Diana K. Darnell

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 ≈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.

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. Developmental Dynamics 235:3156–3165, 2006.

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.

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.

Vertical induction of engrailed-2 and other region-specific markers in the early chick embryo.

We investigated the role of vertical signals in the regulation of Engrailed‐2, a regionally restricted (mesencephalon/metencephalon) neuroectodermal marker, using epiblast grafted from prospective neuroectoderm or prospective trunk mesoderm at mid‐stage 3 in the gastrulating chick embryo. Grafts that were isolated from the rostral (prospective neuroectodermal) epiblast and placed rostral to or at the future mesencephalon/metencephalon level, between the endoderm and epiblast of stage 3d to stage 8 host embryos, expressed Engrailed‐2 after 24 hr in culture, whereas these same grafts failed to express this marker when placed at a more caudal level. Grafts from caudal (prospective trunk mesodermal) epiblast, which would ordinarily not express Engrailed‐2, also expressed this marker when placed at the mesencephalon/metencephalon level, and failed to express it when grafted more caudally. The expression of four other markers, L5, Fgf8, Wnt‐1, and paraxis, were also evaluated. Collectively, our results show that regionally restricted vertical signals are capable of inducing neuroectoderm from naive tissue, and of patterning epiblast to express some but not all mesencephalon/metencephalon isthmus markers. Experiments using grafts taken from older embryos indicated that the competence of prospective neuroectoderm to become regionally patterned by vertical signals is gradually lost between stage 3c and stage 7. Similarly, prospective mesoderm from the caudal epiblast becomes unable to respond to vertical, neural‐inductive signals at these stages. These observations support a role for vertical signals in the induction and patterning of the neuroectoderm at gastrula and early neurula stages. Dev. Dyn. 209:45–58, 1997.

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. Developmental Dynamics 235:3213–3221, 2006.

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. Developmental Dynamics 239:1879–1887, 2010.

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.

Cell interactions underlying notochord induction and formation in the chick embryo.

The development of the notochord in the chick is traditionally associated with Hensens node (the avian equivalent of the organizer). However, recent evidence has shown that two areas outside the node (called the inducer and responder) are capable of interacting after ablation of Hensens node to form a notochord. It was not clear from these studies what effect (if any) signals from these areas had on normal notochord formation. A third area, the postnodal region, may also contribute to notochord formation, although this has also been questioned. Using transection and grafting experiments, we have evaluated the timing and cellular interactions involved in notochord induction and formation in the chick embryo. Our results indicate that the rostral primitive streak, including the node, is not required for formation of the notochord in rostral blastoderm isolates transected at stages 3a/b. In addition, neither the postnodal region nor the inducer is required for the induction and formation of the most rostral notochordal cells. However, inclusion of the inducer results in considerable elongation of the notochord in this experimental paradigm. Our results also demonstrate that the responder per se is not required for notochord formation, provided that at least the inducer and postnodal region are present, although in the absence of the responder, formation of the notochord occurs far less frequently. We also show that the node is not specified to form notochord until stage 4 and concomitant with this, the inducer loses its ability to induce notochord from the responder. The coincident timing of these changes in the node and inducer suggests that notochord specification and the activity of the inducer are regulated through a negative feedback loop. We propose a model relating our results to the induction of head and trunk organizer activity in the node.

Anteroposterior and Dorsoventral Patterning

The predominant method of achieving a patterned vertebrate nervous system involves responses to signaling from an asymmetrical source. This instigates the activation of new transcription factors in the responding tissue, which leads to a cascade of cellular changes that generate additional asymmetry and cell differentiation. Several of the signaling sources have been identified, including the neural organizer/node, the anterior neural ridge, the isthmus, and the caudal mesoderm along the AP axis, and the notochord and epidermal ectoderm in the DV axis. Signals induce changes in target cells depending on concentration, and antagonists or distance protect other cells from responding, generating diverse cell types. These general concepts and in many cases the specific genetic networks for patterning have been conserved for hundreds of millions of years.