Cis-regulatory properties of medaka synexpression groups.

During embryogenesis, tissue specification is triggered by the expression of a unique combination of developmental genes and their expression in time and space is crucial for successful development. Synexpression groups are batteries of spatiotemporally co-expressed genes that act in shared biological processes through their coordinated expression. Although several synexpression groups have been described in numerous vertebrate species, the regulatory mechanisms that orchestrate their common complex expression pattern remain to be elucidated. Here we performed a pilot screen on 560 genes of the vertebrate model system medaka (Oryzias latipes) to systematically identify synexpression groups and investigate their regulatory properties by searching for common regulatory cues. We find that synexpression groups share DNA motifs that are arranged in various combinations into cis-regulatory modules that drive co-expression. In contrast to previous assumptions that these genes are located randomly in the genome, we discovered that genes belonging to the same synexpression group frequently occur in synexpression clusters in the genome. This work presents a first repertoire of synexpression group common signatures, a resource that will contribute to deciphering developmental gene regulatory networks.

The normal development of Platynereis dumerilii (Nereididae, Annelida).

BACKGROUND: The polychaete annelid Platynereis dumerilii is an emerging model organism for the study of molecular developmental processes, evolution, neurobiology and marine biology. Annelids belong to the Lophotrochozoa, the so far understudied third major branch of bilaterian animals besides deuterostomes and ecdysozoans. P. dumerilii has proven highly relevant to explore ancient bilaterian conditions via comparison to the deuterostomes, because it has accumulated less evolutionary change than conventional ecdysozoan models. Previous staging was mainly referring to hours post fertilization but did not allow matching stages between studies performed at (even slightly) different temperatures. To overcome this, and to provide a first comprehensive description of P. dumerilii normal development, a temperature-independent staging system is needed. RESULTS: Platynereis dumerilii normal development is subdivided into 16 stages, starting with the zygote and ending with the death of the mature worms after delivering their gametes. The stages described can be easily identified by conventional light microscopy or even by dissecting scope. Developmental landmarks such as the beginning of phototaxis, the visibility of the stomodeal opening and of the chaetae, the first occurrence of the ciliary bands, the formation of the parapodia, the extension of antennae and cirri, the onset of feeding and other characteristics are used to define different developmental stages. The morphology of all larval stages as well as of juveniles and adults is documented by light microscopy. We also provide an overview of important steps in the development of the nervous system and of the musculature, using fluorescent labeling techniques and confocal laser-scanning microscopy. Timing of each developmental stage refers to hours post fertilization at 18 ± 0.1°C. For comparison, we determined the pace of development of larvae raised at 14°C, 16°C, 20°C, 25°C, 28°C and 30°C. A staging ontology representing the comprehensive list of developmental stages of P. dumerilii is available online. CONCLUSIONS: Our atlas of Platynereis dumerilii normal development represents an important resource for the growing Platynereis community and can also be applied to other nereidid annelids.

4DXpress: a database for cross-species expression pattern comparisons.

In the major animal model species like mouse, fish or fly, detailed spatial information on gene expression over time can be acquired through whole mount in situ hybridization experiments. In these species, expression patterns of many genes have been studied and data has been integrated into dedicated model organism databases like ZFIN for zebrafish, MEPD for medaka, BDGP for Drosophila or GXD for mouse. However, a central repository that allows users to query and compare gene expression patterns across different species has not yet been established. Therefore, we have integrated expression patterns for zebrafish, Drosophila, medaka and mouse into a central public repository called 4DXpress (expression database in four dimensions). Users can query anatomy ontology-based expression annotations across species and quickly jump from one gene to the orthologues in other species. Genes are linked to public microarray data in ArrayExpress. We have mapped developmental stages between the species to be able to compare developmental time phases. We store the largest collection of gene expression patterns available to date in an individual resource, reflecting 16 505 annotated genes. 4DXpress will be an invaluable tool for developmental as well as for computational biologists interested in gene regulation and evolution. 4DXpress is available at http://ani.embl.de/4DXpress.

Development of the Minimum Information Specification for In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE).

We describe the creation process of the Minimum Information Specification for In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE). Modeled after the existing minimum information specification for microarray data, we created a new specification for gene expression localization experiments, initially to facilitate data sharing within a consortium. After successful use within the consortium, the specification was circulated to members of the wider biomedical research community for comment and refinement. After a period of acquiring many new suggested requirements, it was necessary to enter a final phase of excluding those requirements that were deemed inappropriate as a minimum requirement for all experiments. The full specification will soon be published as a version 1.0 proposal to the community, upon which a more full discussion must take place so that the final specification may be achieved with the involvement of the whole community.

MEPD: a Medaka gene expression pattern database.

The Medaka Expression Pattern Database (MEPD) stores and integrates information of gene expression during embryonic development of the small freshwater fish Medaka (Oryzias latipes). Expression patterns of genes identified by ESTs are documented by images and by descriptions through parameters such as staining intensity, category and comments and through a comprehensive, hierarchically organized dictionary of anatomical terms. Sequences of the ESTs are available and searchable through BLAST. ESTs in the database are clustered upon entry and have been blasted against public data-bases. The BLAST results are updated regularly, stored within the database and searchable. The MEPD is a project within the Medaka Genome Initiative (MGI) and entries will be interconnected to integrated genomic map databases. MEPD is accessible through the WWW at http://medaka.dsp.jst.go.jp/MEPD.

GSD: a genetic screen database.

The systematic assignment of gene function to a sequenced genome is one of the outstanding challenges in the post-genomic era. Large-scale systematic mutagenesis screens are important tools for reaching this goal. Here we describe GSD, a software package that allows storage and integration of data from genetic screens. GSD was initially developed for a large-scale F3 mutagenesis screen for developmental mutants of medaka (Oryzias latipes). The version presented here supports a wide range of different screens (mutagenesis, RNAi, morpholinos, transgenesis and others) using different organisms. Data are stored in a relational database and can be made accessible through web interfaces. Researchers can enter data describing their screened embryos: They can track statistics, submit images and describe the resulting phenotypes using a phenotype classification ontology. We developed a fish phenotype classification ontology of medaka and zebrafish for this software package and made it available to the public. In addition, a list of genetic lines resulting from each screen can be generated. These lines (mutant alleles, transgenic lines) can be described and categorized in the same ways as the screened individuals. Raw data from the screen can be integrated to describe these lines. A query module that searches this list can be used to publish the screen results on the Internet. A test version is available at and the software can be downloaded from this site.

Large-scale expression screening by automated whole-mount in situ hybridization.

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.

Mutations affecting the formation of posterior lateral line system in Medaka, Oryzias latipes.

We performed a systematic screen for mutations affecting the trajectory of axons visualized by immunohistochemical staining of Medaka embryos with anti-acetylated tubulin antibody. Among the mutations identified, yanagi (yan) and kazura (kaz) mutations caused specific defects in projection of the posterior lateral line (PLL) nerve. In yan and kaz mutant embryos, the PLL nerve main bundle was misrouted ventrally and dorsally or anteriorly. Medaka semaphorin3A, sdf1, and cxcr4 cDNA fragments were cloned to allow analysis of these mutants. There were no changes in semaphorin3A or sdf1 expression in mutant embryos, suggesting that the tissues expressing semaphorin3A or sdf1 that are involved in PLL nerve guidance are present in these mutant embryos. Double staining revealed that the mislocated PLL primordium and growth cone of the ectopically projected PLL nerve were always colocalized in both yan and kaz mutant embryos, suggesting that migration of PLL primordia and PLL nerve growth cones are not uncoupled in these mutants. Although homozygous yan larvae showed incomplete migration of the PLL primordium along the anteroposterior axis, ventral proneuromast migration was complete, suggesting that ventral migration of the proneuromast does not require the signaling affected in yan mutants. In addition to the PLL system, the distribution of primordial germ cells (PGCs) was also affected in both yan and kaz mutant embryos, indicating that yan and kaz genes are required for the migration of both PLL primordia and PGCs. Genetic linkage analysis indicated that kaz is linked to cxcr4, but yan is not linked to sdf1 or cxcr4. These mutations will provide genetic clues to investigate the molecular mechanism underlying formation of the PLL system.

Mutations affecting retinotectal axonal pathfinding in Medaka, Oryzias latipes.

We screened for mutations affecting retinotectal axonal projection in Medaka, Oryzias latipes. In wild-type Medaka embryos, all the axons of retinal ganglion cells (RGCs) project to the contralateral tectum, such that the topological relationship of the retinal field is maintained. We labeled RGC axons using DiI/DiO at the nasodorsal and temporoventral positions of the retina, and screened for mutations affecting the pattern of stereotypic projections to the tectum. By screening 184 mutagenized haploid genomes, seven mutations in five genes causing defects in axonal pathfinding were identified, whereas mutations affecting the topographic projection of RGC axons were not found. The mutants were grouped into two classes according to their phenotypes. In mutants of Class I, a subpopulation of the RGC axons branched out either immediately after leaving the eye or after reaching the midline, and this axonal subpopulation projected to the ipsilateral tectum. In mutants of Class II, subpopulations of RGC axons branched out after crossing the midline and projected aberrantly. These mutants will provide clues to understanding the functions of genes essential for axonal pathfinding, which may be conserved or partly divergent among vertebrates.

Mutations affecting thymus organogenesis in Medaka, Oryzias latipes.

The thymus is an organ for T lymphocyte maturation and is indispensable for the establishment of a highly developed immune system in vertebrates. In order to genetically dissect thymus organogenesis, we carried out a large-scale mutagenesis screening for Medaka mutations affecting recombination activating gene 1 (rag1) expression in the developing thymus. We identified 24 mutations, defining at least 13 genes, which led to a marked reduction of rag1 expression in the thymus. As thymus development depends on pharyngeal arches, we classified those mutations into three classes according to the defects in the pharyngeal arches. Class 1 mutants had no or slight morphological abnormalities in the pharyngeal arches, implying that the mutations may include defects in such thymus-specific events as lymphocyte development and thymic epithelial cell maturation. Class 2 mutants had abnormally shaped pharyngeal arches. Class 3 mutants showed severely attenuated pharyngeal arch development. In Class 2 and Class 3 mutants, the defects in thymus development may be due to abnormal pharyngeal arch development. Those mutations are expected to be useful for identifying the molecular mechanisms underlying thymus organogenesis.

Mutations affecting early distribution of primordial germ cells in Medaka (Oryzias latipes) embryo.

The development of germ cells has been intensively studied in Medaka (Oryzias latipes). We have undertaken a large-scale screen to identify mutations affecting the development of primordial germ cells (PGCs) in Medaka. Embryos derived from mutagenized founder fish were screened for an abnormal distribution or number of PGCs at embryonic stage 27 by RNA in situ hybridization for the Medaka vasa homologue (olvas). At this stage, PGCs coalesce into two bilateral vasa-expressing foci in the ventrolateral regions of the trunk after their migration and group organization. Nineteen mutations were identified from a screen corresponding to 450 mutagenized haploid genomes. Eleven of the mutations caused altered PGC distribution. Most of these alterations were associated with morphological abnormalities and could be grouped into four phenotypic classes: Class 1, PGCs dispersed into bilateral lines; Class 2, PGCs dispersed in a region more medial than that in Class 1; Class 3, PGCs scattered laterally and over the yolk sac area; and Class 4, PGCs clustered in a single median focus. Eight mutations caused a decrease in the number of PGCs. This decrease was observed in the offspring of heterozygous mothers, indicating the contribution of a maternal factor in determining PGC abundance. Taken together, these mutations should prove useful in identifying molecular mechanisms underlying the early PGC development and migration.

Mutations affecting gonadal development in Medaka, Oryzias latipes.

A gonad is formed from germ cells and somatic mesodermal cells through their interactions. Its development is coupled with the determination and differentiation of the sex and sex-associated traits. We carried out a large-scale screening of Medaka mutants in which gonadal development is affected. Screening was performed on larvae at 8 days posthatching for abnormal abundance and/or distribution of germ cells detected by the in situ hybridization for olvas (Medaka vasa). We describe here 16 mutants of 13 genes, which are classified into four groups. Group 1, consisting of four mutants of three genes kon, tot) characterised by an increase in germ cell number. An adult tot homozygote fish has the characteristic feature of possessing hypertrophic gonads filled with immature oocytes. Group 2, represented by a single gene (zen) mutant characterized by a gradual loss of germ cells. Group 3, consisting of four mutants of distinct genes (eko, eki, sht, ano) showing irregular clustering of germ cells. Group 4, consisting of seven mutants of five genes (arr, hyo, mzr, hdr, fbk) showing fragmented clusters of germ cells. In some mutants belonging to Groups 1, 3 and 4, the expression level of ftz-f1 (sf-1/Ad4BP) in gonadal somatic cells significantly decreased, suggesting that interaction between somatic and germ cells is affected.

Mutations affecting somite formation in the Medaka (Oryzias latipes).

The metameric structure of the vertebrate trunk is generated by repeated formation of somites from the unsegmented presomitic mesoderm (PSM). We report the initial characterization of nine different mutants affecting segmentation that were isolated in a large-scale mutagenesis screen in Medaka (Oryzias latipes). Four mutants were identified that show a complete or partial absence of somites or somite boundaries. In addition, five mutations were found that cause fused somites or somites with irregular sizes and shapes. In situ hybridization analysis using specific markers involved in the segmentation clock and antero-posterior (A-P) polarity of somites revealed that the nine mutants can be compiled into two groups. In group 1, mutants exhibit defects in tailbud formation and PSM prepatterning, whereas A-P identity in the somites is defective in group 2 mutants. Three mutants (planlos, pll; schnelles ende, sne; samidare, sam) have characteristic phenotypes that are similar to those in zebrafish mutants affected in the Delta/Notch signaling pathway. The majority of mutants, however, exhibit somitic phenotypes distinct from those found in zebrafish, such as individually fused somites and irregular somite sizes. Thus, these Medaka mutants can be expected to provide clues to uncovering novel components essential for somitogenesis.

Genetic dissection of the formation of the forebrain in Medaka, Oryzias latipes.

The forebrain, consisting of the telencephalon and diencephalon, is essential for processing sensory information. To genetically dissect formation of the forebrain in vertebrates, we carried out a systematic screen for mutations affecting morphogenesis of the forebrain in Medaka. Thirty-three mutations defining 25 genes affecting the morphological development of the forebrain were grouped into two classes. Class 1 mutants commonly showing a decrease in forebrain size, were further divided into subclasses 1A to 1D. Class 1A mutation (1 gene) caused an early defect evidenced by the lack of bf1 expression, Class 1B mutations (6 genes) patterning defects revealed by the aberrant expression of regional marker genes, Class 1C mutation (1 gene) a defect in a later stage, and Class 1D (3 genes) a midline defect analogous to the zebrafish one-eyed pinhead mutation. Class 2 mutations caused morphological abnormalities in the forebrain without considerably affecting its size, Class 2A mutations (6 genes) caused abnormalities in the development of the ventricle, Class 2B mutations (2 genes) severely affected the anterior commissure, and Class 2C (6 genes) mutations resulted in a unique forebrain morphology. Many of these mutants showed the compromised sonic hedgehog expression in the zona-limitans-intrathalamica (zli), arguing for the importance of this structure as a secondary signaling center. These mutants should provide important clues to the elucidation of the molecular mechanisms underlying forebrain development, and shed new light on phylogenically conserved and divergent functions in the developmental process.

Mutations affecting retina development in Medaka.

In a large scale mutagenesis screen of Medaka we identified 60 recessive zygotic mutations that affect retina development. Based on the onset and type of phenotypic abnormalities, the mutants were grouped into five categories: the first includes 11 mutants that are affected in neural plate and optic vesicle formation. The second group comprises 15 mutants that are impaired in optic vesicle growth. The third group includes 18 mutants that are affected in optic cup development. The fourth group contains 13 mutants with defects in retinal differentiation. 12 of these have smaller eyes, whereas one mutation results in enlarged eyes. The fifth group consists of three mutants with defects in retinal pigmentation. The collection of mutants will be used to address the molecular genetic mechanisms underlying vertebrate eye formation.

Mutations affecting liver development and function in Medaka, Oryzias latipes, screened by multiple criteria.

We report here mutations affecting various aspects of liver development and function identified by multiple assays in a systematic mutagenesis screen in Medaka. The 22 identified recessive mutations assigned to 19 complementation groups fell into five phenotypic groups. Group 1, showing defective liver morphogenesis, comprises mutations in four genes, which may be involved in the regulation of growth or patterning of the gut endoderm. Group 2 comprises mutations in three genes that affect the laterality of the liver; in kendama mutants of this group, the laterality of the heart and liver is uncoupled and randomized. Group 3 includes mutations in three genes altering bile color, indicative of defects in hemoglobin-bilirubin metabolism and globin synthesis. Group 4 consists of mutations in three genes, characterized by a decrease in the accumulation of fluorescent metabolite of a phospholipase A(2) substrate, PED6, in the gall bladder. Lipid metabolism or the transport of lipid metabolites may be affected by these mutations. Mutations in Groups 3 and 4 may provide animal models for relevant human diseases. Group 5 mutations in six genes affect the formation of endoderm, endodermal rods and hepatic bud from which the liver develops. These Medaka mutations, identified by morphological and metabolite marker screens, should provide clues to understanding molecular mechanisms underlying formation of a functional liver.

A systematic genome-wide screen for mutations affecting organogenesis in Medaka, Oryzias latipes.

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.

Evolutionary diversification of MCM3 genes in Xenopus laevis and Danio rerio.

Embryonic cell cycles of amphibians are rapid and lack zygotic transcription and checkpoint control. At the mid-blastula transition, zygotic transcription is initiated and cell divisions become asynchronous. Several cell cycle-related amphibian genes retain 2 distinct forms, maternal and zygotic, but little is known about the functional differences between these 2 forms of proteins. The minichromosome maintenance (MCM) 2-7 complex, consisting of 6 MCM proteins, plays a central role in the regulation of eukaryotic DNA replication. Almost all eukaryotes retain just a single MCM gene for each subunit. Here we report that Xenopus and zebrafish have 2 copies of MCM3 genes, one of which shows a maternal and the other a zygotic expression pattern. Phylogenetic analysis shows that the Xenopus and zebrafish zygotic MCM3 genes are more similar to their mammalian MCM3 ortholog, suggesting that maternal MCM3 was lost during evolution in most vertebrate lineages. Maternal MCM3 proteins in these 2 species are functionally different from zygotic MCM3 proteins because zygotic, but not maternal, MCM3 possesses an active nuclear localization signal in its C-terminal region, such as mammalian MCM3 orthologs do. mRNA injection experiments in zebrafish embryos show that overexpression of maternal MCM3 impairs proliferation and causes developmental defects, whereas zygotic MCM3 has a much weaker effect. This difference is brought about by the difference in their C-terminal regions, which contain putative nuclear localization signals; swapping the C-terminal region between maternal and zygotic genes diminishes the developmental defects. This study suggests that evolutionary diversification has occurred in MCM3 genes, leading to distinct functions, possibly as an adaption to the rapid DNA replication required for early development of Xenopus and zebrafish.

Minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE).

One purpose of the biomedical literature is to report results in sufficient detail that the methods of data collection and analysis can be independently replicated and verified. Here we present reporting guidelines for gene expression localization experiments: the minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE). MISFISHIE is modeled after the Minimum Information About a Microarray Experiment (MIAME) specification for microarray experiments. Both guidelines define what information should be reported without dictating a format for encoding that information. MISFISHIE describes six types of information to be provided for each experiment: experimental design, biomaterials and treatments, reporters, staining, imaging data and image characterizations. This specification has benefited the consortium within which it was developed and is expected to benefit the wider research community. We welcome feedback from the scientific community to help improve our proposal.

New genes in the evolution of the neural crest differentiation program.

BACKGROUND: Development of the vertebrate head depends on the multipotency and migratory behavior of neural crest derivatives. This cell population is considered a vertebrate innovation and, accordingly, chordate ancestors lacked neural crest counterparts. The identification of neural crest specification genes expressed in the neural plate of basal chordates, in addition to the discovery of pigmented migratory cells in ascidians, has challenged this hypothesis. These new findings revive the debate on what is new and what is ancient in the genetic program that controls neural crest formation. RESULTS: To determine the origin of neural crest genes, we analyzed Phenotype Ontology annotations to select genes that control the development of this tissue. Using a sequential blast pipeline, we phylogenetically classified these genes, as well as those associated with other tissues, in order to define tissue-specific profiles of gene emergence. Of neural crest genes, 9% are vertebrate innovations. Our comparative analyses show that, among different tissues, the neural crest exhibits a particularly high rate of gene emergence during vertebrate evolution. A remarkable proportion of the new neural crest genes encode soluble ligands that control neural crest precursor specification into each cell lineage, including pigmented, neural, glial, and skeletal derivatives. CONCLUSION: We propose that the evolution of the neural crest is linked not only to the recruitment of ancestral regulatory genes but also to the emergence of signaling peptides that control the increasingly complex lineage diversification of this plastic cell population.