Advanced optical imaging in living embryos

Developmental biology investigations have evolved from static studies of embryo anatomy and into dynamic studies of the genetic and cellular mechanisms responsible for shaping the embryo anatomy. With the advancement of fluorescent protein fusions, the ability to visualize and comprehend how thousands to millions of cells interact with one another to form tissues and organs in three dimensions (xyz) over time (t) is just beginning to be realized and exploited. In this review, we explore recent advances utilizing confocal and multi-photon time-lapse microscopy to capture gene expression, cell behavior, and embryo development. From choosing the appropriate fluorophore, to labeling strategy, to experimental set-up, and data pipeline handling, this review covers the various aspects related to acquiring and analyzing multi-dimensional data sets. These innovative techniques in multi-dimensional imaging and analysis can be applied across a number of fields in time and space including protein dynamics to cell biology to morphogenesis

Evaluation of the current knowledge limitations in breast cancer research: a gap analysis

BACKGROUND A gap analysis was conducted to determine which areas of breast cancer research, if targeted by researchers and funding bodies, could produce the greatest impact on patients. METHODS Fifty-six Breast Cancer Campaign grant holders and prominent UK breast cancer researchers participated in a gap analysis of current breast cancer research. Before, during and following the meeting, groups in seven key research areas participated in cycles of presentation, literature review and discussion. Summary papers were prepared by each group and collated into this position paper highlighting the research gaps, with recommendations for action. RESULTS Gaps were identified in all seven themes. General barriers to progress were lack of financial and practical resources, and poor collaboration between disciplines. Critical gaps in each theme included: (1) genetics (knowledge of genetic changes, their effects and interactions); (2) initiation of breast cancer (how developmental signalling pathways cause ductal elongation and branching at the cellular level and influence stem cell dynamics, and how their disruption initiates tumour formation); (3) progression of breast cancer (deciphering the intracellular and extracellular regulators of early progression, tumour growth, angiogenesis and metastasis); (4) therapies and targets (understanding who develops advanced disease); (5) disease markers (incorporating intelligent trial design into all studies to ensure new treatments are tested in patient groups stratified using biomarkers); (6) prevention (strategies to prevent oestrogen-receptor negative tumours and the long-term effects of chemoprevention for oestrogen-receptor positive tumours); (7) psychosocial aspects of cancer (the use of appropriate psychosocial interventions, and the personal impact of all stages of the disease among patients from a range of ethnic and demographic backgrounds). CONCLUSION Through recommendations to address these gaps with future research, the long-term benefits to patients will include: better estimation of risk in families with breast cancer and strategies to reduce risk; better prediction of drug response and patient prognosis; improved tailoring of treatments to patient subgroups and development of new therapeutic approaches; earlier initiation of treatment; more effective use of resources for screening populations; and an enhanced experience for people with or at risk of breast cancer and their families. The challenge to funding bodies and researchers in all disciplines is to focus on these gaps and to drive advances in knowledge into improvements in patient care

Molecular profiling of cervical cancer progression

Most cancer patients die of metastatic or recurrent disease, hence the importance to identify target genes upregulated in these lesions. Although a variety of gene signatures associated with metastasis or poor prognosis have been identified in various cancer types, it remains a critical problem to identify key genes as candidate therapeutic targets in metastatic or recurrent cancer. The aim of our study was to identify genes consistently upregulated in both lymph node micrometastases and recurrent tumours compared to matched primary tumours in human cervical cancer. Taqman Low-Density Arrays were used to analyse matched tumour samples, obtained after laser-capture microdissection of tumour cell islands for the expression of 96 genes known to be involved in tumour progression. Immunohistochemistry was performed for a panel of up- and downregulated genes. In lymph node micrometastases, most genes were downregulated or showed expressions equal to the levels found in primary tumours. In more than 50% of lymph node micrometastases studied, eight genes (AKT, BCL2, CSFR1, EGFR1, FGF1, MMP3, MMP9 and TGF-β) were upregulated at least two-fold. Some of these genes (AKT and MMP3) are key regulators of epithelial–mesenchymal transition in cancer. In recurrent tumours, almost all genes were upregulated when compared to the expression profiles of the matched primary tumours, possibly reflecting their aggressive biological behaviour. The two genes showing a consistent downregulated expression in almost all lymph node metastases and recurrent tumours were BAX and APC. As treatment strategies are very limited for metastatic and recurrent cervical cancer, the upregulated genes identified in this study are potential targets for new molecular treatment strategies in metastatic or recurrent cervical cancer

Genome-Wide Search Reveals the Existence of a Limited Number of Thyroid Hormone Receptor Alpha Target Genes in Cerebellar Neurons

Thyroid hormone (T3) has a major influence on cerebellum post-natal development. The major phenotypic landmark of exposure to low levels of T3 during development (hypothyroidism) in the cerebellum is the retarded inward migration of the most numerous cell type, granular neurons. In order to identify the direct genetic regulation exerted by T3 on cerebellar neurons and their precursors, we used microarray RNA hybridization to perform a time course analysis of T3 induced gene expression in primary cultures of cerebellar neuronal cell. These experiments suggest that we identified a small set of genes which are directly regulated, both in vivo and in vitro, during cerebellum post-natal development. These modest changes suggest that T3 does not acts directly on granular neurons and mainly indirectly influences the cellular interactions taking place during development

Ror2 Enhances Polarity and Directional Migration of Primordial Germ Cells

The trafficking of primordial germ cells (PGCs) across multiple embryonic structures to the nascent gonads ensures the transmission of genetic information to the next generation through the gametes, yet our understanding of the mechanisms underlying PGC migration remains incomplete. Here we identify a role for the receptor tyrosine kinase-like protein Ror2 in PGC development. In a Ror2 mouse mutant we isolated in a genetic screen, PGC migration and survival are dysregulated, resulting in a diminished number of PGCs in the embryonic gonad. A similar phenotype in Wnt5a mutants suggests that Wnt5a acts as a ligand to Ror2 in PGCs, although we do not find evidence that WNT5A functions as a PGC chemoattractant. We show that cultured PGCs undergo polarization, elongation, and reorientation in response to the chemotactic factor SCF (secreted KitL), whereas Ror2 PGCs are deficient in these SCF-induced responses. In the embryo, migratory PGCs exhibit a similar elongated geometry, whereas their counterparts in Ror2 mutants are round. The protein distribution of ROR2 within PGCs is asymmetric, both in vitro and in vivo; however, this asymmetry is lost in Ror2 mutants. Together these results indicate that Ror2 acts autonomously to permit the polarized response of PGCs to KitL. We propose a model by which Wnt5a potentiates PGC chemotaxis toward secreted KitL by redistribution of Ror2 within the cell

Fragile X Mental Retardation Protein Regulates Proliferation and Differentiation of Adult Neural Stem/Progenitor Cells

Fragile X syndrome (FXS), the most common form of inherited mental retardation, is caused by the loss of functional fragile X mental retardation protein (FMRP). FMRP is an RNA–binding protein that can regulate the translation of specific mRNAs. Adult neurogenesis, a process considered important for neuroplasticity and memory, is regulated at multiple molecular levels. In this study, we investigated whether Fmrp deficiency affects adult neurogenesis. We show that in a mouse model of fragile X syndrome, adult neurogenesis is indeed altered. The loss of Fmrp increases the proliferation and alters the fate specification of adult neural progenitor/stem cells (aNPCs). We demonstrate that Fmrp regulates the protein expression of several components critical for aNPC function, including CDK4 and GSK3β. Dysregulation of GSK3β led to reduced Wnt signaling pathway activity, which altered the expression of neurogenin1 and the fate specification of aNPCs. These data unveil a novel regulatory role for Fmrp and translational regulation in adult neurogenesis

Bioaccumulation and ecotoxicity of carbon nanotubes

Carbon nanotubes (CNT) have numerous industrial applications and may be released to the environment. In the aquatic environment, pristine or functionalized CNT have different dispersion behavior, potentially leading to different risks of exposure along the water column. Data included in this review indicate that CNT do not cross biological barriers readily. When internalized, only a minimal fraction of CNT translocate into organism body compartments. The reported CNT toxicity depends on exposure conditions, model organism, CNT-type, dispersion state and concentration. In the ecotoxicological tests, the aquatic organisms were generally found to be more sensitive than terrestrial organisms. Invertebrates were more sensitive than vertebrates. Single-walled CNT were found to be more toxic than double-/multi-walled CNT. Generally, the effect concentrations documented in literature were above current modeled average environmental concentrations. Measurement data are needed for estimation of environmental no-effect concentrations. Future studies with benchmark materials are needed to generate comparable results. Studies have to include better characterization of the starting materials, of the dispersions and of the biological fate, to obtain better knowledge of the exposure/effect relationships

Microbial Fuel Cells and Microbial Ecology: Applications in Ruminant Health and Production Research

Microbial fuel cell (MFC) systems employ the catalytic activity of microbes to produce electricity from the oxidation of organic, and in some cases inorganic, substrates. MFC systems have been primarily explored for their use in bioremediation and bioenergy applications; however, these systems also offer a unique strategy for the cultivation of synergistic microbial communities. It has been hypothesized that the mechanism(s) of microbial electron transfer that enable electricity production in MFCs may be a cooperative strategy within mixed microbial consortia that is associated with, or is an alternative to, interspecies hydrogen (H2) transfer. Microbial fermentation processes and methanogenesis in ruminant animals are highly dependent on the consumption and production of H2in the rumen. Given the crucial role that H2 plays in ruminant digestion, it is desirable to understand the microbial relationships that control H2 partial pressures within the rumen; MFCs may serve as unique tools for studying this complex ecological system. Further, MFC systems offer a novel approach to studying biofilms that form under different redox conditions and may be applied to achieve a greater understanding of how microbial biofilms impact animal health. Here, we present a brief summary of the efforts made towards understanding rumen microbial ecology, microbial biofilms related to animal health, and how MFCs may be further applied in ruminant research

Improving metabolic health in obese male mice via diet and exercise restores embryo development and fetal growth

Paternal obesity is now clearly associated with or causal of impaired embryo and fetal development and reduced pregnancy rates in humans and rodents. This appears to be a result of reduced blastocyst potential. Whether these adverse embryo and fetal outcomes can be ameliorated by interventions to reduce paternal obesity has not been established. Here, male mice fed a high fat diet (HFD) to induce obesity were used, to determine if early embryo and fetal development is improved by interventions of diet (CD) and/or exercise to reduce adiposity and improve metabolism. Exercise and to a lesser extent CD in obese males improved embryo development rates, with increased cell to cell contacts in the compacting embryo measured by E-cadherin in exercise interventions and subsequently, increased blastocyst trophectoderm (TE), inner cell mass (ICM) and epiblast cell numbers. Implantation rates and fetal development from resulting blastocysts were also improved by exercise in obese males. Additionally, all interventions to obese males increased fetal weight, with CD alone and exercise alone, also increasing fetal crown-rump length. Measures of embryo and fetal development correlated with paternal measures of glycaemia, insulin action and serum lipids regardless of paternal adiposity or intervention, suggesting a link between paternal metabolic health and subsequent embryo and fetal development. This is the first study to show that improvements to metabolic health of obese males through diet and exercise can improve embryo and fetal development, suggesting such interventions are likely to improve offspring health.Nicole O. McPherson, Hassan W. Bakos, Julie A. Owens, Brian P. Setchell, Michelle Lan