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through its channel activities
Ishikawa, Masaki; Yamada, Yoshihiko (NIH/NIDCR, USA)
Pannexin 3 (Panx3), a new member of the pannexin gap junction family, is expressed in the perichondrium/periosteum and in osteoblast. Previously, we reported that Panx3 is induced in the transition stage from proliferation and differentiation of
osteoprogenitor cells and promotes osteoblast differentiation through its hemichannel, endoplasmic reticulum (ER) Ca2+ channel, and
gap junction. Canonical Wnt/ β-catenin signaling is essential for osteoprogenitor cell proliferation. Here we show that Panx3 inhibits proliferation and promotes cell cycle exit of osteogenic C2C12 cells, primary calvarial cells, and newborn calvaria explants.
Overexpression of Panx3 reduced osteoprogenitor cell proliferation, whereas the inhibition of endogenous Panx3 increased the proliferation. We found that this inhibition was mediated through reduced Wnt signaling by β-catenin degradation and GSK3β activation, which was caused by reduced cAMP/PKA signaling through the Panx3 hemichannel. The Panx3 hemichannel also contributes to the inhibition of proliferation, by reducing cAMP/PKA/CREB signaling which induces the expression of genes such as cyclinD1 for cell progression. Furthermore, the released ATP promotes PI3K/Akt signaling, which activates the Panx3 ER Ca2+
channel to increase intracellular Ca2+ that increases p21 transcription and phosphorylation by promoting Smad signaling through the
calmodulin signaling pathway activated by Panx3 ER Ca2+ channel. Our results revealed multi-functional roles of Panx3 for
osteoprogenitor proliferation and cell cycle exit. Thus, Panx3 plays a critical role in switching from proliferation to differentiation of osteoblasts.
Program/Abstract # 439
Identification and expression of novel Wnt signaling-associated protein kinases
Park, Edmond; Shin, Eun-Young (Korea Basic Science Inst., Rep. of Korea); Shin, Ju-Hyun (Chungnam National Univ. Hosp., Rep. of Korea); Kim, Gun-Hwa (Korea Basic Science Inst., Rep. of Korea)
The Wnt pathway is an evolutionarily conserved signaling network that is critical for mammalian development and adult tissue maintenance. In addition, aberrant activation of the Wnt signaling is implicated in driving the formation of various human cancers, particularly those of the digestive tract. Inhibition of aberrant Wnt pathway activity in cancer cell lines efficiently blocks their growth, highlighting the great potential of therapeutics designed to achieve this in cancer patients. In this study, we try to identify novel protein kinases that are associated with canonical Wnt signaling pathway by using TOPflash reporter assay system and human protein kinase (~500 genes) library. As the result, we identified numbers of protein kinases that positively regulate Wnt signaling pathway. The novel protein kinases would be possible and valuable target for regulating Wnt pathway in cancer and mammalian development.
Program/Abstract # 440
Axin-stimulated Wnt signaling in mouse embryogenesis and intestinal progenitor cells
Parrish, Angela; Mahaffey, James; Anderson, Kathryn (Sloan-Kettering Institute, USA)
The Wnt signaling pathway is essential for embryonic development and adult stem cell maintenance and is misregulated in many cancer types. Although the pathway has been extensively studied, the mechanism of pathway activation remains controversial. In the early mouse embryo, Wnt signals specify and maintain the primitive streak (the site of gastrulation) and progenitor cells in the streak and tail bud. Axin and Axin2 are considered to be negative regulators of the canonical Wnt pathway. However, we previously showed that a protein-stabilizing Axin2 point mutation (canopus) leads to a decrease of Wnt signaling in the head but an increase in the late primitive streak, assessed by the expression of the transgenic Wnt reporter Topgal. To confirm elevated canonical Wnt signaling in the primitive streak, we are examining the intracellular localization of β-catenin in the heads and tails of Axin-stabilized embryos. We find that limiting Wnt production with small molecules blocks the increase in Wnt signaling caused by Axin stabilization; genetic experiments to confirm that Axin-dependent pathway activation depends on ligand are in progress. Elevated Wnt signaling has a critical role in colon cancer through activation of intestinal stem/progenitor fates. We find that adult canopus heterozygotes show an increase in the number of β-catenin+ cells in small intestinal crypts, suggesting that stabilized Axin2 can also increase Wnt signaling in intestinal progenitor cells. We conclude that Axin proteins can tissue specifically activate or repress Wnt signaling, and this will be important for potential use of Axin-stabilizing drugs to treat cancer.
Program/Abstract # 441
Early Endocytic Trafficking in Control of Developmental Signaling
Gerstner, Norman; Zimyanin, Vitaly; Wieffer, Marnix; Zerial, Marino (MPI-CBG, Germany)
During gastrulation, cells simultaneously process different signaling inputs to pattern the early embryonic body axis. Several conserved signaling pathways, fundamental for morphogenesis and other developmental functions, are regulated by endocytosis. Ligand-activated receptors are internalized via different entry routes and compartmentalized into early endosomes. We have
previously performed a genome-wide screen on endocytosis (Collinet et al., Nature 2010) and identified several genes that selectively regulate transport of signaling cargo to different types of endosomes, including early endosomes involved in signal transduction. We hypothesize that cargo sorting between distinct early endosomes is essential for the precise regulation of developmental signaling. By interfering with a novel Rab5-effector that regulates the transport of signaling cargo between endosomal compartments, we
investigated the role of endosomal trafficking in modulating signaling strength and specificity during zebrafish gastrulation. Since Wnt/beta-catenin signaling and D/V patterning were strongly affected upon such perturbation, we focused on the Wnt/beta-catenin pathway and trafficking of Wnt-receptor complexes. We quantified trafficking and signaling defects at the level of the whole embryo,
127 cells and sub-cellular endosomal compartments. Our results suggest that the balance of Wnt-signaling components between distinct early endosomes determines signaling strength and specificity as part of the regulatory mechanisms underlying gastrulation.
Program/Abstract # 442
Detection of BMP signaling in pre-implantation mouse embryos
Reyes de Mochel, Nabora Soledad; Javier, Anna; Chiang, Michael; Luong, Mui Nhuc; Cinquin, Olivier (UC- Irvine, USA)
Mammalian development begins with fertilization, followed by multiple cell cleavage events and the transition from maternal to zygotic transcription. All these events work in concert to establish the first and thus most significant developmental event, lineage differentiation. This lineage specification gives rise to the trophectoderm (TE), the precursor for extraembryonic structures such as the placenta and yolk sac, and the Inner Cell Mass (ICM), which becomes the embryo proper. While the regulatory interactions and morphological contributions of transcription factors such as OCt3/4, Nanog, Sox2, TEAD4, and Cdx2 are extensively studied, surprisingly little is known about the roles of secreted growth factors during the first lineage differentiation. The presence of bone morphogenetic proteins (BMPs) in early mouse embryogenesis suggests a functional role for BMP signaling. In order to determine the functional significance of BMP signaling during early mouse embryogenesis, we investigate the onset of active BMP signaling and its possible functional role. Here, we report the detailed characterization of active BMP signaling in the pre-implantation mouse embryo.
Program/Abstract # 443
TGF-beta/Smad Signaling Maintains Cardiac Homeostasis by Down-regulating miRNAs Inducing Cardiac Hypertrophy
Yang, Xiao; Wang, Jian (Beijing Inst. of Biotechnology, China)
Heart failure (HF) is one of the most frequent causes of death worldwide. The underlying causes of HF are diverse but often relate to cardiac hypertrophy, which is an adaptive enlargement of the myocardium in response to altered stress or injury. The role of TGF-b signaling in cardiac hypertrophy has been extremely contradictory due to the complexity of TGF-β activation as well as its diverse effects on different type of cells. We have previously demonstrated that the endogenous cardiomyocyte Smad4-dependent TGF-b pathway protects heart from cardiac hypertrophy and fibrosis. Recently, we have revealed that the function of endogenous TGF- b/Smad signaling in maintaining cardiac homeostasis involves the downregulation of miRNAs inducing cardiac hypertrophy. We show that TGF-b1 inhibits the expression of miR-23a/miR-27a/miR-24-2 and miR-23b/miR-27b/miR-24-1 clusters at the transcriptional level. Transgenic mice with cardiomyocyte-specific overexpression of miR-27b exhibit cardiac hypertrophy and dysfunction by directly targeting the peroxisome proliferator-activated receptor-γ (PPAR-γ). Most importantly, in vivo silencing of miR-27b using a specific antagomir or adenovirus expressing anti-sense miR-27b in a pressure-overload-induced mouse model of HF attenuates cardiac hypertrophy and dysfunction. The function and mechanisms of other miRNAs regulated by TGF-b/Smad signaling in cardiac hypertrophy will also be discussed. All these results provide critical genetic evidence showing that TGF-b/Smad signaling maintains cardiac homeostasis by down-regulating miRNAs inducing cardiac hypertrophy, which might serve as efficient therapeutic targets for cardiac diseases.
Program/Abstract # 444
Identification and Expression Analysis of Two Homologs from Xenopus laevis of the Tumorhead Putative Binding Protein, FBXO30
Traverso, Edwin E.; Zbinden, Theodor; Flores, Noelia; Núñez, Dariana; Ayala, Jesús (UPuerto Rico-Humacao, USA); Hernández, Josué; García-Arrarás, José (U Puerto Rico-Río Piedras, USA)
Tumorhead (TH) is a maternal factor that regulates cell proliferation during early embryogenesis in Xenopus laevis. To understand how TH functions at the molecular level, we have been studying its relationship with the novel F-Box containing protein FBXO30, found in a two-hybrid screen for TH binding proteins. Using primers based on the sequence we obtained, along with primers based on the 5’ and 3’ UTRs of the Xenopus tropicalis FBXO3O mRNA, we obtained RT-PCR products with total RNA samples from eggs and embryos at early developmental stages. Using this approach, we have uncovered the presence of two FBXO30 homolog genes in X.
laevis, FBXO30-A and FBXO30-B, which encode proteins that are 91% identical to each other. The FBXO30-A and FBXO30-B
proteins share 64% and 63% identity with their Homo sapiens protein homolog, respectively. FBXO30 proteins contain very
conserved Traf-like zinc finger-containing domains at their N-terminus, and F-Box domains at their C-terminus, while the internal part of the proteins diverge extensively. We have found through RT-PCR that FBXO30-A and FBXO30-B are maternal factors as their messages are present in the unfertilized egg. Their mRNAs persist during the cleavage stages, decrease dramatically once gastrulation starts, and reappear at the mid-tailbud stages. The FBXO30-A protein has been detected in the nuclei of cells at the gastrula (st. 12) stage. Our studies show the presence of two homologs of FBXO30 in X. laevis that are maternally expressed, which could be key regulators of early development working with TH to promote cell proliferation.
Program/Abstract # 445
The Effect of Calcium Activity Perturbation on Gene Expression in the Developing Nervous System of Xenopus
Rabe, Brian A.; Herbst, Wendy A.; Saha, Margaret (The College of William and Mary, USA)
Calcium ions serve as ubiquitous secondary messengers in a wide array of cellular processes, particularly during early neural development. While spontaneous calcium transients have been implicated in neural specification and differentiation, little is known regarding the mechanisms by which different patterns of calcium activity influence phenotype. In order to address this question, we
128 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 up- regulated 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
129 accumulation is decreased. This work was supported in part by grants 104350 from Consejo Nacional de Ciencia y Tecnología and PICDS08-8 from ICyTDF (Mexico). CJLV is a graduate student supported by the PICDS08-8 scholarship from ICyTDF.
Program/Abstract # 449
Importance of Intersectin1 isoforms during proper embryonic development of Xenopus laevis
Cheng, Cheng; Jimenez, Oscar; Thorn, Judith (Knox College, USA)
Intersectin 1 (ITSN1), located on the human chromosome 21, is associated with neurodegenerative diseases such as Down Syndrome, Alzheimer disease and Hungtington disease. Itsn1 interacts with many proteins, forming complexes implicated in endocytic and mitogenic pathways important in neurogenesis and maintenance. In this study, we reported the abnormality caused by itsn1 depletion and overexpression during the early Xenopus development. We microinjected the embryos at 1 cell stage with translation blocking itsn1 morpholino, in which no abnormal phenotype was observed; however, the itsn1 morpholino injection in the oocytes followed by host transfer results in the slow of blastopore closure, abnormal pigmentatation and a general shortening of the embryo axis. Similar phenotype is also observed in embryos microinjected with Intersectin1-short (itsn1-S) mRNA at the 2-cell stage. Our analysis of the relative expression of itsn1 during embryonic development along with our microinjection results indicates that sufficient itsn1 protein is necessary pre-zygotically for early embryonic development, and regulating cell movement. Further research is going to be focusing on the overexpression of itsn1 long isoform (itsn1-L) and the causality of the axis defects due to protein overexpression and depletion.
Program/Abstract # 450
Chromatin state transitions and epigenetic constraints during early Xenopus embryogenesis
Veenstra, Gert Jan (Radboud Univ, Netherlands)
Chromatin state is essential for pluripotency, competence and cell lineage commitment. It specifies how genes are marked for activation or repression by epigenetic mechanisms. Little is known however, about the developmental origins of chromatin state and its regulation. We have generated chromatin state maps of Xenopus tropicalis embryos by ChIP-sequencing to explore the
developmental origins of chromatin state with respect to inheritance, sequence features and molecular mechanisms. To assess chromatin state dynamics we profiled promoter histone modifications, enhancer histone modifications, facultative and constitutive heterochromatin modifications and DNA methylation at multiple stages of development. We find blastula stage marking of promoters and enhancers by histone H3 lysine 4 tri- and mono methylation (H3K4me3, H3K4me1) respectively, followed by dynamic
commisioning of enhancers by the enhancer-bound p300 co-activator during gastrulation and subsequent development. The Polycomb Repressor Complex 2 (PRC2) binds widely to enhancers, but the Polycomb mark H3K27me3 is only deposited at a small subset of these sites. This mark is newly deposited from blastula stages onward within constrained domains lacking prior DNA methylation. Unmethylated regions represent two epigenetically different loci: Polycomb-regulated genes and constitutive house-keeping unmethylated promoters which gain H3K4me3 but not H3K27me3. These loci can be differentiated on the basis of specific DNA sequence signatures which are conserved between humans, frogs and fish. The results imply a genetic-default model in which genomic sequence is the major determinant of unmethylated regions. Unmethylated DNA triggers H3K27me3 deposition by an allosteric loop when not opposed by transcriptional activation. The sequence signature involved provides an epigenetically marked but genetically inheritable constraint on Polycomb regulation and serves as a scaffold to guide deposition of H3K27me3 during exit of pluripotency.