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.

Whole mount in situ hybridization detection of mRNAs using short LNA containing DNA oligonucleotide probes.

In situ hybridization is widely used to visualize transcribed sequences in embryos, tissues, and cells. For whole mount detection of mRNAs in embryos, hybridization with an antisense RNA probe is followed by visual or fluorescence detection of target mRNAs. A limitation of this approach is that a cDNA template of the target RNA must be obtained in order to generate the antisense RNA probe. Here we investigate the use of short (12-24 nucleotides) locked nucleic acid (LNA) containing DNA probes for whole mount in situ hybridization detection of mRNAs. Following extensive protocol optimization, we show that LNA probes can be used to localize several mRNAs of varying abundances in chicken embryos. LNA probes also detected alternatively spliced exons that are processed in a tissue specific manner. The use of LNA probes for whole mount in situ detection of mRNAs will enable in silico design and chemical synthesis and will expand the general use of in situ hybridization for studies of transcriptional regulation and alternative splicing.

Accelerated evolution of 3'avian FOXE1 genes, and thyroid and feather specific expression of chicken FoxE1.

BACKGROUND: The forkhead transcription factor gene E1 (FOXE1) plays an important role in regulation of thyroid development, palate formation and hair morphogenesis in mammals. However, avian FOXE1 genes have not been characterized and as such, codon evolution of FOXE1 orthologs in a broader evolutionary context of mammals and birds is not known. RESULTS: In this study we identified the avian FOXE1 gene in chicken, turkey and zebra finch, all of which consist of a single exon. Chicken and zebra finch FOXE1 are uniquely located on the sex-determining Z chromosome. In situ hybridization shows that chicken FOXE1 is specifically expressed in the developing thyroid. Its expression is initiated at the placode stage and is maintained during the stages of vesicle formation and follicle primordia. Based on this expression pattern, we propose that avian FOXE1 may be involved in regulating the evagination and morphogenesis of thyroid. Chicken FOXE1 is also expressed in growing feathers. Sequence analysis identified two microdeletions in the avian FOXE1 genes, corresponding to the loss of a transferable repression domain and an engrailed homology motif 1 (Eh1) C-terminal to the forkhead domain. The avian FOXE1 proteins exhibit a significant sequence divergence of the C-terminus compared to those of amphibian and mammalian FOXE1. The codon evolution analysis (dN/dS) of FOXE1 shows a significantly increased dN/dS ratio in the avian lineages, consistent with either a relaxed purifying selection or positive selection on a few residues in avian FOXE1 evolution. Further site specific analysis indicates that while relaxed purifying selection is likely to be a predominant cause of accelerated evolution at the 3'-region of avian FOXE1, a few residues might have evolved under positive selection. CONCLUSIONS: We have identified three avian FOXE1 genes based on synteny and sequence similarity as well as characterized the expression pattern of the chicken FOXE1 gene during development. Our evolutionary analyses suggest that while a relaxed purifying selection is likely to be the dominant force driving accelerated evolution of avian FOXE1 genes, a few residues may have evolved adaptively. This study provides a basis for future genetic and comparative biochemical studies of FOXE1.

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.

Student engagement, assessed using heart rate, shows no reset following active learning sessions in lectures.

Heart rate can be used as a measure of cognitive engagement. We measured average student heart rates during medical school lecture classes using wristwatch-style monitors. Analysis of 42 classes showed a steady decline in heart rate from the beginning to end of a lecture class. Active learning sessions (peer-discussion based problem solving) resulted in a significant uptick in heart rate, but this returned to the average level immediately following the active learning period. This is the first statistically robust assessment of changes in heart rate during the course of college lecture classes and indicates that personal heart rate monitors may be useful tools for assessment of different teaching modalities. The key findings suggest that the value of active learning within the classroom resides in the activity itself and not in an increase in engagement or reset in attention during the didactic period following an active learning session.

LNA-based in situ hybridization detection of mRNAs in embryos.

In situ hybridization (ISH) in embryos allows the visualization of specific RNAs as a readout of gene expression during normal development or after experimental manipulations. ISH using short DNA probes containing locked nucleic acid nucleotides (LNAs) holds the additional advantage of allowing the detection of specific RNA splice variants or of closely related family members that differ in only short regions, creating new diagnostic and detection opportunities. Here we describe methods for using short (14-24 nt) DNA probes containing LNA nucleotides to detect moderately to highly expressed RNAs in whole chick embryos during the first 5 days of embryonic development. The protocol is easily adaptable for use with embryos of other vertebrate species.

Developmental expression of chicken FOXN1 and putative target genes during feather development.

FOXN1 is a member of the forkhead box family of transcription factors. FOXN1 is crucial for hair outgrowth and thymus differentiation in mammals. Unlike the thymus, which is found in all amniotes, hair is an epidermal appendage that arose after the last shared common ancestor between mammals and birds, and hair and feathers differ markedly in their differentiation and gene expression. Here, we show that FOXN1 is expressed in embryonic chicken feathers, nails and thymus, demonstrating an evolutionary conservation that goes beyond obvious homology. At embryonic day (ED) 12, FOXN1 is expressed in some feather buds and at ED13 expression extends along the length of the feather filament. At ED14 FOXN1 mRNA is restricted to the proximal feather filament and is not detectable in distal feather shafts. At the base of the feather, FOXN1 is expressed in the epithelium of the feather sheath and distal barb and marginal plate, whereas in the midsection FOXN1 transcripts are mainly detected in the barb plates of the feather filament. FOXN1 is also expressed in claws; however, no expression was detected in skin or scales. Despite expression of FOXN1 in developing feathers, examination of chick homologs of five putative mammalian FOXN1 target genes shows that, while these genes are expressed in feathers, there is little similarity to the FOXN1 expression pattern, suggesting that some gene regulatory networks may have diverged during evolution of epidermal appendages.

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.

Latrophilin-2 is a novel component of the epithelial-mesenchymal transition within the atrioventricular canal of the embryonic chicken heart.

Endothelial cells in the atrioventricular canal of the heart undergo an epithelial-mesenchymal transition (EMT) to form heart valves. We surveyed an on-line database (http://www.geisha.arizona.edu/) for clones expressed during gastrulation to identify novel EMT components. One gene, latrophilin-2, was identified as expressed in the heart and appeared to be functional in EMT. This molecule was chosen for further examination. In situ localization showed it to be expressed in both the myocardium and endothelium. Several antisense DNA probes and an siRNA for latrophilin-2 produced a loss of EMT in collagen gel cultures. Latrophilin-2 is a putative G-protein-coupled receptor and we previously identified a pertussis toxin-sensitive G-protein signal transduction pathway. Microarray experiments were performed to examine whether these molecules were related. After treatment with antisense DNA against latrophilin-2, expression of 1,385 genes and ESTs was altered. This represented approximately 12.5% of the microarray elements. In contrast, pertussis toxin altered only 103 (0.9%) elements of the array. There appears to be little overlap between the two signal transduction pathways. Latrophilin-2 is thus a novel component of EMT and provides a new avenue for investigation of this cellular process.

Differences in vertebrate microRNA expression.

MicroRNAs (miRNAs) attenuate gene expression by means of translational inhibition and mRNA degradation. They are abundant, highly conserved, and predicted to regulate a large number of transcripts. Several hundred miRNA classes are known, and many are associated with cell proliferation and differentiation. Many exhibit tissue-specific expression, which aids in evaluating their functions, and it has been assumed that their high level of sequence conservation implies a high level of expression conservation. A limited amount of data supports this, although discrepancies do exist. By comparing the expression of approximately 100 miRNAs in medaka and chicken with existing data for zebrafish and mouse, we conclude that the timing and location of miRNA expression is not strictly conserved. In some instances, differences in expression are associated with changes in miRNA copy number, genomic context, or both between species. Variation in miRNA expression is more pronounced the greater the differences in physiology, and it is enticing to speculate that changes in miRNA expression may play a role in shaping the physiological differences produced during animal development.