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A–D Expression pattern of clone 554-37-b02. A,B Dorsal views; left anterior. C,D Lateral views; left anterior. A,C 12-somite stage, st. 23. B,D 18-/19-somite stage, st. 25
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To rapidly isolate genes specifically expressed during medaka development we generated a cDNA library enriched for genes expressed in the head region of the developing embryo. Clones were spotted on filters automatically and preselected for abundantly expressed genes by hybridizing them with a probe derived from RNA of undifferentiated totipotent c...
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The identification of marker chromosomes in clinical and tumor cytogenetics by chromosome banding analysis can create problems. In this study, we present a strategy to define minute chromosomal rearrangements by multicolor fluorescence in situ hybridization (FISH) with whole chromosome painting probes derived from chromosome-specific DNA libraries...
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... Publications on developmental gene expression atlases containing data from high throughput ISH projects appeared a few years later. In 2000 it was shown that whole-mount ISH techniques could be adapted to perform high-throughput gene expression analyses on mouse (Neidhardt et al., 2000) and medaka (Henrich and Wittbrodt, 2000). In 2003 and 2004 similar high-throughput techniques resulted in mouse atlases like GENSAT, covering the nervous system at different developmental and post natal stages (Gong et al., 2003) and GenePaint.org, ...
Hartontwikkeling is een complex proces. Razendsnel transformeert het hart in wording van een buisvormige structuur naar een hart met vier kamers. Dit is mogelijk door toevoeging van cellen aan de instroom- en uitstroomzijde en door lokale verschillen in celdeling. Bouke de Boer kon met de genexpressiepatronen van twaalf genen in een 3D-model van het hart achttien ruimtelijke domeinen definiëren, elk met een uniek genexpressieprofiel. Twee van die gebieden leidden tot de ontdekking van een nieuwe geninteractie. Ook zag De Boer grote verschillen in celdelingssnelheid tussen de verschillende delen van het hart.
... Histological sections of some of the samples showed that, in most cases that are classified as embryonic body, the staining is restricted to the epidermis of the embryo region (Fig. 6C 0 ). This staining pattern has not been previously reported for similar in situ screenings in either zebrafish or medaka (Kudoh et al., 2001;Henrich and Wittbrodt, 2000;Nguyen et al., 2001;Henrich et al., 2003). At present, the reason for frequent appearance of this staining pattern is unknown, and we confirmed that sense probes of some of these clones did not show any positive staining. ...
The medaka is becoming an attractive model organism for the study of vertebrate early development and organogenesis and large-scale mutagenesis projects that are aimed at creating developmentally defective mutants are now being conducted by several groups in Japan. To strengthen the study of medaka developmental genetics, we have conducted a large-scale isolation of ESTs from medaka embryos and developed tools that facilitate mutant analysis. In this study, we have characterized a total of 132,082 sequences from both ends of cloned insert cDNAs from libraries generated at different stages of medaka embryo development. Clustering analysis with 3-prime sequences finally identified a total of 12,429 clusters. As a pilot analysis, 924 clusters were subjected to in situ hybridization to determine the spatial localization of their transcripts. Using EST sequence data generated in the present study, a 60-mer oligonucleotide microarray with 8,091 unigenes (Medaka Microarray 8K) was constructed and tested for its usefulness in expression profiling. Furthermore, we have developed a rapid and reliable mutant mapping system using a set of mapped EST markers (M-marker 2003) that covers the entire medaka genome. These resources will accelerate medaka mutant analyses and make an important contribution to the medaka genome project.
... Since, even at later stages of development, the embryos are still highly transparent, a unique opportunity is given to look at genes expressed in internal organs during organogenesis. In medaka (Wittbrodt et al., 2002), in situ screens have been reported (Henrich and Wittbrodt, 2000; Nguyen et al., 2001) and large-scale whole-mount in situ screens are in progress (reviewed in Shima et al., 2003 and the present work). Here, we describe an efficient strategy for screening a large number of genes for their expression pattern by wholemount in situ hybridization in medaka embryos. ...
Gene expression profiling is an important component of functional genomics. We present a time and cost efficient high-throughput whole-mount in situ technique to perform a large-scale gene expression analysis in medaka fish (Oryzias latipes) embryos. Medaka is a model system ideally suited for the study of molecular genetics of vertebrate development. Random cDNA clones from an arrayed stage 20 medaka plasmid library were analyzed by whole-mount in situ hybridization on embryos of three representative stages of medaka development. cDNA inserts were colony PCR amplified in a 384-format. The PCR products were used to generate over 2000 antisense RNA digoxigenin probes in a high-throughput process. Whole-mount in situ hybridization was carried out in a robot and a broad range of expression patterns was observed. Partial cDNA sequences and expression patterns were documented with BLAST results, cluster analysis, images and descriptions, respectively; collectively this information was entered into a web-based database, "MEPD" (http://www.embl-heidelberg.de/mepd/), that is publicly accessible.
... While Wnt8 has been shown to be a good candidate for the posteriorizing factor in zebrafish (Fekany-Lee et al., 2000; Erter et al., 2001; Lekven et al., 2001), Wnt8 may rather be required in non-axial mesoderm than in neuroectoderm (Momoi et al., in press). Systematic screening for genes that show a similar expression pattern, designated as a synexpression group (Niehrs and Pollet, 1999), has been successful to define novel signaling pathways (Gawantka et al., 1998; Henrich and Wittbrodt, 2000; Sagerstrom et al., 2001; Nguyen et al., 2001). To gain insights into the molecular mechanism underlying the induction of the posterior CNS, we screened at mid-gastrulation for genes specifically expressed in the posterior tissues, including nonaxial mesoderm and overlying posterior neuroectoderm. ...
The posterior nervous system, including the hindbrain and the spinal cord, has been shown to be formed by the transformation of neural plate of anterior character by signals derived from non-axial mesoderm. Although secreted factors, such as fibroblast growth factors (FGFs), Wnts, retinoic acid (RA) and Nodal, have been proposed to be the posteriorizing factors, the mechanism how neural tissue of posterior character is induced and subsequently specified along the anteroposterior axis remains elusive. To identify intercellular signaling molecules responsible for posteriorization of the neural plate as well as to find molecules induced intracellularly by the posteriorizing signal in the caudal neural plate, we screened by in situ hybridization for genes specifically expressed in posterior tissues, including the posterior neural plate and non-axial mesoderm when posteriorization of the neural plate takes place. From a subtracted library differentiating anterior versus posterior neural plate, 420 cDNA clones were tested, out of which 76 cDNA fragments showed expression restricted to the posterior tissue. These clones turned out to represent 32 different genes, including one novel secreted factor and one transmembrane protein. Seven genes were induced by non-axial mesodermal implants and bFGF beads, suggesting that these are among the early-response genes of the posteriorizing signal. Thus, our approach employing cDNA subtraction and subsequent expression pattern screening allows us to clone candidate genes involved in a novel signaling pathway contributing to the formation of the posterior nervous system.
... The medaka is a freshwater teleost fish that became an additional model in developmental biology complementary to zebrafish (Ishikawa, 2000; Wittbrodt et al., 2002 ). Since it has a short life cycle and has transparent embryos, it has recently been used with success in in situ screens for genes expressed in the developing embryo (Henrich and Wittbrodt, 2000) and for genes controlling cell proliferation in the optic tectum (Nguyen et al., 2001). To the best of our knowledge, it has not been used in the past to study pancreatic development. ...
In the present work, pancreatic organogenesis has been studied in the medaka (Oryzias latipes), a teleost fish with several advantages as an experimental system in developmental biology. We demonstrated that the pancreas develops from three primordia budding from the dorsal and ventral faces of the gut epithelium. Such buds then fuse to form a single endocrine islet surrounded by exocrine tissue. Interestingly, the endocrine tissue forms only from the dorsal bud. We next analyzed a collection of medakas that had been hybridized with cDNAs derived from an anterior brain library. We found new clones expressed in the pancreatic region demonstrating that the medaka can be used to define new genes expressed in the pancreatic region that follow a specific spatial and temporal pattern of expression.
... This finding is further supported by mass spectrometry analysis, which showed that this peptide's molecular mass (6676.6 Da) is very similar to that of 40S Rp S30 from medaka fish (6660 Da) [15]. Moreover, preliminary experiments performed in our laboratory have identified antibacterial activity in ribonucleoprotein particles obtained from liver cells of rainbow trout (data not shown) and we are Fig. 1. ...
An antimicrobial peptide was purified from skin secretions and epithelial cells of rainbow trout by cation exchange and reversed phase chromatography. Partial N-terminal amino acid sequence of the purified peptide revealed 100% identity with the first 11 residues of a 40S ribosomal peptide from medaka fish. Its molecular mass, determined by matrix-associated laser desorption/ionisation mass spectrometry, was found to be 6676.6Da. These results indicate that this antimicrobial peptide is likely to be the 40S ribosomal protein S30. It is active at submicromolar concentrations, with an effective 50% reduction concentration of 0.02-0.04 microM against Planococcus citreus. Thus, in addition to its conventional function in the cell as part of the small ribosomal subunit, this peptide may play a role in protection against intracellular or extracellular pathogens.
... All our data are freely available to the scientific community through interactive web pages; these pages will continue to develop and will allow for more sophisticated mining of the growing dataset. Similar large-scale gene-expression studies have been carried out in other model organisms including Xenopus [33], mouse [34], medaka [35] and Caenorhabditis elegans [36]. Our database is, to our knowledge, the first to use non-redundant expressed sequence tag (EST) collections with the aim of determining and systematically annotating the expression patterns of all genes in an organism. ...
Cell-fate specification and tissue differentiation during development are largely achieved by the regulation of gene transcription.
As a first step to creating a comprehensive atlas of gene-expression patterns during Drosophila embryogenesis, we examined 2,179 genes by in situ hybridization to fixed Drosophila embryos. Of the genes assayed, 63.7% displayed dynamic expression patterns that were documented with 25,690 digital photomicrographs of individual embryos. The photomicrographs were annotated using controlled vocabularies for anatomical structures that are organized into a developmental hierarchy. We also generated a detailed time course of gene expression during embryogenesis using microarrays to provide an independent corroboration of the in situ hybridization results. All image, annotation and microarray data are stored in publicly available database. We found that the RNA transcripts of about 1% of genes show clear subcellular localization. Nearly all the annotated expression patterns are distinct. We present an approach for organizing the data by hierarchical clustering of annotation terms that allows us to group tissues that express similar sets of genes as well as genes displaying similar expression patterns.
Analyzing gene-expression patterns by in situ hybridization to whole-mount embryos provides an extremely rich dataset that can be used to identify genes involved in developmental processes that have been missed by traditional genetic analysis. Systematic analysis of rigorously annotated patterns of gene expression will complement and extend the types of analyses carried out using expression microarrays.
... This species possesses oral and pharyngeal teeth (Parenti 1987; Miyake & Hall 1994) and an extensive repertoire of experimental techniques has been worked out for the analysis of its development (Ishikawa 2000). Particularly promising are mutagenic screens (Ishikawa 2000), which may reveal genes with e¡ects on subsets of the dentition, and large-scale in situ hybridization screens (Henrich & Wittbrot 2000), which may reveal genes expressed during the development of certain types of teeth but not others. Transgenic reporter analysis of cis-regulatory regions of genes involved in tooth development represents an experimental approach that should prove useful in the analysis of modularity. ...
The construction of organisms from units that develop under semi-autonomous genetic control (modules) has been proposed to be an important component of their ability to undergo adaptive phenotypic evolution. The organization of the vertebrate dentition as a system of repeated parts provides an opportunity to study the extent to which phenotypic modules, identified by their evolutionary independence from other such units, are related to modularity in the genetic control of development. The evolutionary history of vertebrates provides numerous examples of both correlated and independent evolution of groups of teeth. The dentition itself appears to be a module of the dermal exoskeleton, from which it has long been under independent genetic control. Region-specific tooth loss has been a common trend in vertebrate evolution. Novel deployment of teeth and reacquisition of lost teeth have also occurred, although less frequently. Tooth shape differences within the dentition may be discontinuous (referred to as heterodonty) or graded. The occurrence of homeotic changes in tooth shape provides evidence for the decoupling of tooth shape and location in the course of evolution. Potential mechanisms for region-specific evolutionary tooth loss are suggested by a number of mouse gene knockouts and human genetic dental anomalies, as well as a comparison between fully-developed and rudimentary teeth in the dentition of rodents. These mechanisms include loss of a tooth-type-specific initiation signal, alterations of the relative strength of inductive and inhibitory signals acting at the time of tooth initiation and the overall reduction in levels of proteins required for the development of all teeth. Ectopic expression of tooth initiation signals provides a potential mechanism for the novel deployment or reacquisition of teeth; a single instance is known of a gene whose ectopic expression in transgenic mice can lead to ectopic teeth. Differences in shape between incisor and molar teeth in the mouse have been proposed to be controlled by the region-specific expression of signalling molecules in the oral epithelium. These molecules induce the expression of transcription factors in the underlying jaw mesenchyme that may act as selectors of tooth type. It is speculated that shifts in the expression domains of the epithelial signalling molecules might be responsible for homeotic changes in tooth shape. The observation that these molecules are regionally restricted in the chicken, whose ancestors were not heterodont, suggests that mammalian heterodonty may have evolved through the use of patterning mechanisms already acting on skeletal elements of the jaws. In general, genetic and morphological approaches identify similar types of modules in the dentition, but the data are not yet sufficient to identify exact correspondences. It is speculated that modularity may be achieved by gene expression differences between teeth or by differences in the time of their development, causing mutations to have cumulative effects on later-developing teeth. The mammalian dentition, for which virtually all of the available developmental genetic data have been collected, represents a small subset of the dental diversity present in vertebrates as a whole. In particular, teleost fishes may have a much more extensive dentition. Extension of research on the genetic control of tooth development to this and other vertebrate groups has great potential to further the understanding of modularity in the dentition.
... It should be noted that this mode of growth is quite exceptional in the vertebrate CNS; to the best of our knowledge, the only other system exhibiting a similar growth mode is the retina of amphibians and ®shes (Straznicky and Gaze, 1971; Johns, 1977; Johns and Easter, 1977), which also grows by addition of new cells at the ciliary marginal zone, a peripheral ring of undifferentiated cells located at its periphery. This peculiar process has indeed been dissected by cloning and ®nely mapping the expression of known neurogenic genes (Harris and Perron, 1998;) disation analysis have proved to be a successful approach to isolate differentially expressed genes (Gawantka et al., 1998; Henrich and Wittbrodt, 2000; Thisse et al., 2000). Indeed this approach allows direct access to the DNA sequence and re¯ects endogenous gene expression. ...
... Forebrain melanogaster (Kopczynski et al., 1998), Xenopus (Gawantka et al., 1998), zebra®sh (Thisse et al., 2000) and medaka (Henrich and Wittbrodt, 2000). These screens were performed in different organisms and at various developmental stages. ...
The optic tectum is a dorsal, prominent and well corticalised structure of the fish brain. It grows according to a pattern exceptional in the vertebrate central nervous system, by addition of radial columns of cells at its periphery. We took advantage of this peculiar feature to readily identify genes differentially expressed in the tectal proliferative (marginal) vs. post-mitotic (central) zones. Out of 500 medaka cDNA clones screened by WMISH, more than 100 were expressed in one or the other of these zones. Unexpectedly, we also identified a small class of genes expressed between these two zones. All the characterised genes of this class encode down regulators of the cell cycle. Therefore, such a screening strategy allows in particular cases to raise testable hypotheses on the involvement of genes in the control of the cell cycle, in addition to characterising unknown genes with patterned expression related to cell proliferation.
... Recent advances in molecular biology are providing a snapshot of the multitude of gene changes, including their temporal kinetics, involved in complex physiological processes, including growth regulation, differentiation, response to chemotherapy, and tumor progression (Schena et al., 1995Schena, 1996;Shalon et al., 1996;Dong et al., 1997;Kang et al., 1998;Fambrough et al., 1999;Hiorns et al., 1999;Huang et al., 1999aHuang et al., , 1999bIyer et al., 1999;Leimeister et al., 1999;Glienke et al., 2000;Henrich & Wittbrodt, 2000;Scherf et al., 2000). These approaches, including subtraction hybridization, differential RNA display, reciprocal subtraction differential RNA display, high-throughput cDNA oligonucleotide microarrays, serial analysis of gene expression, and rapid subtraction hybridization (RaSH), are identifying novel genes that play a role in cancer growth and physiology (Jiang & Fisher, 1993;Reddy et al., 1993;Jiang et al., 1994bJiang et al., , 1995aJiang et al., , 1995bJiang et al., , 1996aJiang et al., , 1996bJiang et al., , 2000Schena et al., 1995Schena et al., , 1996Velculescu et al., 1995;Bonaldo et al., 1996;Schena, 1996;Shalon et al., 1996;Su et al., 1997Su et al., , 1998Su et al., , 1999Zhang et al., 1997;Carulli et al., 1998;Kang et al., 1998;Pardinas et al., 1998;Powell, 1998;Watson et al., 1998;Welford et al., 1998;Huang et al., 1999aHuang et al., , 1999bNilsson et al., 1999). ...
Current cancer therapies are highly toxic and often nonspecific. A potentially less toxic approach to treating this prevalent disease employs agents that modify cancer cell differentiation, termed ‘differentiation therapy.’ This approach is based on the tacit assumption that many neoplastic cell types exhibit reversible defects in differentiation, which upon appropriate treatment, results in tumor reprogramming and a concomitant loss in proliferative capacity and induction of terminal differentiation or apoptosis (programmed cell death). Laboratory studies that focus on elucidating mechanisms of action are demonstrating the effectiveness of ‘differentiation therapy,’ which is now beginning to show translational promise in the clinical setting.
