Independent polled mutations leading to complex gene expression differences in cattle

The molecular regulation of horn growth in ruminants is still poorly understood. To investigate this process, we collected 1019 hornless (polled) animals from different cattle breeds. High-density SNP genotyping confirmed the presence of two different polled associated haplotypes in Simmental and Holstein cattle co-localized on BTA 1. We refined the critical region of the Simmental polled mutation to 212 kb and identified an overlapping region of 932 kb containing the Holstein polled mutation. Subsequently, whole genome sequencing of polled Simmental and Holstein cows was used to determine polled associated genomic variants. By genotyping larger cohorts of animals with known horn status we found a single perfectly associated insertion/deletion variant in Simmental and other beef cattle confirming the recently published possible Celtic polled mutation. We identified a total of 182 sequence variants as candidate mutations for polledness in Holstein cattle, including an 80 kb genomic duplication and three SNPs reported before. For the first time we showed that hornless cattle with scurs are obligate heterozygous for one of the polled mutations. This is in contrast to published complex inheritance models for the bovine scurs phenotype. Studying differential expression of the annotated genes and loci within the mapped region on BTA 1 revealed a locus (LOC100848215), known in cow and buffalo only, which is higher expressed in fetal tissue of wildtype horn buds compared to tissue of polled fetuses. This implicates that the presence of this long noncoding RNA is a prerequisite for horn bud formation. In addition, both transcripts associated with polledness in goat and sheep (FOXL2 and RXFP2), show an overexpression in horn buds confirming their importance during horn development in cattle.

High tumor budding stratifies breast cancer with metastatic properties.

Tumor budding refers to single or small cluster of tumor cells detached from the main tumor mass. In colon cancer high tumor budding is associated with positive lymph nodes and worse prognosis. Therefore, we investigated the value of tumor budding as a predictive feature of lymph node status in breast cancer (BC). Whole tissue sections from 148 surgical resection specimens (SRS) and 99 matched preoperative core biopsies (CB) with invasive BC of no special type were analyzed on one slide stained with pan-cytokeratin. In SRS, the total number of intratumoral (ITB) and peripheral tumor buds (PTB) in ten high-power fields (HPF) were counted. A bud was defined as a single tumor cell or a cluster of up to five tumor cells. High tumor budding equated to scores averaging >4 tumor buds across 10HPFs. In CB high tumor budding was defined as ≥10 buds/HPF. The results were correlated with pathological parameters. In SRS high PTB stratified BC with lymph node metastases (p ≤ 0.03) and lymphatic invasion (p ≤ 0.015). In CB high tumor budding was significantly (p = 0.0063) associated with venous invasion. Pathologists are able, based on morphology, to categorize BC into a high and low risk groups based in part on lymph node status. This risk assessment can be easily performed during routine diagnostics and it is time and cost effective. These results suggest that high PTB is associated with loco-regional metastasis, highlighting the possibility that this tumor feature may help in therapeutic decision-making.

Unearthing the roots of degradation of Quercus pyrenaica coppices: A root-to-shoot imbalance caused by historical management?

Slow growth, branch dieback and scarce acorn yield are visible symptoms of decay in abandoned Quercus pyrenaica coppices. A hypothetical root-to-shoot (R:S) imbalance provoked by historical coppicing is investigated as the underlying driver of stand degradation. After stem genotyping, 12 stems belonging to two clones covering 81 and 16 m2 were harvested and excavated to measure above- and below-ground biomass and nonstructural carbohydrate (NSC) pools. To study root system functionality, root connections and root longevity were assessed by radiocarbon analysis. Seasonality of NSC was monitored on five additional clones. NSC pools, R:S biomass ratio and fine roots-to-foliage ratio were higher in the large clone, whose centennial root system, estimated to be 550 years old, maintained large amounts of sapwood (51.8%) for NSC storage. 248 root connections were observed within the large clone, whereas the small clone showed comparatively simpler root structure (26 connections). NSC concentrations were higher in spring (before bud burst) and autumn (before leaf fall), and lower in summer (after complete leaf expansion); they were always higher in roots than in stems or twigs. The persistence of massive and highly inter-connected root systems after coppicing may lead to increasing R:S biomass ratios and root NSC pools over time. We highlight the need of surveying belowground organs to understand aboveground dynamics of Q. pyrenaica, and suggest that enhanced belowground NSC storage and consumption reflect a trade-off between clonal vegetative resilience and aboveground performance.

The Osr1 and Osr2 genes act in the pronephric anlage downstream of retinoic acid signaling and upstream of Wnt2b to maintain pectoral fin development.

Vertebrate odd-skipped related genes (Osr) have an essential function during the formation of the intermediate mesoderm (IM) and the kidney structures derived from it. Here, we show that these genes are also crucial for limb bud formation in the adjacent lateral plate mesoderm (LPM). Reduction of zebrafish Osr function impairs fin development by the failure of tbx5a maintenance in the developing pectoral fin bud. Osr morphant embryos show reduced wnt2b expression, and increasing Wnt signaling in Osr morphant embryos partially rescues tbx5a expression. Thus, Osr genes control limb bud development in a non-cell-autonomous manner, probably through the activation of Wnt2b. Finally, we demonstrate that Osr genes are downstream targets of retinoic acid (RA) signaling. Therefore, Osr genes act as a relay within the genetic cascade of fin bud formation: by controlling the expression of the signaling molecule Wnt2ba in the IM they play an essential function transmitting the RA signaling originated in the somites to the LPM.

Larix decidua and other larches in Europe: distribution, habitat, usage and threats

The European larch (Larix decidua Mill.) is a pioneer, very long-lived, fast-growing coniferous tree, which occurs in the central and eastern mountains of Europe, forming open forests or pasture woods at the upper tree limits. Larch is the only deciduous conifer in Europe as an adaptation to continental alpine climates. In fact, it is able to tolerate very cold temperatures during winter and, by losing its needles, avoids foliage desiccation. It is a transitional species, colonising open terrain after natural disturbances. It forms pure stands but more often it is found with other alpine tree species, which tend to replace it if no other disturbances occur. Thanks to its adaptability and the durability of its wood, the European larch represents an important silvicultural tree species in the alpine regions, planted even outside its natural ranges. Its wood is largely used for carpentry, furniture and pulp for paper. In lower altitudes or with high precipitation rates, larch is more susceptible to fungal diseases. Outbreaks of insect defoliators, principally caused by the larch bud moth (Zeiraphera diniana), can limit tree development, with economic losses in plantations, but they rarely lead to the death of the trees.

Analysis of BMP signalling through the receptor type IA in embryogenesis using conditional gene inactivation in mice

Although bone morphogenetic proteins (BMPs) were initially identified for their potent bone-inducing activity, their precise roles in processes of endochondral and intramembranous bone formation are far from being clear. Tissue-specific loss-of-function experiments using the BMP receptor type IA (BMPR-IA) are particularly attractive since this receptor is thought to be essential for signaling by the closely related BMPs -2, 4, and 7. To ablate signaling through this receptor during chondrogenesis, we have generated transgenic mice expressing Cre recombinase under the control of the collagen type II (Col2a1) gene regulatory sequences. Mice lacking BMPR-IA function in chondrocytes display a number of skeletal abnormalities, including defects in bones of the chondrocranium, abnormal dorsal vertebral processes, scapulae with severe hypoplasia of dorsal elements, and shortening of the long bones. Alterations in the growth plate of long bones in mutants suggest that BMPR-IA is not required for early steps of the chondrocyte specification, but is rather important in regulation of terminal differentiation. Molecular analysis revealed noticeable downregulation of the Ihh/Ptch signalling pathway, decreased chondrocyte proliferation rate and deregulation of hypertrophy. ^ In order to elucidate the role of BMP signalling in development of the limb and intramembranous ossification, we have used mice expressing Cre recombinase under control of the Prx1 (MHox) regulatory elements (M. Logan, pers comm.). Cre activity was found in those mice in the developing limb bud mesenchyme, as well as in a subset of cranial neural crest cells. Prx1-Cre-induced conditional mutants display prominent defects in distal limb outgrowth, as well as ossification defects in a number of neural crest-derived calvarial bones. Intriguingly, mutant limbs displayed alterations in patterning along all three axes. Molecular analysis revealed ectopic anterior Shh/Ptch signalling pathway activation and expression of some Hox genes. Observed loss of Msx1 and Msx2 expression in the progress zone correlates with downregulation of Cyclin D1 and decreased distal outgrowth. Abnormal ventral localization of Lmx1b-expressing cells along with observed later morphological abnormalities suggest a novel role for BMP signalling in establishment or maintaining of the dorso-ventral polarity in the limb mesoderm. ^

The role of Lmx1b in the control of dorsoventral compartmentalization in the distal mesenchyme of the developing mouse limb

Lmx1b encodes a LIM-homeodomain transcription factor required for dorso-ventral (D-V) patterning in the mesenchyme of the vertebrate limb. In the absence of Lmx1b function, limbs exhibit a bi-ventral pattern indicating that Lmx1b is required for cells to adopt a dorsal cell fate. However, how Lmx1b specifies dorsal cell fates in the mesenchyme of the distal limb is unknown. Lmx1b is initially expressed throughout the dorsal and ventral limb bud mesenchyme, then becomes dorsally restricted around E10.5. At this stage, there is a sharp boundary between Lmx1b expressing and Lmx1b non-expressing cells. How the dorso-ventral Lmx1b expression boundary is formed and maintained is currently unknown. One mechanism that may contribute to establishing and/or maintaining the Lmx1b expression boundary is if the dorsal mesenchyme is a lineage-based compartment, where different groups of non-mingling cells are separated. Compartment formation has been proposed to rely on compartment-specific selector gene activity which functions to restrict cells to a compartment and specifies the identity of cells within that compartment. Based on the evidence that the dorsal limb identity relies on the expression of Lmx1b in the dorsal half of the limb mesenchyme, we hypothesized that Lmx1b might function as a dorsal limb bud mesenchyme selector gene to set up a dorsal compartment. To test this hypothesis, we developed an inducible CreERT2/ loxP based fate mapping approach that permanently marks Lmx1b wild-type and mutant cells and examined the distribution of their descendents in the developing limb. Our data is the first to show that dorso-ventral lineage compartments exist in the limb bud mesenchyme. Furthermore, Lmx1b is required for maintenance of the dorso-ventral compartment lineage boundary. The behavior of Lmx1b mutant cells that cross into the ventral mesenchyme, as well as previous chimera analysis in which mutant cells spread evenly in the ventral limb and form patches in the dorsal side, suggest that cell affinity differences prevent intermingling of dorsal and ventral cells. ^

Coordination of murine limb muscle, skeleton and connective tissue development by Lmx1b

The underlying genetic defects of a congenital disease Nail-Patella Syndrome are loss-of-function mutations in the LMX1B gene. Lmx1b encodes a LIM-homeodomain transcription factor that is expressed specifically in the dorsal limb bud mesenchyme. Gain- and loss-of-function experiments suggest that Lmx1b is both necessary and sufficient to specify dorsal limb patterning. However, how Lmx1b coordinates patterning of the dorsal tissues in the limb, including muscle, skeleton and connective tissues, remains unknown. One possibility is that each tissue specifies its own pattern cell-autonomously, i.e., Lmx1b is expressed in tissues in which it functions and different tissues do not communicate with each other. Another possibility is that tissues that express Lmx1b interact with adjacent tissues and provide patterning information thereby directing the development of tissues non-cell-autonomously. Previous results showed that Lmx1b is expressed in limb connective tissue and skeleton, but is not expressed in muscle tissue. Moreover, muscles and muscle connective tissue are closely associated during development. Therefore, we hypothesize that Lmx1b controls limb muscle dorsal-ventral (DV) patterning through muscle connective tissue, but regulates skeleton and tendon/ligament development cell-autonomously. ^ To test this hypothesis, we first examined when and where the limb dorsal-ventral asymmetry is established during development. Subsequently, conditional knockout and overexpression experiments were performed to delete or activate Lmx1b in different tissues within the limb. Our results show that deletion of Lmx1b from whole limb mesenchyme results in all dorsal tissues, including muscle, tendon/ligament and skeleton, transforming into ventral structures. Skeleton-specific knockout of Lmx1b led to the dorsal duplication of distal sesamoid and metacarpal bones, but did not affect the pattern formation of other tissues, suggesting that Lmx1b controls skeleton development cell-autonomously. In addition, this skeleton-specific pattern alteration only occurs in distal limb tissues, not proximal limb tissues, indicating different regulatory mechanisms operate along the limb proximal-distal axis. Moreover, skeleton-specific ectopic expression of Lmx1b reveals a complementary skeletal-specific dorsalized phenotype. This result supports a cell-autonomous role for Lmx1b in dorsal-ventral skeletal patterning. This study enriched our understanding of limb development, and the insights from this research may also be applicable for the development of other organs. ^

Targeted Deletion Of Functionally Validated Enhancers Defines Their Role in Mouse Limb Development

Transcriptional enhancers are genomic DNA sequences that contain clustered transcription factor (TF) binding sites. When combinations of TFs bind to enhancer sequences they act together with basal transcriptional machinery to regulate the timing, location and quantity of gene transcription. Elucidating the genetic mechanisms responsible for differential gene expression, including the role of enhancers, during embryological and postnatal development is essential to an understanding of evolutionary processes and disease etiology. Numerous methods are in use to identify and characterize enhancers. Several high-throughput methods generate large datasets of enhancer sequences with putative roles in embryonic development. However, few enhancers have been deleted from the genome to determine their roles in the development of specific structures, such as the limb. Manipulation of enhancers at their endogenous loci, such as the deletion of such elements, leads to a better understanding of the regulatory interactions, rules and complexities that contribute to faithful and variant gene transcription – the molecular genetic substrate of evolution and disease. To understand the endogenous roles of two distinct enhancers known to be active in the mouse embryo limb bud we deleted them from the mouse genome. I hypothesized that deletion of these enhancers would lead to aberrant limb development. The enhancers were selected because of their association with p300, a protein associated with active transcription, and because the human enhancer sequences drive distinct lacZ expression patterns in limb buds of embryonic day (E) 11.5 transgenic mice. To confirm that the orthologous mouse enhancers, mouse 280 and 1442 (M280 and M1442, respectively), regulate expression in the developing limb we generated stable transgenic lines, and examined lacZ expression. In M280-lacZ mice, expression was detected in E11.5 fore- and hindlimbs in a region that corresponds to digits II-IV. M1442-lacZ mice exhibited lacZ expression in posterior and anterior margins of the fore- and hindlimbs that overlapped with digits I and V and several wrist bones. We generated mice lacking the M280 and M1442 enhancers by gene targeting. Intercrosses between M280 -/+ and M1442 -/+, respectively, generated M280 and M1442 null mice, which are born at expected Mendelian ratios and manifest no gross limb malformations. Quantitative real-time PCR of mutant E11.5 limb buds indicated that significant changes in transcriptional output of enhancer-proximal genes accompanied the deletion of both M280 and M1442. In neonatal null mice we observed that all limb bones are present in their expected positions, an observation also confirmed by histology of E18.5 distal limbs. Fine-scale measurement of E18.5 digit bone lengths found no differences between mutant and control embryos. Furthermore, when the developmental progression of cartilaginous elements was analyzed in M280 and M1442 embryos from E13.5-E15.5, transient development defects were not detected. These results demonstrate that M280 and M1442 are not required for mouse limb development. Though M280 is not required for embryonic limb development it is required for the development and/or maintenance of body size – adult M280 mice are significantly smaller than control littermates. These studies highlight the importance of experiments that manipulate enhancers in situ to understand their contribution to development.

The yeast STM1 protein binds G*G multiplex nucleic acids in vitro and associates with ribosomes in vivo

The formation of triple helical, or triplex DNA has been suggested to occur in several cellular processes such as transcription, replication, and recombination. Our laboratory previously found proteins in HeLa nuclear extracts and in S. cerevisiae whole cell extracts that avidly bound a Purine-motif (Pu) triplex probe in gel shift assays, or EMSA. In order to identify a triplex DNA-binding protein, we used conventional and affinity chromatography to purify the major Pu triplex-binding protein in yeast. Peptide microsequencing and data base searches identified this protein as the product of the STM1 gene. Confirmation that Stm1p is a Pu triplex-binding protein was obtained by EMSA using both recombinant Stm1p and whole cell extracts from stm1Δ yeast. Stm1p had previously been identified as G4p2, a G-quartet DNA- and RNA-binding protein. To study the cellular role and identify the nucleic acid ligand of Stm1p in vivo, we introduced an HA epitope at either the N- or C-terminus of Stm1p and performed immunoprecipitations with the HA.11 mAb. Using peptide microsequencing and Northern analysis, we positively identified a subset of both large and small subunit ribosomal proteins and all four rRNAs as associating with Stm1p. DNase I treatment did not affect the association of Stm1p with ribosomal components, but RNase A treatment abolished the association with all ribosomal proteins and RNA, suggesting this association is RNA-dependent. Sucrose gradient fractionation followed by Western and EMSA analysis confirmed that Stm1p associates with intact 80S monosomes, but not polysomes. The presence of additional, unidentified RNA in the Stm1p-immunoprecipitate, and the absence of tRNAs and elongation factors suggests that Stm1p binds RNA and could be involved in the regulation of translation. Immunofluorescence microscopy data showed Stm1p to be located throughout the cytoplasm, with a specific movement to the bud during the G2 phase of the cell cycle. A dramatically flocculent, large cell phenotype is observed when Stm1p has a C-terminal HA tag in a protease-deficient strain background. When STM1 is deleted in this background, the same phenotype is not observed and the deletion yeast grow very slowly compared to the wild-type. These data suggest that STM1 is not essential, but plays a role in cell growth by interacting with an RNP complex that may contain G*G multiplex RNA. ^

Performance of Gibson Golden Delicious on Dwarfing Rootstocks

To evaluate the adaptability and performance of new and promising apple rootstocks in the dwarfing size-control category, a NC-140 regional rootstock trial was established in 2003 at 14 sites in the United States (AR, CA, IA, GA, KY, ME, MI, NY, OH, PA, UT, WI), Canada (BC), and Mexico. The Iowa planting, located at the ISU Horticulture Research Station, includes 23 rootstocks with new selections from the Cornell-Geneva breeding program (G, CG), Russia (Bud), Czech Republic (J-TE), Japan (JM), and Germany (PiAu) with M.26, M.9 Pajam 2, and M.9 T337 serving as industry standards. These rootstocks are being evaluated with Gibson Golden Delicious serving as the test cultivar. This report summarizes the tree-growth and production characteristics through the 2011 growing season.

Second Year Performance of Honeycrisp on 31 Dwarfing Rootstocks in the Iowa Planting of the NC-140 2010 Regional Apple Rootstock Trial

To evaluate the adaptability and performance of new and promising apple rootstocks in the dwarfing size-control category, a NC-140 regional rootstock trial was established in 2010 at 12 sites in the United States (CO, IA, IL, IN, MA, MI, MN, NJ, NY, OH, UT, WI), two sites in Canada (BC, NS), and one site in Mexico (CHIH) with Honeycrisp serving as the test cultivar. The Iowa planting, located at the ISU Horticulture Research Station, includes 31 rootstocks with new selections from the Cornell-Geneva breeding program (G, CG.), Russia (Bud), Germany (PiAu), and Japan (Supp), with M.26, M.9 Pajam 2, and M.9 T337 serving as industry standards. Tissue cultured propagated (TC) rootstocks of G.41, G.202, and G.935 were included for comparison with normal (N) stool bed propagated rootstocks. This report summarizes the tree-growth characteristics of the Iowa planting during the 2011 growing season.

Pollen and macroremains from sediment core Aschenhütte

The Upper Pleistocene sediments of the Aschenhütte sink-hole (west of Herzberg am Harz, Lower Saxony) enable one to make interesting correlations between palynological and geological results. The sequence is composed of limnic-telmatic deposits (Eemain to Lower Weichselian) and loess with paleosoils (Weichselian). Sedimentation started during the hornbeam-dominated phase of the Eemian interglacial period and continued throughout the Eemian, the Weichselian Brörup interstadial (sensu Andersen) and parts of the preceding and the following stadial phases, the Herning and the Rederstall stadials. As opposed to most of the known Eemian sites spruce was a major tree species during the hornbeam-dominated phase of the Eemian. The vegetational development during the interstadial phase does not show a period of climatic deterioration as is the case for the Brörup interstadial when considering regions with a more demanding vegetation or regions close to the natural boundaries of the tree species concerned. Pollen or seeds of Bruckenthalia and Picea omoricoides have not been found in the Aschenhütte cores. The limnic-telmatic sediments interlock with loess-paleosoils (Eemian soil and Lower Weichselian bleaching soils) at the lake shore. They are overlaid by loess paleosoils of the Stillfried-B interstadial (Hattorf soil and Lohne soil). Lake level fluctuations were determined by means of the facies distribution and isochrones as defined by pollen analysis. A relatively high stand of the lake level existed after the end of the Eemian interglacial and during the Brörup interstadial periods. In the course of the Herning stadial period the water level dropped, whereas during the Rederstall stadial phase the lake basin was covered by sediments and therefore dried up.

Comparación de materiales y épocas de plantación para Populus sp. : Mendoza (Argentina)

Este ensayo tuvo como objetivo determinar el material y la época de plantación más adecuada para la instalación en sitio definitivo de un cultivo de álamos con los clones Populus euramericana Conti 12 y Populus deltoides Harvard en las condiciones ambientales de la provincia de Mendoza. La plantación se realizó en dos épocas distintas: otoño y primavera, utilizando como material de plantación en ambos casos: a. barbados: plantas con raíz y brote de 1 año (R1T1); b. plantas recepadas: plantas con raíz de 1 año y brote del año cortado a 30 cm de nivel de suelo (R1T0); c. estacas: 1.5-2.5 cm de diámetro y 30-40 cm de longitud. Los resultados se expresaron en porcentaje de plantas prendidas y en volumen de madera producida por hectárea a los siete años en cada una de las variantes. Para los materiales y condiciones del ensayo el mejor resultado fue para el clon Conti 12 plantación en otoño utilizando barbados (plantas R1T1) o plantación en primavera utilizando estacas, barbados (R1T1) o plantas recepadas (R1T0). Para el clon Harvard, plantación en primavera utilizando planta recepada (R1T0) o estacas.

Las yemas, principal reservorio de levaduras entre los órganos aéreos vegetativos y reproductivos de la vid (Vitis vinifera var. Malbec).

El objetivo de esta investigación fue suministrar nueva evidencia acerca del modelo de permanencia de las levaduras en el ciclo natural de la vid. Se efectuó la observación, la medición del número de levaduras y la descripción morfológica de los diferentes órganos aéreos de la vid. Se procedió a la recolección aséptica de muestras a campo, en yema en actividad, yema en reposo, hoja joven, hoja adulta, ritidomis, zarcillo, capullo floral, flor y fruto. Los resultados revelaron dos momentos de máxima población de levaduras: en yema cerrada a fines de otoño y en yema terminal abierta a mediados de verano. La evolución de las levaduras en función de la superficie del fruto mostró poca relación entre ambas variables, por lo que el valor a considerar sería la cantidad de levaduras por baya como unidad. La ritidomis exhibió valores muy uniformes a lo largo del ciclo vegetativo, asumiendo desde esta perspectiva el papel de reservorio de moderada importancia.