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-α) or pharmacologically blocking TNF-α expression prevented neuroinflammation and ameliorated the behavioral phenotype associated with tinnitus in mice with NIHL. Conversely, infusion of TNF-α into AI resulted in behavioral signs of tinnitus in both wild-type and TNF-α 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-α expression. These results implicate neuroinflammation as a therapeutic target for treating tinnitus and other hearing loss-related disorders.

Blocking Tumor Necrosis Factor-Alpha Expression Prevents Blast-Induced Excitatory/Inhibitory Synaptic Imbalance and Parvalbumin-Positive Interneuron Loss in the Hippocampus.

Traumatic brain injury (TBI) is a major cause of neurological disorder and death in civilian and military populations. It comprises two components-direct injury from the traumatic impact and secondary injury from ensuing neural inflammatory responses. Blocking tumor necrosis factor-alpha (TNF-α), a central regulator of neural inflammation, has been shown to improve functional recovery after TBI. However, the mechanisms underlying those therapeutic effects are still poorly understood. Here, we examined effects of 3,6'-dithiothalidomide (dTT), a potentially therapeutic TNF-α inhibitor, in mice with blast-induced TBI. We found that blast exposure resulted in elevated expression of TNF-α, activation of microglial cells, enhanced excitatory synaptic transmission, reduced inhibitory synaptic transmission, and a loss of parvalbumin-positive (PV+) inhibitory interneurons. Administration of dTT for 5 days after the blast exposure completely suppressed blast-induced increases in TNF-α transcription, largely reversed blasted-induced synaptic changes, and prevented PV+ neuron loss. However, blocking TNF-α expression by dTT failed to mitigate blast-induced microglial activation in the hippocampus, as evidenced by their non-ramified morphology. These results indicate that TNF-α plays a major role in modulating neuronal functions in blast-induced TBI and that it is a potential target for treatment of TBI-related brain disorders.

Gastrulation EMT Is Independent of P-Cadherin Downregulation.

Epithelial-mesenchymal transition (EMT) is an evolutionarily conserved process during which cells lose epithelial characteristics and gain a migratory phenotype. Although downregulation of epithelial cadherins by Snail and other transcriptional repressors is generally considered a prerequisite for EMT, recent studies have challenged this view. Here we investigate the relationship between E-cadherin and P-cadherin expression and localization, Snail function and EMT during gastrulation in chicken embryos. Expression analyses show that while E-cadherin transcripts are detected in the epiblast but not in the primitive streak or mesoderm, P-cadherin mRNA and protein are present in the epiblast, primitive and mesoderm. Antibodies that specifically recognize E-cadherin are not presently available. During EMT, P-cadherin relocalizes from the lateral surfaces of epithelial epiblast cells to a circumferential distribution in emerging mesodermal cells. Cells electroporated with an E-cadherin expression construct undergo EMT and migrate into the mesoderm. An examination of Snail function showed that reduction of Slug (SNAI2) protein levels using a morpholino fails to inhibit EMT, and expression of human or chicken Snail in epiblast cells fails to induce EMT. In contrast, cells expressing the Rho inhibitor peptide C3 rapidly exit the epiblast without activating Slug or the mesoderm marker N-cadherin. Together, these experiments show that epiblast cells undergo EMT while retaining P-cadherin, and raise questions about the mechanisms of EMT regulation during avian gastrulation.

GEISHA: an evolving gene expression resource for the chicken embryo.

GEISHA (Gallus Expression In Situ Hybridization Analysis; http://geisha.arizona.edu) is an in situ hybridization gene expression and genomic resource for the chicken embryo. This update describes modifications that enhance its utility to users. During the past 5 years, GEISHA has undertaken a significant restructuring to more closely conform to the data organization and formatting of Model Organism Databases in other species. This has involved migrating from an entry-centric format to one that is gene-centered. Database restructuring has enabled the inclusion of data pertaining to chicken genes and proteins and their orthologs in other species. This new information is presented through an updated user interface. In situ hybridization data in mouse, frog, zebrafish and fruitfly are integrated with chicken genomic and expression information. A resource has also been developed that integrates the GEISHA interface information with the Online Mendelian Inheritance in Man human disease gene database. Finally, the Chicken Gene Nomenclature Committee database and the GEISHA database have been integrated so that they draw from the same data resources.

Embryonic expression of the transforming growth factor beta ligand and receptor genes in chicken.

BACKGROUND: Transforming growth factor-beta (TGFß) signaling regulates a myriad of biological processes during embryogenesis, in the adult, and during the manifestation of disease. TGFß signaling is propagated through one of three TGFß ligands interacting with Type I and Type II receptors, and Type III co-receptors. Although TGFß signaling is regulated partly by the combinatorial expression patterns of TGFß receptors and ligands, a comprehensive gene expression analysis has not been published. RESULTS: Here we report the embryonic mRNA expression patterns in chicken embryos of the canonical TGFß ligands (TGFB1, TGFB2, and TGFB3) and receptors (TGFBR1, TGFBR2, TGFBR3), plus the Activin A receptor, type 1 (ACVR1) and co receptor Endoglin (ENG) that also transduce TGFß signaling. CONCLUSIONS: TGFB ligands and receptors show dynamic and frequently overlapping expression patterns in numerous embryonic cell layers and structures. Integrating expression information identifies combinations of ligands and receptors that are involved in specific developmental processes including somitogenesis, cardiogenesis and vasculogenesis.

Fibroblast growth factor (FGF) signaling during gastrulation negatively modulates the abundance of microRNAs that regulate proteins required for cell migration and embryo patterning.

FGF signaling plays a pivotal role in regulating cell movements and lineage induction during gastrulation. Here we identify 44 microRNAs that are expressed in the primitive streak region of gastrula stage chicken embryos. We show that the primary effect of FGF signaling on microRNA abundance is to negatively regulate the levels of miR-let-7b, -9, -19b, -107, -130b, and -218. LIN28B inhibits microRNA processing and is positively regulated by FGF signaling. Gain- and loss-of-function experiments show that LIN28B negatively regulates the expression of miR-19b, -130b, and let-7b, whereas negative modulation of miR-9, -107, and -218 appears to be independent of LIN28B function. Predicted mRNA targets of the FGF-regulated microRNAs are over-represented in serine/threonine and tyrosine kinase receptors, including ACVR1, ACVR2B, PDGFRA, TGFBR1, and TGFBR3. Luciferase assays show that these and other candidates are targeted by FGF-regulated microRNAs. PDGFRA, a receptor whose activity is required for cell migration through the primitive streak, is a target of miR-130b and -218 in vivo. These results identify a novel mechanism by which FGF signaling regulates gene expression by negatively modulating microRNA abundance through both LIN28B-dependent and LIN28B-independent pathways.

Wnt signaling and a Smad pathway blockade direct the differentiation of human pluripotent stem cells to multipotent neural crest cells.

Neural crest stem cells can be isolated from differentiated cultures of human pluripotent stem cells, but the process is inefficient and requires cell sorting to obtain a highly enriched population. No specific method for directed differentiation of human pluripotent cells toward neural crest stem cells has yet been reported. This severely restricts the utility of these cells as a model for disease and development and for more applied purposes such as cell therapy and tissue engineering. In this report, we use small-molecule compounds in a single-step method for the efficient generation of self-renewing neural crest-like stem cells in chemically defined media. This approach is accomplished directly from human pluripotent cells without the need for coculture on feeder layers or cell sorting to obtain a highly enriched population. Critical to this approach is the activation of canonical Wnt signaling and concurrent suppression of the Activin A/Nodal pathway. Over 12-14 d, pluripotent cells are efficiently specified along the neuroectoderm lineage toward p75(+) Hnk1(+) Ap2(+) neural crest-like cells with little or no contamination by Pax6(+) neural progenitors. This cell population can be clonally amplified and maintained for >25 passages (>100 d) while retaining the capacity to differentiate into peripheral neurons, smooth muscle cells, and mesenchymal precursor cells. Neural crest-like stem cell-derived mesenchymal precursors have the capacity for differentiation into osteocytes, chondrocytes, and adipocytes. In sum, we have developed methods for the efficient generation of self-renewing neural crest stem cells that greatly enhance their potential utility in disease modeling and regenerative medicine.

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

BACKGROUND: 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. RESULTS: 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. SNAI2 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. CONCLUSIONS: 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.

Embryonic expression of the chicken Krüppel-like (KLF) transcription factor gene family.

The Krüppel-like transcription factors (KLF) are zinc finger proteins that activate and suppress target gene transcription. Although KLF factors have been implicated in regulating many developmental processes, a comprehensive gene expression analysis has not been reported. Here we present the chicken KLF gene family and expression during the first five days of embryonic development. Fourteen chicken KLF genes or expressed sequences have been previously identified. Through synteny analysis and cDNA mapping, we have identified the KLF9 gene and determined that the gene presently named KLF1 is the true ortholog of KLF17 in other species. In situ hybridization expression analyses show that in general KLFs are broadly expressed in multiple cell and tissue types. Expression of KLFs 3, 7, 8, and 9, is widespread at all stages examined. KLFs 2, 4, 5, 6, 10, 11, 15, and 17 show more restricted patterns that suggest multiple functions during early stages of embryonic development.

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 species; this 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/.

Non-canonical Wnt signaling through Wnt5a/b and a novel Wnt11 gene, Wnt11b, regulates cell migration during avian gastrulation.

Knowledge of the molecular mechanisms regulating cell ingression, epithelial-mesenchymal transition and migration movements during amniote gastrulation is steadily improving. In the frog and fish embryo, Wnt5 and Wnt11 ligands are expressed around the blastopore and play an important role in regulating cell movements associated with gastrulation. In the chicken embryo, although Wnt5a and Wnt5b are expressed in the primitive streak, the known Wnt11 gene is expressed in paraxial and intermediate mesoderm, and in differentiated myocardial cells, but not in the streak. Here, we identify a previously uncharacterized chicken Wnt11 gene, Wnt11b, that is orthologous to the frog Wnt11 and zebrafish Wnt11 (silberblick) genes. Chicken Wnt11b is expressed in the primitive streak in a pattern similar to chicken Wnt5a and Wnt5b. When non-canonical Wnt signaling is blocked using a Dishevelled dominant-negative protein, gastrulation movements are inhibited and cells accumulate in the primitive streak. Furthermore, disruption of non-canonical Wnt signaling by overexpression of full-length or dominant-negative Wnt11b or Wnt5a constructions abrogates normal cell migration through the primitive streak. We conclude that non-canonical Wnt signaling, mediated in part by Wnt11b, is important for regulation of gastrulation cell movements in the avian embryo.

Myocardin expression during avian embryonic heart development requires the endoderm but is independent of BMP signaling.

Myocardin, a serum response factor cofactor, plays an important role in regulating heart and smooth muscle development. To investigate myocardin function during early stages of heart development, we isolated the chicken orthologue of myocardin and characterized its expression between Hamburger and Hamilton stages 3 and 15. At stage 4, myocardin transcripts are detected in the lateral and extraembryonic mesoderm, become progressively localized to the precardiac mesoderm and the differentiated myocardium and are also seen in smooth muscle cells of the developing vascular plexus. Surprisingly, myocardin expression within the developing chicken embryo precedes that of the homeodomain transcription factor Nkx2.5. Embryonic dissection studies demonstrate that signals from the endoderm are required for myocardin expression within the precardiac mesoderm. However, unlike Nkx2.5, myocardin expression is not regulated by bone morphogenetic protein (BMP) signaling. These results suggest that initial expression of myocardin in the precardiac mesoderm is regulated by a signaling pathway that is parallel to, and independent of, Nkx2.5 expression.

MicroRNA expression during chick embryo development.

MicroRNAs (miRNAs) are small, abundant, noncoding RNAs that modulate protein abundance by interfering with target mRNA translation or stability. miRNAs are detected in organisms from all domains and may regulate 30% of transcripts in vertebrates. Understanding miRNA function requires a detailed determination of expression, yet this has not been reported in an amniote species. High-throughput whole mount in situ hybridization was performed on chicken embryos to map expression of 135 miRNA genes including five miRNAs that had not been previously reported in chicken. Eighty-four miRNAs were detected before day 5 of embryogenesis, and 75 miRNAs showed differential expression. Whereas few miRNAs were expressed during formation of the primary germ layers, the number of miRNAs detected increased rapidly during organogenesis. Patterns highlighted cell-type, organ or structure-specific expression, localization within germ layers and their derivatives, and expression in multiple cell and tissue types and within sub-regions of structures and tissues. A novel group of miRNAs was highly expressed in most tissues but much reduced in one or a few organs, including the heart. This study presents the first comprehensive overview of miRNA expression in an amniote organism and provides an important foundation for investigations of miRNA gene regulation and function.

Hedgehog signaling is essential for endothelial tube formation during vasculogenesis.

During embryonic development, the first blood vessels are formed through the aggregation and subsequent assembly of angioblasts (endothelial precursors) into a network of endothelial tubes, a process known as vasculogenesis. These first vessels generally form in mesoderm that is adjacent to endodermal tissue. Although specification of the angioblast lineage is independent of endoderm interactions, a signal from the endoderm is necessary for angioblasts to assemble into a vascular network and to undergo vascular tube formation. In this study, we show that endodermally derived sonic hedgehog is both necessary and sufficient for vascular tube formation in avian embryos. We also show that Hedgehog signaling is required for vascular tube formation in mouse embryos, and for vascular cord formation in cultured mouse endothelial cells. These results demonstrate a previously uncharacterized role for Hedgehog signaling in vascular development, and identify Hedgehog signaling as an important component of the molecular pathway leading to vascular tube formation.

Regulation of Hex gene expression and initial stages of avian hepatogenesis by Bmp and Fgf signaling.

The vertebrate liver and heart arise from adjacent cell layers in the anterior lateral (AL) endoderm and mesoderm of late gastrula embryos, and the earliest stages of liver and heart development are interrelated through reciprocal tissue interactions. Although classical embryological studies performed several decades ago in chick and quail defined the timing of hepatogenic induction in birds and the important role for cardiogenic mesoderm in this process, almost nothing is known about the molecular aspects of avian liver development. Here we use in vivo and explantation assays to investigate tissue interactions and signaling pathways regulating Hex, a homeobox gene required for liver development, and the earliest stages of hepatogenesis in the chick embryo. We find that explants of late gastrula anterior lateral endoderm plus mesoderm, which have been used extensively for studies relating to heart development, also produce albumin-expressing hepatoblasts. Expression of Hex, the earliest known molecular marker for the hepatogenic endoderm, and albumin, indicative of early committed hepatoblasts, requires both autocrine Bmp signaling and a specific paracrine signal from the cardiogenic (anterior lateral) mesoderm. Endodermal expression of Fox2a, in contrast, requires the mesoderm but is independent of Bmp signaling. In vivo induction assays show that the ability of BMP2 to activate Hex expression in the endoderm is restricted to a region that is only slightly larger than the endogenous domain of Hex expression. Although Fgfs can substitute for the cardiogenic mesoderm to support the expression of Hex and albumin in the endoderm, several Fgf genes are expressed in the anterior lateral endoderm but an Fgf expressed predominantly in the mesoderm was not identified. Studies also showed that Fgf gene expression in the endoderm does not require a signal from the mesoderm. Mechanisms regulating endodermal signaling pathways activated by Fgfs may therefore be more complex than previously appreciated.

GEISHA, a whole-mount in situ hybridization gene expression screen in chicken embryos.

Despite the increasing quality and quantity of genomic sequence that is available to researchers, predicting gene function from sequence information remains a challenge. One method for obtaining rapid insight into potential functional roles of novel genes is through gene expression mapping. We have performed a high throughput whole-mount in situ hybridization (ISH) screen with chick embryos to identify novel, differentially expressed genes. Approximately 1,200 5' expressed sequence tags (ESTs) were generated from cDNA clones of a Hamburger and Hamilton (HH) stage 4-7 (late gastrula) chick embryo endoderm-mesoderm library. After screening to remove ubiquitously expressed cDNAs and internal clustering and after comparison to GenBank sequences, remaining cDNAs (representing both characterized and uncharacterized genes) were screened for expression in HH stage 3-14 embryos by automated high throughput ISH. Of 786 cDNAs for which ISH was successfully performed, approximately 30% showed ubiquitous expression, 40% were negative, and approximately 30% showed a restricted expression pattern. cDNAs were identified that showed restricted expression in every embryonic region, including the primitive streak, somites, developing cardiovascular system and neural tube/neural crest. A relational database was developed to hold all EST sequences, ISH images, and corresponding BLAST report information, and to enable browsing and querying of data. A user interface is freely accessible at http://geisha.biosci.arizona.edu. Results show that high throughput whole-mount ISH provides an effective approach for identifying novel genes that are differentially expressed in the developing chicken embryo.

Regulation of Hex gene expression by a Smads-dependent signaling pathway.

The homeobox gene Hex is expressed in multiple cell types during embryogenesis and is required for liver and monocyte development. Hex is expressed in the foregut region of late gastrula avian and mammalian embryos in a pattern that overlaps with expression of bone morphogenetic proteins (BMPs). Here we investigate the relationship between BMP signaling and Hex gene expression. We find that Hex expression in avian anterior lateral endoderm is regulated by autocrine BMP signaling. Characterization of the mouse Hex gene promoter identified a 71-nucleotide BMP-responsive element (BRE) that is required for up-regulation of Hex by an activated BMP signaling pathway. The Hex BRE binds Smad4 and Smad1-Smad4 complexes in vitro, and in transfection assays, it is responsive to Smad1 and Smad4 but not to Smad2 and Smad4 or Smad3 and Smad4. The BRE contains two copies of a GCCGnCGC-like motif that in Drosophila is the binding site for Mad and Madea followed by two CAGAG boxes that are similar to sequences required for transforming growth factor-beta/activin responsiveness of several vertebrate genes. Mutation of the GC elements, but not the two CAGAG boxes, abolishes Smads responsiveness in the intact Hex promoter, whereas mutations in both the GC elements and CAGAG boxes show that they act cooperatively to confer Smads responsiveness to the Hex promoter. The Hex BRE can confer Smads responsiveness to a heterologous promoter, and in this context, both the GC-rich elements and the CAGAG boxes are required for Smads-dependent promoter activity. An element almost identical to the Hex BRE is present within the BMP-responsive Nkx2-5 gene promoter, suggesting that the Hex BRE represents a common response element for genes regulated by BMP signaling in the foregut region of the embryo.

Precocious expression of cardiac troponin T in early chick embryos is independent of bone morphogenetic protein signaling.

Cardiac troponin T (cTNT) is a component of the troponin complex, which confers calcium sensitivity to contraction in skeletal and cardiac muscle. Although it is thought that most components of the contractile myofibril are expressed exclusively in differentiated muscle cells, we observed that mRNAs coding for cTNT were detectable in explanted late gastrula mesoderm at least 12 hr before cardiac myocyte differentiation. We therefore conducted a detailed analysis of cTNT gene expression in the early chick embryo. Whole-mount in situ hybridization studies showed that by Hamburger and Hamilton stage 5, cTNT mRNAs are detectable in lateral mesoderm and, by stage 6, are observed throughout the lateral embryonic and extraembryonic mesoderm in a distribution that is much broader than the recognized heart field. As myocardial cell differentiation commences, cTNT transcripts become progressively localized to the forming heart and, by stage 14, are completely restricted to heart muscle cells. Western blot analyses demonstrated that cTNT protein expression is under translational control, as cTNT protein is not detectable until stage 9, concomitant with myocardial cell differentiation. Removal of endoderm at stage 5 had no effect on cTNT mRNA levels, and the bone morphogenetic protein (BMP) inhibitor noggin failed to block cTNT expression, even in the heart-forming region and in cases where heart formation was inhibited. Implantation of noggin-expressing CHO cells at the anterior midline of stage 7 embryos resulted in cardia bifida. These findings demonstrate the precocious, BMP-independent expression of a gene coding for a myofibrillar protein and suggest that an additional regulatory pathway exists for activation of some cardiogenic genes.