Vidya Chandrasekaran

The Genomics Education Partnership: Successful Integration of Research into Laboratory Classes at a Diverse Group of Undergraduate Institutions

Genomics is not only essential for students to understand biology but also provides unprecedented opportunities for undergraduate research. The goal of the Genomics Education Partnership (GEP), a collaboration between a growing number of colleges and universities around the country and the Department of Biology and Genome Center of Washington University in St. Louis, is to provide such research opportunities. Using a versatile curriculum that has been adapted to many different class settings, GEP undergraduates undertake projects to bring draft-quality genomic sequence up to high quality and/or participate in the annotation of these sequences. GEP undergraduates have improved more than 2 million bases of draft genomic sequence from several species of Drosophila and have produced hundreds of gene models using evidence-based manual annotation. Students appreciate their ability to make a contribution to ongoing research, and report increased independence and a more active learning approach after participation in GEP projects. They show knowledge gains on pre- and postcourse quizzes about genes and genomes and in bioinformatic analysis. Participating faculty also report professional gains, increased access to genomics-related technology, and an overall positive experience. We have found that using a genomics research project as the core of a laboratory course is rewarding for both faculty and students.

Bone morphogenetic protein-7 stimulates initial dendritic growth in sympathetic neurons through an intracellular fibroblast growth factor signaling pathway

Bone morphogenetic protein‐7 (BMP‐7), a member of the transforming growth factor (TGF)‐β superfamily of signaling cytokines, induces dendritic growth in rat sympathetic neurons. In this study, we present evidence that the recently discovered integrative nuclear FGFR1 signaling (INFS) pathway is involved in dendrite outgrowth mediated by BMP‐7. Immunocytochemical analysis of expressed fibroblast growth factors (FGFs) showed that little FGF‐2 was detected in control neurons, but the expression of this molecule in the cytoplasm and nucleus increased within 6 h after BMP‐7 treatment. In contrast, FGF‐1 was constitutively present in the peripheral cytoplasm and in neurites under control conditions, and its distribution did not change with BMP‐7 exposure. The high‐affinity receptor FGFR1 was present in low amounts in control neurons and was associated with the cytoplasm, the plasma membrane, and the nucleus. Twenty‐four hours of␣BMP‐7 treatment elicited an increase in FGFR1 nuclear localization. Overexpressed constructs of FGFR1 that lack the␣tyrosine kinase domain, and have been shown to act in a␣dominant‐negative manner on FGFR1 signaling, inhibited BMP‐7 mediated initial dendrite outgrowth in transfected neurons by ≈ 50%. However, targeted inhibition of extracellular FGF‐2 by overexpression of a secreted receptor mutant FGFR1(TM–) lacking the transmembrane domain failed to affect BMP‐7 induced dendritic growth, as did treatment with the extracellular FGFR antagonist inositol hexakisphosphate. These results suggest that the INFS, which has already been␣implicated in a broad range of activities in other cell types, may also be required for BMP‐7 to stimulate dendritic development.

A Course-Based Research Experience: How Benefits Change with Increased Investment in Instructional Time

While course-based research in genomics can generate both knowledge gains and a greater appreciation for how science is done, a significant investment of course time is required to enable students to show gains commensurate to a summer research experience. Nonetheless, this is a very cost-effective way to reach larger numbers of students.

Cerebrospinal fluid contains biologically active bone morphogenetic protein-7.

Bone morphogenetic proteins (BMPs) regulate the development and function of many types of neurons. However, little is known of the actual concentrations of BMPs in the various parts of the brain. In this study, we considered the possibility that BMPs might be present in cerebrospinal fluid (CSF). Western blot analysis of normal adult bovine CSF revealed the presence of dimeric and monomeric forms of BMP-7, and the concentration of this molecule was found to be approximately 12 ng/ml in a radioimmunoassay. Since BMP-7 is known to induce dendritic growth in rat sympathetic neurons, this was used as a bioassay to examine the biological activity of the BMP-7 present in CSF. Addition of normal bovine CSF to cultures of sympathetic neurons produced a dose-dependent increase in dendritic growth and the magnitude of this response approximated that obtained with maximally effective concentrations of exogenous BMP-7. Moreover, CSF-induced dendritic growth was inhibited by follistatin, a protein that can sequester BMPs, and by either of two monoclonal antibodies that react with BMP-7. These results show that, unlike most other neurotrophic factors, BMP-7 is a constituent of normal CSF and is present at concentrations sufficient to elicit a near maximal biological response.

Retinoic acid regulates the morphological development of sympathetic neurons

Interactions between all-trans-retinoic acid (RA) and bone morphogenetic proteins (BMPs) affect the expression of neurotrophin receptors in sympathetic neurons (Kobayashi et al., 1998). In this study, we examined the possibility that similar interactions might regulate the morphological development of these neurons. Under control conditions, embryonic rat sympathetic neurons formed axons but not dendrites; cells exposed to RA had a similar appearance. Profuse dendritic growth was observed upon exposure to BMP-7, and this was reduced by approximately 70% by RA. This inhibitory effect of RA was mediated primarily by retinoic acid receptors (RARs) and it exhibited substantial specificity because it was not associated with changes in either axonal elongation or cell survival. Moreover, mRNAs for enzymes required for synthesis of RA were expressed in the sympathetic neurons and retinoid activity was released from superior cervical ganglia. These observations suggest that retinoids may function as endogenous morphogens and regulate neural cell shape and polarity in developing sympathetic ganglia.

A Central Support System Can Facilitate Implementation and Sustainability of a Classroom-Based Undergraduate Research Experience (CURE) in Genomics

There have been numerous calls to engage students in science as science is done. A survey of 90-plus faculty members explores barriers and incentives when developing a research-based genomics course. The results indicate that a central core supporting a national experiment can help overcome local obstacles.

Reactive oxygen species are involved in BMP-induced dendritic growth in cultured rat sympathetic neurons.

Previous studies have shown that bone morphogenetic proteins (BMPs) promote dendritic growth in sympathetic neurons; however, the downstream signaling molecules that mediate the dendrite promoting activity of BMPs are not well characterized. Here we test the hypothesis that reactive oxygen species (ROS)-mediated signaling links BMP receptor activation to dendritic growth. In cultured rat sympathetic neurons, exposure to any of the three mechanistically distinct antioxidants, diphenylene iodinium (DPI), nordihydroguaiaretic acid (NGA) or desferroxamine (DFO), blocked de novo BMP-induced dendritic growth. Addition of DPI to cultures previously induced with BMP to extend dendrites caused dendritic retraction while DFO and NGA prevented further growth of dendrites. The inhibition of the dendrite promoting activity of BMPs by antioxidants was concentration-dependent and occurred without altering axonal growth or neuronal cell survival. Antioxidant treatment did not block BMP activation of SMAD 1,5 as determined by nuclear localization of these SMADs. While BMP treatment did not cause a detectable increase in intracellular ROS in cultured sympathetic neurons as assessed using fluorescent indicator dyes, BMP treatment increased the oxygen consumption rate in cultured sympathetic neurons as determined using the Seahorse XF24 Analyzer, suggesting increased mitochondrial activity. In addition, BMPs upregulated expression of NADPH oxidase 2 (NOX2) and either pharmacological inhibition or siRNA knockdown of NOX2 significantly decreased BMP-7 induced dendritic growth. Collectively, these data support the hypothesis that ROS are involved in the downstream signaling events that mediate BMP7-induced dendritic growth in sympathetic neurons, and suggest that ROS-mediated signaling positively modulates dendritic complexity in peripheral neurons.

The effects of energy beverages on cultured cells

The popularity and prevalence of energy beverages makes it essential to examine the interactions between the ingredients and their effects on the safety of these beverages. In this study, we used in vitro assays to examine the effects of two energy beverages on mesenchymal, epithelial and neuronal cells. Our results showed that treatment of epithelial and mesenchymal cells with either energy beverage resulted in a dose dependent delay in wound closure, in a scratch wound healing assay. In rat embryonic fibroblasts, treatment with the energy beverages led to decreased lamellipodia formation and decreased proliferation/viability; whereas in MDCK cells, energy beverage treatment resulted in actin disorganization without any effects on cell proliferation. This suggests that the mechanisms underlying delayed wound healing might be different in the two cell types. Interestingly, the delays in both cell types could not be mimicked by treatment of caffeine, taurine and glucose alone or in combinations. Furthermore, treatment of chick forebrain neuronal cultures with energy beverages resulted in a dose dependent inhibition of neurite outgrowth. The cellular assays used in this study provide a consistent, qualitative and quantitative system for examining the combinatorial effects of the various ingredients used in energy beverages.

Opportunities and challenges for using the zebrafish to study neuronal connectivity as an endpoint of developmental neurotoxicity

HIGHLIGHTSGene‐environment interactions influence risk for neurodevelopmental disorders.Zebrafish may be useful for identifying relevant gene‐environment interactions.Altered neuronal connectivity is associated with many neurodevelopmental disorders.Neuronal connectivity has not been integrated into zebrafish screening efforts.We review approaches for quantifying neuronal connectivity in developing zebrafish. ABSTRACT Chemical exposures have been implicated as environmental risk factors that interact with genetic susceptibilities to influence individual risk for complex neurodevelopmental disorders, including autism spectrum disorder, schizophrenia, attention deficit hyperactivity disorder and intellectual disabilities. Altered patterns of neuronal connectivity represent a convergent mechanism of pathogenesis for these and other neurodevelopmental disorders, and growing evidence suggests that chemicals can interfere with specific signaling pathways that regulate the development of neuronal connections. There is, therefore, a growing interest in developing screening platforms to identify chemicals that alter neuronal connectivity. Cell‐cell, cell‐matrix interactions and systemic influences are known to be important in defining neuronal connectivity in the developing brain, thus, a systems‐based model offers significant advantages over cell‐based models for screening chemicals for effects on neuronal connectivity. The embryonic zebrafish represents a vertebrate model amenable to higher throughput chemical screening that has proven useful in characterizing conserved mechanisms of neurodevelopment. Moreover, the zebrafish is readily amenable to gene editing to integrate genetic susceptibilities. Although use of the zebrafish model in toxicity testing has increased in recent years, the diverse tools available for imaging structural differences in the developing zebrafish brain have not been widely applied to studies of the influence of gene by environment interactions on neuronal connectivity in the developing zebrafish brain. Here, we discuss tools available for imaging of neuronal connectivity in the developing zebrafish, review what has been published in this regard, and suggest a path forward for applying this information to developmental neurotoxicity testing.