Jay H. Konieczka

GEISHA: an in situ hybridization gene expression resource for the chicken embryo

An important and ongoing focus of biomedical and agricultural avian research is to understand gene function, which for a significant fraction of genes remains unknown. A first step is to determine when and where genes are expressed during development and in the adult. Whole mount in situ hybridization gives precise spatial and temporal resolution of gene expression throughout an embryo, and a comprehensive analysis and centralized repository of in situ hybridization information would provide a valuable research tool. The GEISHA project (<X_Underline>g</X_Underline>allus <X_Underline>e</X_Underline>xpression <X_Underline>i</X_Underline>n <X_Underline>s</X_Underline>itu <X_Underline>h</X_Underline>ybridization <X_Underline>a</X_Underline>nalysis) was initiated to explore the utility of using high-throughput in situ hybridization as a means for gene discovery and annotation in chicken embryos, and to provide a unified repository for in situ hybridization information. This report describes the design and implementation of a new GEISHA database and user interface (www.geisha.arizona.edu), and illustrates its utility for researchers in the biomedical and poultry science communities. Results obtained from a high throughput screen of microRNA expression in chicken embryos are also presented.

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

BackgroundFGF 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.ResultsWe 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.ConclusionsIn 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.

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

BackgroundSystems 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 users Cytoscape environment. Until recently however, chicken and other eukaryotic model systems had little interaction data available.ResultsVersion 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.ConclusionBioNetBuilder 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/.

Genomic Resources for Chicken

The recent sequencing and draft assembly of a chicken genome has provided biologists with an invaluable research tool that complements a growing list of additional avian genomic resources. For many researchers, finding and using these resources is challenging, because information is presented through an increasing number of Web sites and browser navigation frequently requires specific knowledge and expertise. This primer provides an overview of online genomic resources for the chicken, including the Ensembl, UCSC, and NCBI annotated chicken genome browsers; expressed sequence tag and in situ hybridization databases; and sources for microarrays, cDNAs, and bacterial artificial chromosomes (BACs). Several short tutorials oriented toward the biologist with limited bioinformatics skills outline how to retrieve several types of commonly needed information and reagents. Developmental Dynamics 232:877–882, 2005.

Arsenic Exposure Perturbs Epithelial-Mesenchymal Cell Transition and Gene Expression In a Collagen Gel Assay

Arsenic is a naturally occurring metalloid and environmental contaminant. Arsenic exposure in drinking water is reported to cause cancer of the liver, kidneys, lung, bladder, and skin as well as birth defects, including neural tube, facial, and vasculogenic defects. The early embryonic period most sensitive to arsenic includes a variety of cellular processes. One key cellular process is epithelial-mesenchymal transition (EMT) where epithelial sheets develop into three-dimensional structures. An embryonic prototype of EMT is found in the atrioventricular (AV) canal of the developing heart, where endothelia differentiate to form heart valves. Effects of arsenic on this cellular process were examined by collagen gel invasion assay (EMT assay) using explanted AV canals from chicken embryo hearts. AV canals treated with 12.5-500 ppb arsenic showed a loss of mesenchyme at 12.5 ppb, and mesenchyme formation was completely inhibited at 500 ppb. Altered gene expression in arsenic-treated explants was investigated by microarray analysis. Genes whose expression was altered consistently at exposure levels of 10, 25, and 100 ppb were identified, and results showed that 25 ppb in vitro was particularly effective. Three hundred and eighty two genes were significantly altered at this exposure level. Cytoscape analysis of the microarray data using the chicken interactome identified four clusters of altered genes based on published relationships and pathways. This analysis identified cytoskeleton and cell adhesion-related genes whose disruption is consistent with an altered ability to undergo EMT. These studies show that EMT is sensitive to arsenic and that an interactome-based approach can be useful in identifying targets.

ArrayIDer : automated structural re-annotation pipeline for DNA microarrays

BackgroundSystems biology modeling from microarray data requires the most contemporary structural and functional array annotation. However, microarray annotations, especially for non-commercial, non-traditional biomedical model organisms, are often dated. In addition, most microarray analysis tools do not readily accept EST clone names, which are abundantly represented on arrays. Manual re-annotation of microarrays is impracticable and so we developed a computational re-annotation tool (ArrayIDer) to retrieve the most recent accession mapping files from public databases based on EST clone names or accessions and rapidly generate database accessions for entire microarrays.ResultsWe utilized the Fred Hutchinson Cancer Research Centre 13K chicken cDNA array – a widely-used non-commercial chicken microarray – to demonstrate the principle that ArrayIDer could markedly improve annotation. We structurally re-annotated 55% of the entire array. Moreover, we decreased non-chicken functional annotations by 2 fold. One beneficial consequence of our re-annotation was to identify 290 pseudogenes, of which 66 were previously incorrectly annotated.ConclusionArrayIDer allows rapid automated structural re-annotation of entire arrays and provides multiple accession types for use in subsequent functional analysis. This information is especially valuable for systems biology modeling in the non-traditional biomedical model organisms.

A Genome-Wide Analysis of FRT-Like Sequences in the Human Genome

Efficient and precise genome manipulations can be achieved by the Flp/FRT system of site-specific DNA recombination. Applications of this system are limited, however, to cases when target sites for Flp recombinase, FRT sites, are pre-introduced into a genome locale of interest. To expand use of the Flp/FRT system in genome engineering, variants of Flp recombinase can be evolved to recognize pre-existing genomic sequences that resemble FRT and thus can serve as recombination sites. To understand the distribution and sequence properties of genomic FRT-like sites, we performed a genome-wide analysis of FRT-like sites in the human genome using the experimentally-derived parameters. Out of 642,151 identified FRT-like sequences, 581,157 sequences were unique and 12,452 sequences had at least one exact duplicate. Duplicated FRT-like sequences are located mostly within LINE1, but also within LTRs of endogenous retroviruses, Alu repeats and other repetitive DNA sequences. The unique FRT-like sequences were classified based on the number of matches to FRT within the first four proximal bases pairs of the Flp binding elements of FRT and the nature of mismatched base pairs in the same region. The data obtained will be useful for the emerging field of genome engineering.

Network Elucidation Template: A framework for human-guided network inference

Network elucidation is the problem of inferring all parameters of a network from a subset of those parameters. We introduce the Network Elucidation Template (NET), which provides a framework upon which algorithms for such problems can be built. NET algorithms take advantage of novel methods for collaboration between human operators and computers. They use visualizations of the peculiar structures that appear in optimal solutions to aid the parameter search. By design, NET is at a high enough level of abstraction to describe a class of algorithms, as opposed to a single algorithm. Given a problem, and the structure of that problem, an effective instantiation of the template into an algorithm can be created. We describe one such instantiation: using a network flow framework to implement a NET algorithm for uncovering smuggling networks; as well as the general template.

18-P005 A novel bioinformatics approach to developing a preliminary network model of cardiac myogenesis

Krüppel (Kr) is a segmentation gene which plays one of the key morphogenetic roles in early development of Drosophila. In order to better elucidate the regulatory role of this gene, we analyzed quantitative expression patterns of other segmentation genes in homozygous Kr mutants. During cleavage cycle 14A the posterior domain of giant (gt) and even-skipped (eve) stripe 7 are significantly shifted to the anterior relative to their position in wild-type embryos. We did not detect this difference in positions until 13 and 26 min from the beginning of cycle 14A for gt posterior domain and eve stripe 7, respectively. During the latter part of cycle 14A, these domains shift by 12 and 5% embryo length as compared with wild-type. As zygotic gap proteins first appear at cleavage cycle 12, our results point on the existence of a significant delay in the influence of absence of Kr protein on the behavior of gene expression domains. This suggests that zygotic gap–gap cross-regulation does not play a role in the positioning of segmentation gene expression domains at early times and comes into effect only in cycle 14A. Moreover, we have detected that by the end of cycle 14A in Kr mutants gt posterior domain shifts to the position of neighbouring knirps (kni) domain and does not move further to the anterior, so that none of the genes occupies the position of missing Kr. We propose that in Kr mutants the positions of posterior domains of gt and kni are set by hunchback (hb) which functions as a strong repressor.