FGF signalling through RAS/MAPK and PI3K pathways regulates cell movement and gene expression in the chicken primitive streak without affecting E-cadherin expression

<p>Abstract</p> <p>Background</p> <p>FGF signalling regulates numerous aspects of early embryo development. During gastrulation in amniotes, epiblast cells undergo an epithelial to mesenchymal transition (EMT) in the primitive streak to form the mesoderm and endoderm. In mice lacking FGFR1, epiblast cells in the primitive streak fail to downregulate E-cadherin and undergo EMT, and cell migration is inhibited. This study investigated how FGF signalling regulates cell movement and gene expression in the primitive streak of chicken embryos.</p> <p>Results</p> <p>We find that pharmacological inhibition of FGFR activity blocks migration of cells through the primitive streak of chicken embryos without apparent alterations in the level or intracellular localization of E-cadherin. E-cadherin protein is localized to the periphery of epiblast, primitive streak and some mesodermal cells. FGFR inhibition leads to downregulation of a large number of regulatory genes in the preingression epiblast adjacent to the primitive streak, the primitive streak and the newly formed mesoderm. This includes members of the FGF, NOTCH, EPH, PDGF, and canonical and non-canonical WNT pathways, negative modulators of these pathways, and a large number of transcriptional regulatory genes. <it>SNAI2 </it>expression in the primitive streak and mesoderm is not altered by FGFR inhibition, but is downregulated only in the preingression epiblast region with no significant effect on E-cadherin. Furthermore, over expression of SNAIL has no discernable effect on E-cadherin protein levels or localization in epiblast, primitive streak or mesodermal cells. FGFR activity modulates distinct downstream pathways including RAS/MAPK and PI3K/AKT. Pharmacological inhibition of MEK or AKT indicate that these downstream effectors control discrete and overlapping groups of genes during gastrulation. FGFR activity regulates components of several pathways known to be required for cell migration through the streak or in the mesoderm, including RHOA, the non-canonical WNT pathway, PDGF signalling and the cell adhesion protein N-cadherin.</p> <p>Conclusions</p> <p>In chicken embryos, FGF signalling regulates cell movement through the primitive streak by mechanisms that appear to be independent of changes in E-cadherin expression or protein localization. The positive and negative effects on large groups of genes by pharmacological inhibition of FGF signalling, including major signalling pathways and transcription factor families, indicates that the FGF pathway is a focal point of regulation during gastrulation in chicken.</p

BioNetBuilder2.0: bringing systems biology to chicken and other model organisms

BACKGROUND:Systems Biology research tools, such as Cytoscape, have greatly extended the reach of genomic research. By providing platforms to integrate data with molecular interaction networks, researchers can more rapidly begin interpretation of large data sets collected for a system of interest. BioNetBuilder is an open-source client-server Cytoscape plugin that automatically integrates molecular interactions from all major public interaction databases and serves them directly to the user's Cytoscape environment. Until recently however, chicken and other eukaryotic model systems had little interaction data available.RESULTS:Version 2.0 of BioNetBuilder includes a redesigned synonyms resolution engine that enables transfer and integration of interactions across speciesthis engine translates between alternate gene names as well as between orthologs in multiple species. Additionally, BioNetBuilder is now implemented to be part of the Gaggle, thereby allowing seamless communication of interaction data to any software implementing the widely used Gaggle software. Using BioNetBuilder, we constructed a chicken interactome possessing 72,000 interactions among 8,140 genes directly in the Cytoscape environment. In this paper, we present a tutorial on how to do so and analysis of a specific use case.CONCLUSION:BioNetBuilder 2.0 provides numerous user-friendly systems biology tools that were otherwise inaccessible to researchers in chicken genomics, as well as other model systems. We provide a detailed tutorial spanning all required steps in the analysis. BioNetBuilder 2.0, the tools for maintaining its data bases, standard operating procedures for creating local copies of its back-end data bases, as well as all of the Gaggle and Cytoscape codes required, are open-source and freely available at http://err.bio.nyu.edu/cytoscape/bionetbuilder/ webcite.This item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at [email protected]

Neuroinflammation mediates noise-induced synaptic imbalance and tinnitus in rodent models

Hearing loss is a major risk factor for tinnitus, hyperacusis, and central auditory processing disorder. Although recent studies indicate that hearing loss causes neuroinflammation in the auditory pathway, the mechanisms underlying hearing loss-related pathologies are still poorly understood. We examined neuroinflammation in the auditory cortex following noise-induced hearing loss (NIHL) and its role in tinnitus in rodent models. Our results indicate that NIHL is associated with elevated expression of proinflammatory cytokines and microglial activation-two defining features of neuroinflammatory responses-in the primary auditory cortex (AI). Genetic knockout of tumor necrosis factor alpha (TNF-alpha) or pharmacologically blocking TNF-alpha expression prevented neuroinflammation and ameliorated the behavioral phenotype associated with tinnitus in mice with NIHL. Conversely, infusion of TNF-alpha into AI resulted in behavioral signs of tinnitus in both wild-type and TNF-alpha knockout mice with normal hearing. Pharmacological depletion of microglia also prevented tinnitus in mice with NIHL. At the synaptic level, the frequency of miniature excitatory synaptic currents (mEPSCs) increased and that of miniature inhibitory synaptic currents (mIPSCs) decreased in AI pyramidal neurons in animals with NIHL. This excitatory-to-inhibitory synaptic imbalance was completely prevented by pharmacological blockade of TNF-alpha expression. These results implicate neuroinflammation as a therapeutic target for treating tinnitus and other hearing loss-related disorders.National Institute of Health [DC009259, DC014335]; Department of Defense [W81XWH-15-1-0028, W81XWH-15-1-0356, W81XWH-15-1-0357]; Food and Health Bureau of Hong Kong Special Administrative Region Government [04150076]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Gastrulation EMT Is Independent of P-Cadherin Downregulation - Fig 2

<p>Co localization of E-cad and P-cad in using a P-cad-specific antibody prepared against a peptide from the extracellular domain of P-cad that shares only three amino acids with the corresponding sequence in E-cad (A), and a commercially obtained antibody raised against the E-cad intracellular domain that recognizes both P-cad and E-cad. (B-D) Transverse section through the mid streak region of a HH stage 4 embryos, showing immunolocalization E-cad and/or P-cad (B), or P-cad (C), or both (D).</p

C3 induces EMT without activating Slug or upregulating N-cad.

<p>(A,B) Transverse sections showing the same microscopic field four hours following electroporation with the C3 expression vector. While cells expressing C3 (green) in the primitive streak region within the domain of normal Slug expression were also Slug-positive (arrowheads in A, B), C3 expressing cells in more lateral regions that were exiting the epiblast or that had migrated into the mesoderm failed to express detectable levels of Slug (arrows in A, B). (C, D) C3 expressing cells that had undergone EMT and migrated into the mesoderm failed to express N-cad.</p

Protein localization in transverse sections of gastrulating chicken and mouse embryos.

<p>(A-C) Localization of E-cad and/or P-cad, p120-catenin, GM130 and ZO1 in transverse sections of HH stage 4 embryos. (A, B) The same microscopic field showing expression of E-cad and/or P-cad (A) and p120-catenin (B). The proteins colocalize at the periphery of cells in the epiblast, primitive streak and mesoderm. (C) Colocalization of E-cad and/or P-cad, GM130 and ZO1in a transverse section of a HH stage 4 embryo. Cells moving from the epiblast to the mesoderm retain E-cad and/or P-cad at their periphery while exhibiting a reorientation of Golgi that is characteristic of the change in cell polarity associated with EMT. (D) Transverse section through a E7.5 mouse embryo at the level of the primitive streak, visualizing E-cad and/or P-cad proteins. (E) Higher magnification of the boxed area in (D), showing persistence of E-cad and/or P-cad proteins on the surface of cells in the mesoderm below and near the primitive streak (arrow). Asterisks are rounded cells in the mesoderm that retain E-cad and/or P-cad protein.</p

Effects of SNAI2 morpholino on Slug protein expression and cell location.

<p>(A) Reduction of Slug protein in most cells electroporated with morpholino targeting the initiation of translation of the Slug (Snail2) mRNA compared to the control (five base pair mismatch) morpholino. While most cells containing the control morpholino show high levels of Slug protein eight hours following electroporation as assayed by immunofluorescence, almost all cells containing the SNAI2 morpholino showed reduced or undetectable levels of Slug. (B) Relative location of epiblast cells electroporated with control or SNAI2 morpholino eight hours following electroporation, and the percent of cells at each location that are Slug-positive. Numbers of cells are shown above each bar. Whereas the percent of cells containing control morpholinos that are Slug-positive is similar at all locations, a higher percentage of Slug-positive cells containing the SNAI2 morpholinos were observed in the mesoderm away from the streak, indicating that SNAI2 morpholino containing cells lacking detectable Slug expression were preferentially located near the primitive streak.</p

Location of SNAI2 morpholino and control morpholino containing cells in transverse sections of embryos.

<p>A fluorescein-labeled morpholino targeting the initiation of translation of the <i>SNAI2</i> mRNA, or a five base pair mismatch control morpholino, were electroporated into the epiblast of HH stage 3–4 embryos. (A-B) The same microscopic field visualizing cells containing the control morpholino (green) and Slug (red) in (A), or Slug (B). Almost all cells containing the control morpholino also express Slug protein (white arrows). (C-D) The same microscopic field visualizing cells containing the SNAI2 morpholino and Slug (C), or Slug (D). Most cells in the primitive streak containing SNAI2 morpholinos were Slug negative (white arrows). (E,F) Higher magnification views showing cells containing the SNAI2 morpholino, and lacking detectable Slug protein, undergoing EMT (arrows).</p