Congress Abstracts - Society for Developmental Biology

have investigated the effect of calcium activity perturbation on gene expression during neural development using Xenopus primary
cell culture of presumptive neural tissue. Neural plates were dissected, dissociated, and plated in either normal physiological calcium
(2mM Ca++) or elevated calcium (10mM Ca++) which results in significantly increased spiking activity. After culturing these cells
until sibling embryos have reached late neurula, early tailbud, or early swimming tadpole stage, we extracted RNA and performed
microarray analysis to investigate differential gene expression on each of these three stages. We have utilized a number of techniques
to analyze the resulting data using a modified t-test, fold change analysis, and a novel mathematical algorithm employing a
generalized linear model designed for low sample size. Our preliminary results indicate a wide array of genes exhibit differential
expression ranging from genes whose products have known calcium binding activity to genes controlling cell cycle and stress
responses. Each time point reveals a unique set of differentially expressed genes, suggesting a dynamic regulation of response to
increased calcium. To complement these approaches we are also using RNAseq of similarly collected samples as well as an in vivo
approach using a GCaMP.
Program/Abstract # 446
RA induced primitive extraembryonic endoderm leads to increased reactive oxygen species and a shift from aerobic glycolysis
to mitochondrial biogenesis
Hwang, Jason TK; Wen, Jason; Kelly, Gregory (U of Western Ontario, Canada)
Mouse F9 cells are used to recapitulate the epithelial-to-mesenchymal transition (EMT) associated with extraembryonic endoderm
differentiation. F9 cells treated with retinoic acid (RA) form primitive endoderm (PrE) and this is accompanied by an increase in
reactive oxygen species (ROS). Treating cells with H2O2 induces differentiation, while treating either RA- or H2O2-treated cells with
antioxidants inhibits it. Together, these results indicate that ROS are sufficient and necessary for PrE differentiation. Furthermore, that
NADPH oxidase (Nox) genes are up-regulated in F9 cells treated with RA, and the ability of DPI, a Nox inhibitor, to block
differentiation, provide evidence that the ROS source is cytoplasmic in nature. To investigate further and to examine mitochondrial
function during this EMT, the levels of LDHA, PDK1 and phospho-PDH, which are elevated in cells using aerobic glycolysis, were
examined in undifferentiated and RA-treated F9 cells. Results show the levels of these proteins decreased in response to RA.
Furthermore, treating cells with dichloroacetate to increase mitochondrial respiration induced PrE differentiation in the absence of RA.
Together, these results suggest that undifferentiated F9, like stem cells, use aerobic glycolysis to maintain their undifferentiated state.
When induced, however, they undergo a metabolic shift from glycolytic to oxidative phosphorylation, which together with increased
Nox activity would contribute to elevated ROS levels required for PrE differentiation.
Program/Abstract # 447
Distinct roles for isoforms of Regulator of G-protein Signalling 3 (RGS3) throughout neuronal maturation.
Fleenor, Stephen (U of Oxford, UK)
The differentiation and maturation of neurons from the neuroepithelial progenitor state is a fundamental process in nervous system
development. In the forming cranial sensory ganglia, committed neuroblasts delaminate from a progenitor neuroepithelium and
differentiate into mature neurons as they migrate away. Recently Regulator of G-protein Signalling 3 (RGS3) was found to be upregulated
during this process. In the chick, the RGS3 gene produces three distinct isoforms with unique functional domains. Here I
present specific localisation of individual RGS3 isoforms to progressive neuro-differentiation states: from progenitor epithelium, to
immature neuroblast, to mature neuron. shRNA-mediated knockdown of the longest isoform in ovo results in precocious neuronal
differentiation. Consistently, overexpression produces an opposite effect. Excitingly however, distinct phenotypes emerge from
knockdown of all isoforms and from overexpression of individual isoforms, suggesting separate roles for individual isoforms. These
findings suggest roles for specific RGS3 isoforms in driving distinct aspects of neuronal maturation.
Program/Abstract # 448
Regulation of TNFa and COX2 by NFATc1 pathway during adipose commitment.
López-Victorio, Carlos J; Beltrán-Langarica, Alicia; Vellez-delValle, Cristina; Kuri-Harcuch, Walid (Cinvestav IPN, Mexico)
Obesity is defined as an abnormal and excessive accumulation of fat that causes weight gain and and it is considered a risk factor for
several common diseases. During adipogenesis preadipocytes undergo biochemical, morphological and metabolic changes related to
gene expression. The adipogenic conversion has three major steps: commitment, clonal expansion of committed cells, and phenotype
expression. Our group has developed a model to study of adipose differentiation were the 3T3-F442A cell line is stimulated by a
combination of staurosporine and dexamethasone (St/Dex) in absence of adipogenic serum. StDex induces two well-defined stages of
commitment: induction and stabilization. In this study we analyzed the relation between Cn/NFATc1 signaling pathway, TNFα
expression and the inhibition of COX2 pathway by celecoxib during the early events of adipose commitment. It has been reported that
Cn acts as a anti-adipogenic pathway that negatively regulates adipogenesis by preventing the expression of critical pro-adipogenic
transcription factors, this pathway includes NFATc1 participation as a nuclear effector, and Calmodulin A (CaM) as a molecule
needed for Cn activity. We found increased transiently nfatc1 and ptgs2 mRNAs during the induction of commitment, and then it
proceeds with the down regulation in the expression of these, the activation of Cn up regulates ptgs2 and nfatc1 and tnfα, furthermore
the inhibition of Cn phosphatase activity in 3T3-F442A cells down-regulate the expression of TNFα and promoting progression of
adipogenesis. Treatment with celecoxib, a specific COX2 inhibitor, does not change adipose conversion, however triglycerides
128