmalemmal links and to the establishment of cytoplasmic continuity between the 2 cells. The significance of such binucleate pollen grains in the formation of non-haploid embryos is discussed.

Pollen ultrastructure in anther cultures of Datura innoxia. I. Division of the presumptive vegetative cell.

Ultrastructural features of embryogenic pollen in Datura innoxia are described, just prior to, during, and after completion of the first division of the presumptive vegetative cell. In anther cultures initiated towards the end of the microspore phase and incubated at 28 degrees C in darkness, the spores divide within 24 h and show features consistent with those of dividing spores in vivo. Cytokinesis is also normal in most of the spores and the gametophytic cell-plate curves round the presumptive generative nucleus in the usual highly ordered way. Further differentiation of the 2 gametophytic cells does not take place and the pollen either switches to embryogenesis or degenerates. After 48-72 h, the remaining viable pollen shows the vegetative cell in division. The cell, which has a large vacuole and thin layer of parietal cytoplasm carried over from the microspore, divides consistently in a plane parallel to the microspore division. The dividing wall follows a less-ordered course than the gametophytic wall and usually traverses the vacuole, small portions of which are incorporated into the daughter cell adjacent to the generative cell. The only structural changes in the vegetative cell associated with the change in programme appear to be an increase in electron density of both plastids and mitochondria and deposition of an electron-dense material (possibly lipid) on the tonoplast. The generative cell is attached to the intine when the vegetative cell divides. Ribosomal density increases in the generative cell and exceeds that in the vegetative cell. A thin electron-dense layer also appears in the generative-cell wall. It is concluded that embryogenesis commences as soon as the 2 gametophytic cells are laid down. Gene activity associated with postmitotic synthesis of RNA and protein in the vegetative cell is switched off. The data are discussed in relation to the first division of the embryogenic vegetative cells in Nicotiana tabacum.

Effects of corn silage derived from a genetically modified variety containing two Transgenes on feed intake, milk production, and composition, and the absence of detectable Transgenic deoxyribonucleic acid in milk in holstein dairy cows

The objectives were to compare the chemical composition, nutritive value, feed intake, milk production and composition, and presence in milk of transgenic DNA and the encoded protein Cry1Ab when corn silages containing 2 transgenes (2GM: herbicide tolerance: mepsps and insect resistance: cry1Ab) were fed as part of a standard total mixed ration (TMR) compared with a near isogenic corn silage ( C) to 8 multiparous lactating Holstein dairy cows in a single reversal design study. Cows were fed a TMR ration ad libitum and milked twice daily. Diets contained [ dry matter (DM) basis] 45% corn silage, 10% alfalfa hay, and 45% concentrate (1.66 Mcal of net energy for lactation/kg of DM, 15.8% crude protein, 35% neutral detergent fiber, and 4.1% fat). Each period was 28-d long. During the last 4 d of each period, feed intake and milk production data were recorded and milk samples taken for compositional analysis, including the presence of transgenic DNA and Cry1Ab protein. There was no significant difference in the chemical composition between C and 2GM silages, and both were within the expected range (37.6% DM, 1.51 Mcal of net energy for lactation/kg, 8.6% crude protein, 40% neutral detergent fiber, 19.6% acid detergent fiber, pH 3.76, and 62% in vitro DM digestibility). Cows fed the 2GM silage produced milk with slightly higher protein (3.09 vs. 3.00%), lactose ( 4.83 vs. 4.72%) and solids-not-fat (8.60 vs. 8.40%) compared with C. However, the yield (kg/d) of milk (36.5), 3.5% fat-corrected milk (34.4), fat (1.151), protein (1.106), lactose (1.738), and solids-not-fat ( 3.094), somatic cell count (log(10): 2.11), change in body weight (+ 7.8 kg), and condition score (+ 0.09) were not affected by type of silage, indicating no overall production difference. All milk samples were negative for the presence of transgenic DNA from either trait or the Cry1Ab protein. Results indicate that the 2GM silage modified with 2 transgenes did not affect nutrient composition of the silages and had no effect on animal performance and milk composition. No transgenic DNA and Cry1Ab protein were detected in milk.

Difference in competence for in vitro proliferation and ex vitro growth of genetically identical mature and juvenile clones of apomictic Malus species

The apomictic system in Malus wits Used Is a model to examine rejuvenation by generating genetically identical tissue culture lines that had two entirely different developmental origins: either embryo-derived tissues (juvenile clones) or somatic tissue from the adult/mature tree (mature clones). These two lines were then subsequently used to examine in vitro difference between mature (M) and juvenile (J) tissues in potential for shoot, root proliferation and ex vitro (glasshouse) growth. The M clones of M. hupehensis and M. toringoides in vitro had significantly fewer total shoots and shoot more than 2 cm in length per proliferating explant than the J clones and also rooted less efficiently. Ex vitro (glasshouse) juvenile clones had shorter internodes, a greater number of leaves and more dry weight compared to their mature counterparts.

Recent advances in the application of in vitro systems to plant improvement

The horticultural industry was instrumental in the early development and exploitation of genetic techniques over a century ago. This review will describe recent advances in a range of in vitro methods and their application to plant breeding, with especial emphasis on horticultural crops. These methods include improvements in the efficiency of haploid breeding techniques in many fruit and vegetable species using either microspore-derived or ovule-derived plants. Significant molecular information is now available to supplement these essentially empirical approaches and this may enable the more predictable application of these technologies in previously intransigent crops. Similarly there are now improved techniques for isolation of somatic hybrids, by application of either in vitro fertilisation or the culture of excised ovules from interspecific crosses. In addition to examples taken from the traditional scientific literature, emphasis will also be given to the use of patent databases as a valuable source of information on recent novel technologies developed in the commercial world.

Linking molecular and population stress responses in Daphnia magna exposed to cadmium

DNA microarrays can be used to measure environmental stress responses. If they are to be predictive of environmental impact, we need to determine if altered gene expression translates into negative impacts on individuals and populations. A large cDNA microarray (14000 spots) was created to measure molecular stress responses to cadmium in Daphnia magna,the most widely used aquatic indicator species, and relate responses to population growth rate (pgr). We used the array to detect differences in the transcription of genes in juvenile D. magna (24 h old) after 24 h exposure to a control and three cadmium concentrations (6, 20, and 37 mu g Cd2+ L-1). Stress responses at the population level were estimated following a further 8 days exposure. Pgr was approximately linear negative with increasing cadmium concentration over this range. The microarray profile of gene expression in response to acute cadmium exposure begins to provide an overview of the molecular responses of D. magna, especially in relation to growth and development. Of the responding genes, 29% were involved with metabolism including carbohydrate, fat and peptide metabolism, and energy production, 31% were involved with transcription/translation, while 40% of responding genes were associated with cellular processes like growth and moulting, ion transport, and general stress responses (which included oxidative stress). Our production and application of a large Daphnia magna microarray has shown that measured gene responses can be logically linked to the impact of a toxicant such as cadmium on somatic growth and development, and consequently pgr.

Genesis and expansion of metazoan transcription factor gene classes

We know little about the genomic events that led to the advent of a multicellular grade of organization in animals, one of the most dramatic transitions in evolution. Metazoan multicellularity is correlated with the evolution of embryogenesis, which presumably was underpinned by a gene regulatory network reliant on the differential activation of signaling pathways and transcription factors. Many transcription factor genes that play critical roles in bilaterian development largely appear to have evolved before the divergence of cnidarian and bilaterian lineages. In contrast, sponges seem to have a more limited suite of transcription factors, suggesting that the developmental regulatory gene repertoire changed markedly during early metazoan evolution. Using whole- genome information from the sponge Amphimedon queenslandica, a range of eumetazoans, and the choanoflagellate Monosiga brevicollis, we investigate the genesis and expansion of homeobox, Sox, T- box, and Fox transcription factor genes. Comparative analyses reveal that novel transcription factor domains ( such as Paired, POU, and T- box) arose very early in metazoan evolution, prior to the separation of extant metazoan phyla but after the divergence of choanoflagellate and metazoan lineages. Phylogenetic analyses indicate that transcription factor classes then gradually expanded at the base of Metazoa before the bilaterian radiation, with each class following a different evolutionary trajectory. Based on the limited number of transcription factors in the Amphimedon genome, we infer that the genome of the metazoan last common ancestor included fewer gene members in each class than are present in extant eumetazoans. Transcription factor orthologues present in sponge, cnidarian, and bilaterian genomes may represent part of the core metazoan regulatory network underlying the origin of animal development and multicellularity.

The role of Wnt signalling in the development of somites and neural crest

The Wnt family of secreted signalling molecules controls a wide range of developmental processes in all metazoans. In this investigation we concentrate on the role that members of this family play during the development of (1) the somites and (2) the neural crest. (3) We also isolate a novel component of the Wnt signalling pathway called Naked cuticle and investigate the role that this protein may play in both of the previously mentioned developmental processes. (1) In higher vertebrates the paraxial mesoderm undergoes a mesenchymal-to-epithelial transformation to form segmentally organised structures called somites. Experiments have shown that signals originating from the ectoderm overlying the somites or from midline structures are required for the formation of the somites, but their identity has yet to be determined. Wnt6 is a good candidate as a somite epithelialisation factor from the ectoderm since it is expressed in this tissue. In this study we show that injection of Wnt6-producing cells beneath the ectoderm at the level of the segmental plate or lateral to the segmental plate leads to the formation of numerous small epithelial somites. We show that Wnts are indeed responsible for the epithelialisation of somites by applying Wnt antagonists which result in the segmental plate being unable to form somites. These results show that Wnt6, the only member of this family to be localised to the chick paraxial ectoderm, is able to regulate the development of epithelial somites and that cellular organisation is pivotal in the execution of the differentiation programmes. (2) The neural crest is a population of multipotent progenitor cells that arise from the neural ectoderm in all vertebrate embryos and form a multitude of derivatives including the peripheral sensory neurons, the enteric nervous system, Schwann cells, pigment cells and parts of the craniofacial skeleton. The induction of the neural crest relies on an ectodermally derived signal, but the identity of the molecule performing this role in amniotes is not known. Here we show that Wnt6, a protein expressed in the ectoderm, induces neural crest production. (3) The intracellular response to Wnt signalling depends on the choice of signalling cascade activated in the responding cell. Cells can activate either the canonical pathway that modulates gene expression to control cellular differentiation and proliferation, or the non-canonical pathway that controls cell polarity and movement (Pandur et al. 2002b). Recent work has identified the protein Naked cuticle as an intracellular switch promoting the non-canonical pathway at the expense of the canonical pathway. We have cloned chick Naked cuticle-1 (cNkd1) and demonstrate that it is expressed in a dynamic manner during early embryogenesis. We show that it is expressed in the somites and in particular regions where cells are undergoing movement. Lastly our study shows that the expression of cNkd1 is regulated by Wnt expression originating from the neural tube. This study provides evidence that non-canonical Wnt signalling plays a part in somite development.

TGF-beta superfamily members and ovarian follicle development

In recent years, exciting progress has been made towards unravelling the complex intraovarian control mechanisms that, in concert with systemic signals, coordinate the recruitment, selection and growth of follicles from the primordial stage through to ovulation and corpus luteum formation. A plethora of growth factors, many belonging to the transforming growth factor-beta (TGF-beta) superfamily, are expressed by ovarian somatic cells and oocytes in a developmental, stage-related manner and function as intraovarian regulators of folliculogenesis. Two such factors, bone morphogenetic proteins, RMP-4 and BMP-7, are expressed by ovarian stromal cells and/or theca cells and have recently been implicated as positive regulators of the primordial-to-primary follicle transition. In contrast, evidence indicates a negative role for anti-Mullerian hormone (AMH, also known as Mullerian-inhibiting substance) of pre-granulosa/granulosa cell origin in this key event and subsequent progression to the antral stage. Two other TGF-beta superfamily members, growth and differentiation factor-9 (GDF-9) and BMP-15 (also known as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play key roles in promoting follicle growth beyond the primary stage; mice with null mutations in the gdf-9 gene or ewes with inactivating mutations in gdf-9 or bmp-15 genes are infertile with follicle development arrested at the primary stage. Studies on later stages of follicle development indicate positive roles for granulosa cell-derived activin, BMP-2, -5 and -6, theca cell-derived BMP-2, -4 and -7 and oocyte-derived BMP-6 in promoting granulosa cell proliferation, follicle survival and prevention of premature luteinization and/or atresia. Concomitantly, activin, TGF-beta and several BMPs may exert paracrine actions on theca cells to attenuate LH-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection in monovular species may depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Changes in intrafollicular activins, GDF-9, AMH and several BMPs may contribute to this selection process by modulating both FSH- and IGF-dependent signalling pathways in granulosa cells. Activin may also play a positive role in oocyte maturation and acquisition of developmental competence. in addition to its endocrine role to suppress FSH secretion, increased output of inhibin by the selected dominant follicle(s) may upregulate LH-induced androgen secretion that is required to sustain a high level of oestradiol secretion during the pre-ovulatory phase. Advances in our understanding of intraovarian regulatory mechanisms should facilitate the development of new approaches for monitoring and manipulating ovarian function and improving fertility in domesticated livestock, endangered species and man.

The allometry of ornaments and weapons

Exaggerated male traits that have evolved under sexual selection include ornaments to attract mates and weapons to deter rivals. Data from studies of many such traits in diverse kinds of organisms show that they almost universally exhibit positive allometries. Both ornaments and weapons increase disproportionately with overall body size, resulting in scaling exponents within species that are consistently > 1.0 and usually in the range 1.5-2.5. We show how scaling exponents reflect the relative fitness advantages of ornaments vs. somatic growth by using a simple mathematical model of resource allocation during ontogeny. Because the scaling exponents are similar for the different taxonomic groups, it follows that the fitness advantages of investing in ornaments also are similar. The model also shows how selection for ornaments influences body size at first reproduction and explains why interspecific allometries have consistently lower exponents than intraspecific ones.

Expression and regulation of Nkd-1, an intracellular component of Wnt signalling pathway in the chick embryo

The Wnt family of secreted signalling molecules control a wide range of developmental processes in all metazoans. The intracellular response to Wnt signalling depends on the choice of signalling cascade activated in the responding cell. Cells can activate either the canonical pathway that modulates gene expression to control cellular differentiation and proliferation, or the non-canonical pathway that controls cell polarity and movement. Recent work has identified the protein Naked Cuticle to act as an intracellular switch to promote the non-canonical pathway at the expense of the canonical pathway. We have cloned chick Naked Cuticle-1 (cNkd-1) and show that it is expressed in a dynamic manner during early embryogenesis. We show that it is expressed in the somites and in particular regions where cells are undergoing movement. Lastly, we show that the expression of cNkd-1 is regulated by Wnt expression originating from the neural tube. This study provides evidence that non-canonical Wnt signalling plays a part in somite development.

The influence of larval density, food availability and habitat longevity on the life history and population growth rate of the midge Chironomus riparius

Despite long-standing interest in the forms and mechanisms of density dependence, these are still imperfectly understood. However, in a constant environment an increase in density must reduce per capita resource availability, which in turn leads to reduced survival, fecundity and somatic growth rate. Here we report two population experiments examining the density dependent responses under controlled conditions of an important indicator species, Chironomus riparius. The first experiment was run for 35 weeks and was started at low density with replicate populations being fed three different rations. Increased ration reduced generation time and increased population growth rate (pgr) but had no effect on survival, fecundity and female body weight in the first generation. In the second generation there was a six-fold increase in generation time, presumably due to the greatly reduced per capita resource availability as the estimated initial densities of the second generation were 300 times greater than the first. Juvenile survival to emergence, fecundity, adult body weight and pgr declined by 90%, 75%, 35% and 99%, respectively. These large between-generation effects may have obscured the effects of the threefold variation in ration, as only survival to emergence significantly increased with ration in the second generation. These results suggest that some chironomid larvae survive a reduction in resource availability by growing more slowly. In the ephemeral habitats sometimes occupied by C. riparius, the effects of population density may depend crucially on the longevity of the environment. A second experiment was therefore performed to measure pgr from six different starting densities over an eight-week period. The relationship between pgr and density was concave, viewed from above. At densities above 16 larvae per cm(2), less than 1% of the population emerged and no offspring were produced. Under the conditions of experiment 2 - an 8-week habitat lifespan carrying capacity was estimated as 8 larvae per cm(2).