ABSTRACT: Lymphogenous metastasis is an important event in the progression of many human cancers, and is associated with expression of vascular endothelial growth factor-D (VEGF-D). Changes to the lymphatic vasculature can occur during metastasis, and may aid metastatic spread. We investigated the effect of tumour derived VEGFD on the endothelium of the collecting lymphatic vessels draining primary tumors. We used microarrays to detail the changes in gene expression in the collecting lymphatic endothelium of mice with 293EBNA xenografts compared to 293EBNA xenografts overexpressing VEGFD. Mice were injected with 293EBNA cells (transfected with either empty APEX vector, or vector containing VEGFD) and tumours were allowed to grow to size. Mice were sacrificed and collecting lymphatic vessels were dissected. The endothelial cell population was isolated and RNA was extracted and hybridized on Affymetrix microarrays.
Project description:Metastasis to lymph nodes is an early and prognostically important event in the progression of many human cancers, and is associated with expression of vascular endothelial growth factor-D (VEGF-D). Changes to lymph node vasculature occur during metastasis, and may establish a metastatic niche capable of attracting and supporting tumor cells. We used microarrays to characterise the molecular profiles of endothelial cells from lymph nodes draining metastatic (VEGF-D-overexpressing) and non-metastatic tumors, and to identify differentially-expressed genes that might have therapeutic or prognostic potential. Draining lymph nodes of metastatic (VEGF-D-overexpressing) or non-metastatic tumors were pooled from 1-5 mice and enzymatically digested. Lymph nodes draining metastatic tumors were included for the analysis only if macroscopically enlarged, indicating the presence of metastatic cells. After digestion, tumor cells and leukocytes were depleted via immunomagnetic selection, and the resulting lymph node stromal cells were cultured briefly. Podoplanin was then used as a positive immunomagnetic selection marker to enrich for lymphatic and other endothelial cells in the lymph node. RNA was isolated from biological duplicate lymph node endothelial cell (LN EC) preparations and analysed by microarray.
Project description:The QSi5 inbred strain of mice was established from an outbred Quackenbush-Swiss strain by full-sib inbreeding and selection on the basis of increased litter size and shortened inter-litter interval in the Department of Veterinary Physiology (later REPROGEN) , University of Sydney (Holt et al., 2004). The strain has an average litter size of more than 13 pups, and females commonly nurse up to 18 pups with greater than 90% survival to weaning. Along with an increased body weight (BW), these traits are clearly indicative of enhanced lactation performance (Knight et al., 1986). Indeed lactation performance, assessed by a weigh-suckle-weigh method, was 3-fold greater in QSi5 mice than the CBA strain (Riley et al., 2006). In this study, we utilize the divergent phenotypes of QSi5 and CBA/CaH mice to identify genes associated with enhanced mammary gland capacity. Experiment Overall Design: Five replicates from each of the two strains CBA and QSi5 were used for gene expression profiling. Mice were force weaned after 9 days of first lactation and the fourth inguinal mammary glands were collected.
Project description:Lymphogenous metastasis is an important event in the progression of many human cancers, and is associated with expression of vascular endothelial growth factor-D (VEGF-D). Changes to the lymphatic vasculature can occur during metastasis, and may aid metastatic spread. We investigated the effect of tumour derived VEGFD on the endothelium of the collecting lymphatic vessels draining primary tumors. We used microarrays to detail the changes in gene expression in the collecting lymphatic endothelium of mice with 293EBNA xenografts compared to 293EBNA xenografts overexpressing VEGFD. Overall design: Mice were injected with 293EBNA cells (transfected with either empty APEX vector, or vector containing VEGFD) and tumours were allowed to grow to size. Mice were sacrificed and collecting lymphatic vessels were dissected. The endothelial cell population was isolated and RNA was extracted and hybridized on Affymetrix microarrays.
Project description:Arabidopsis thaliana shows hybrid vigour (heterosis) in progeny of crosses between Col and C24 accessions (1). Hybrid vigour was evident as early as the mature seeds and in the seedlings 3 days after sowing (DAS). At 3 DAS genes encoding chloroplast-located proteins were significantly overrepresented (187) among the 724 genes which have greater than mid parent values of expression in the hybrid. Many of these genes are involved in chlorophyll biosynthesis and photosynthesis. The rate of photosynthesis was constant per unit leaf area in parents and hybrids. Larger cell sizes in the hybrids were associated with more chloroplasts per cell, more total chlorophyll and more photosynthesis. The increased transcription of the chloroplast-targeted genes was restricted to the 3 to 7 DAS period. At 10 DAS only 118 genes had expression levels different from the expected mid parent value in the hybrid and only 12 of these genes were differentially expressed at 3 DAS. The early increase in activity of genes involved in photosynthesis and the associated phenomena of increase in cell size and number through development, leading to larger leaf areas of all leaves in the hybrid, suggest a central role for increased photosynthesis in the production of the heterotic biomass. In support of this correlation we found that an inhibitor of photosynthesis eliminated heterosis and higher light intensities enhanced both photosynthesis and heterosis. In hybrids with low level heterosis (Ler x Col) chloroplast-targeted genes were not upregulated and leaf areas were only marginally increased. Whole plants in Col, C24, and their F1 hybrids with two replications
Project description:We compared the expression among three lines, Col, C24, and their hybrids at 10 days after sowing (DAS). Whole plants without root in Col, C24, and their F1 hybrids with three replications.
Project description:The regional specificity and timing of gene activation following chemotherapy, and how this relates to subsequent mucositis development is currently unknown. The aim of the study was therefore to determine the early time course of gene expression changes along the gastrointestinal tract (GIT) of the DA rat following irinotecan treatment, so as to provide an insight into the genetic component of mucositis. We have found that changes in gene expression following irinotecan occur by 1 hour, and persist for at least 72 h after treatment. Overall changes in gene expression are similar along the GIT, however there is temporal variability between regions. Experiment Overall Design: Thirty female DA rats were placed in groups (n = 6) and treated with 200 mg/kg i.p. of irinotecan. Animals were killed at 0, 1, 6, 24 and 72 h and samples of stomach, jejunum and colon collected. These times were chosen to represent very early responses to treatment, up to the time of peak damage and morphological changes. The 0 h group did not receive irinotecan and acted as experimental controls. RNA was extracted from whole tissue samples. RNA was pooled into 2 per group (n = 3) to allow for replication of experiment. Each sample was then hybridised to Affymetrix GeneChip Arrays (Rat 230 2.0).
Project description:Cotton (Gossypium hirsutum L.) fibres are specialised trichomes that extend from the seedcoat. To date only a few genes directly involved in the differentiation of these epidermal cells have been identified. We have identified a HD-ZIP transcription factor, GhHD-1, expressed in trichomes and early fibres that might play a role in cotton fibre initiation. Here we characterise GhHD-1 from G. hirsutum and show, using reporter constructs and qRT-PCR, that they are expressed predominantly in epidermal tissues during early fibre development and in other tissues bearing epidermal trichomes. GhHD-1 silencing caused reduced trichome formation and delayed the timing of fibre initiation whereas constitutive over-expression of GhHD-1 increased the number of fibres initiating on the seed, but did not affect leaf trichomes. Expression of GhHD-1 in transgenic cotton silenced for different fibre MYBs suggest that in ovules it acts downstream of GhMYB25-like. Microarray analysis of silencing and over-expression lines of GhHD-1 indicated that it potentially functions through a WRKY transcription factor and calcium-signalling pathway genes that are shared with some biotic stress reactions. Microarray analysis of 2 biological replicates each of GhHD-1 silenced and over-expression transgenic lines relative to wild-type were used to identify downstream targets of this transcription factor during early fibre development. 0 dpa (Days post anthesis) ovules from GhHD-1 silenced and over-expression lines and wild-type cotton plants were selected for RNA extraction and hybridisation on Affymetrix cotton arrays. Cotton fibre development initiates on the epidermal surface of each ovule within a cotton boll on the day of flowering and GhHD-1 has been shown to be most highly expressed at this stage (0 dpa). Ovules were obtained from 0dpa flowers for hybridisation to cotton arrays to identify genes that may be regulated by GhHD-1 during this early fibre development stage (initiation).
Project description:Water soluble carbohydrates (WSC, composed of mainly fructans, sucrose, glucose and fructose) deposited in wheat stems are important carbon sources for grain filling. Variation in stem WSC concentrations among wheat genotypes is one of the genetic factors influencing grain weight and yield under water-limited environments. Here, we describe the molecular dissection of carbohydrate metabolism in stems, at the WSC accumulation phase, of recombinant inbred SB (Seri/Babax) lines of Triticum aestivum differing in stem WSC concentrations. Affymetrix GeneChip analysis of carbohydrate metabolic enzymes revealed that the mRNA levels of two fructan synthetic enzyme families (sucrose:sucrose 1-fructosyltransferase and sucrose:fructan 6-fructosyltransferase) in the stem were positively correlated with stem WSC and fructan concentrations, while the mRNA levels of enzyme families involved in sucrose hydrolysis (sucrose synthase and soluble acid invertase) were inversely correlated with WSC concentrations. Differential regulation of the mRNA levels of these sucrose hydrolytic enzymes in SB lines resulted in genotypic differences in these enzyme activities. Down-regulation of sucrose synthase and soluble acid invertase in high WSC lines was accompanied by significant decreases in the mRNA levels of enzyme families related to sugar catabolic pathways (fructokinase and mitochondrion pyruvate dehydrogenase complex) and enzyme families involved in diverting UDP-glucose to cell wall synthesis (UDP-glucose 6-dehydrogenase, UDP-glucuronate decarboxylase and cellulose synthase), resulting in a reduction in cell wall polysaccharide contents (mainly hemicellulose) in the stem of high WSC lines. These data suggest that differential carbon partitioning in the wheat stem is one mechanism that contributes to genotypic variation in WSC accumulation. We used Affymetrix GeneChip to dissect genotypic variation in carbohydrate metabolism related to water soluble carbohydrate accumulation in stems of wheat. Experiment Overall Design: 8 genotypes of recombinant inbred lines Seri M82 x Babax with 2 biological replicates per genotype. Grown in the field under rain-fed conditions
Project description:The lymphatic vascular system plays important roles in the maintenance of interstitial fluid pressure, the afferent immune response and the absorption of dietary lipids. However, the molecular mechanisms that control lymphatic vessel network maturation and function remain largely unknown. To identify novel players in lymphatic vessel function, we isolated pure populations of lymphatic and blood vascular endothelial cells from mouse intestine using fluorescence-activated high-speed cell sorting and performed transcriptional profiling. We found that the axonal guidance molecules semaphorin 3A (Sema3A) and Sema3D were specifically expressed by lymphatic vessels. Quantitative PCR of ex vivo isolated cells and immunohistochemical analysis confirmed these results. Importantly, we found that the semaphorin receptor neuropilin-1 (Nrp-1) is expressed on the valves of collecting lymphatic vessels. Treatment of mice in utero (E12.5-E16.5) with an antibody that blocks Sema3A binding to Nrp-1, but not with an antibody that blocks VEGFA binding to Nrp-1, resulted in abnormal development of collecting lymphatic vessels and valves, and aberrant smooth muscle cell coverage. Conversely, Sema3A-deficient mice displayed branching defects of collecting lymphatic vessels as well as impaired valve development. Together, these results reveal an unanticipated role of Sema3A/Nrp-1 signaling in the maturation of the lymphatic vascular network. Colon single-cell suspensions were prepared by a fast protocol that minimizes the RNA degradation. Fluorescence-activated cell sorting (FACS) was used to sort blood vascular endothelial cells (BEC) and lymphatic endothelial cells (LEC). 4 animal-matched pairs of LEC and BEC were chosen based on the quality of extracted and amplified material to provide homogenous groups of biological replicates. This gave 8 samples to analyze. Samples present LEC and BEC isolated from 4 healthy normal mice. The 4 mice used present the 4 biological replicates.