Ruprecht-Karls-Universität Heidelberg
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Tierphysiologie / Entwicklungsbiologie

Prof. Dr. Joachim Wittbrodt

Recent Key Publications

Inoue D*, Stemmer MThumberger T, Ruppert T, Bärenz F, Wittbrodt J*, Gruss OJ*. (2017). Expression of the novel maternal centrosome assembly factor Wdr8 is required for vertebrate embryonic mitoses. Nat Commun. 8:14090. DOI: 10.1038/ncomms14090

*:Co-correspondence

Europe PMC 

Abstract
The assembly of the first centrosome occurs upon fertilisation when male centrioles recruit pericentriolar material (PCM) from the egg cytoplasm. The mechanisms underlying the proper assembly of centrosomes during early embryogenesis remain obscure. We identify Wdr8 as a novel maternally essential protein that is required for centrosome assembly during embryonic mitoses of medaka (Oryzias latipes). By CRISPR-Cas9-mediated knockout, maternal/zygotic Wdr8-null (m/zWdr8(-/-)) blastomeres exhibit severe defects in centrosome structure that lead to asymmetric division, multipolar mitotic spindles and chromosome alignment errors. Via its WD40 domains, Wdr8 interacts with the centriolar satellite protein SSX2IP. Combining targeted gene knockout and in vivo reconstitution of the maternally essential Wdr8-SSX2IP complex reveals an essential link between maternal centrosome proteins and the stability of the zygotic genome for accurate vertebrate embryogenesis. Our approach provides a way of distinguishing maternal from paternal effects in early embryos and should contribute to understanding molecular defects in human infertility.

 

Aghaallaei N, Gruhl F, Schaefer CQ, Wernet T, Weinhardt V, Centanin L, Loosli F, Baumbach T, Wittbrodt J. (2016). Identification, visualization and clonal analysis of intestinal stem cells in fish. Development. 143(19):3470-3480.

Article has an altmetric score of 5

 

Lust K, Sinn R, Pérez Saturnino A, Centanin L, Wittbrodt J. (2016). De novo neurogenesis by targeted expression of atoh7 to Müller glia cells. Development. 143(11):1874-83. DOI:10.1242/dev.135905

Europe PMC 

 

 

Reinhardt R, Centanin L, Tavhelidse T, Inoue D, Wittbrodt B, Concordet JP, Martinez-Morales JR, Wittbrodt J. (2015). Sox2, Tlx, Gli3, and Her9 converge on Rx2 to define retinal stem cells in vivo. EMBO J. 2015 Apr 23. pii: e201490706.

Europe PMC 

 

Heermann S, Schütz L, Lemke S, Krieglstein K, Wittbrodt J. (2015). Eye morphogenesis driven by epithelial flow into the optic cup facilitated by modulation of bone morphogenetic protein. eLIFE. 4:e05216

Europe PMC 

 

Stemmer M, Schuhmacher LN, Foulkes NS, Bertolucci C, Wittbrodt J. (2015). Cavefish eye loss in response to an early block in retinal differentiation progression. Development. 142:743-752. 

Europe PMC 

 

 

Publications last ten years

 


2017

Stemmer M, Thumberger T, Del Sol Keyer M, Wittbrodt J, Mateo JL. (2017). Correction: CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool. PLoS One. 12(4):e0176619.

Abstract
[This corrects the article DOI: 10.1371/journal.pone.0124633.].
 
Pereiro L, Loosli F, Fernández J, Härtel S, Wittbrodt J, Concha ML. (2017). Gastrulation in an annual killifish: Molecular and cellular events during germ layer formation in Austrolebias. 2. Dev Dyn. 2017 Mar 1.
 
 
Shi D*, Tavhelidse T*, Thumberger T, Wittbrodt J, Greb T. (2017). Bifacial stem cell niches in fish and plants. Curr Opin Genet Dev. 45:28-33.
 
 

Inoue D*, Stemmer MThumberger T, Ruppert T, Bärenz F, Wittbrodt J*, Gruss OJ*. (2017). Expression of the novel maternal centrosome assembly factor Wdr8 is required for vertebrate embryonic mitoses. Nat Commun. 8:14090. DOI: 10.1038/ncomms14090

*:Co-correspondence

Europe PMC 

 
 
Kirchmaier S, Lust K, Wittbrodt J. (5. Methods Mol Biol. 2017;1472:157-68. doi: 10.1007/978-1-4939-6343-0_12. ). Generation of DNA Constructs Using the Golden GATEway Cloning Method. 5. Methods Mol Biol. 2017;1472:157-68.
 

 

Article has an altmetric score of 17

Europe PMC 


2016

Gutierrez-Triana JA, Mateo JL, Ibberson D, Ryu S, Wittbrodt J. (2016). iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins. Development. 143(22):4272-4278. DOI:10.1242/dev.139261

Article has an altmetric score of 17
Europe PMC 

 

Nicolás-Pérez M, Kuchling F, Letelier J, Polvillo R, Wittbrodt J, Martínez-Morales JR. (2016). Analysis of cellular behavior and cytoskeletal dynamics reveal a constriction mechanism driving optic cup morphogenesis. Elife. 5. pii: e15797.
 
Aghaallaei N, Gruhl F, Schaefer CQ, Wernet T, Weinhardt V, Centanin L, Loosli F, Baumbach T, Wittbrodt J. (2016). Identification, visualization and clonal analysis of intestinal stem cells in fish. Development. 143(19):3470-3480.
 
Kromm D, Thumberger T, Wittbrodt J. (2016). An eye on light-sheet microscopy. 4. Methods Cell Biol. 2016;133:105-23.
 
Abstract
This chapter introduces the principles and advantages of selective plane illumination microscopy (SPIM) and compares it to commonly used epifluorescence or confocal setups. Due to the low phototoxicity, speed of imaging, high penetration depth, and spatiotemporal resolution, SPIM is predestined for in vivo imaging but can as well be used for in toto analysis of large fixed samples. Key points of light-sheet microscopy are highlighted and discussed priming the investigator to choose the best suitable system from the large collection of possible SPIM setups. Mounting of samples is shown and the demands for data acquisition, processing, handling, and visualization are discussed.
Pubmed 
Lust K, Sinn R, Pérez Saturnino A, Centanin L, Wittbrodt J. (2016). De novo neurogenesis by targeted expression of atoh7 to Müller glia cells. Development. 143(11):1874-83.
Abstract
Regenerative responses in the vertebrate CNS depend on quiescent radial glia stem cells, which re-enter the cell cycle and eventually differentiate into neurons. The entry into the cell cycle and the differentiation into neurons are events of opposite nature, and therefore efforts to force quiescent radial glia into neurons require different factors. Here, we use fish to show that a single neurogenic factor, Atoh7, directs retinal radial glia (Müller glia, MG) into proliferation. The resulting neurogenic clusters differentiate in vivo into various retinal neurons. We use signaling reporters to demonstrate that the Atoh7-induced regeneration-like response of MG cells is mimicked by Notch, resembling the behavior of early progenitors during retinogenesis. Activation of Notch signaling in MG cells is sufficient to trigger proliferation and differentiation. Our results uncover a new role for Atoh7 as a universal neurogenic factor, and illustrate how signaling modules are re-employed in diverse contexts to trigger different biological responses.
Pubmed 
Friedrich J, Sorge S, Bujupi F, Eichenlaub MP, Schulz NG, Wittbrodt J, Lohmann I. (2016). Hox Function Is Required for the Development and Maintenance of the Drosophila Feeding Motor Unit. Cell Rep. 14(4):850-60.
Abstract
Feeding is an evolutionarily conserved and integral behavior that depends on the rhythmic activity of feeding muscles stimulated by specific motoneurons. However, critical molecular determinants underlying the development of the neuromuscular feeding unit are largely unknown. Here, we identify the Hox transcription factor Deformed (Dfd) as essential for feeding unit formation, from initial specification to the establishment of active synapses, by controlling stage-specific sets of target genes. Importantly, we found Dfd to control the expression of functional components of synapses, such as Ankyrin2-XL, a protein known to be critical for synaptic stability and connectivity. Furthermore, we uncovered Dfd as a potential regulator of synaptic specificity, as it represses expression of the synaptic cell adhesion molecule Connectin (Con). These results demonstrate that Dfd is critical for the establishment and maintenance of the neuromuscular unit required for feeding behavior, which might be shared by other group 4 Hox genes.

Pubmed 

 

Alonso-Barba JI, Rahman RUWittbrodt J, Mateo JL. (2016). MEPD: medaka expression pattern database, genes and more. Nucleic Acids Res2016 Jan 4;44(D1):D819-21. doi: 10.1093/nar/gkv1029.

Article has an altmetric score of 3 

Abstract
The Medaka Expression Pattern Database (MEPD; http://mepd.cos.uni-heidelberg.de/) is designed as a repository of medaka expression data for the scientific community. In this update we present two main improvements. First, we have changed the previous clone-centric view for in situ data to a gene-centric view. This is possible because now we have linked all the data present in MEPD to the medaka gene annotation in ENSEMBL. In addition, we have also connected the medaka genes in MEPD to their corresponding orthologous gene in zebrafish, again using the ENSEMBL database. Based on this, we provide a link to the Zebrafish Model Organism Database (ZFIN) to allow researches to compare expression data between these two fish model organisms. As a second major improvement, we have modified the design of the database to enable it to host regulatory elements, promoters or enhancers, expression patterns in addition to gene expression. The combination of gene expression, by traditional in situ, and regulatory element expression, typically by fluorescence reporter gene, within the same platform assures consistency in terms of annotation. In our opinion, this will allow researchers to uncover new insights between the expression domain of genes and their regulatory landscape.
Pubmed 

 


2015

Lust K, Wittbrodt J. (2015) Hold your breath. Elife. 2015 Dec 16;4:e12523. doi: 10.7554/eLife.12523. 

Abstract
Reactive oxygen species produced in response to changes in the level of oxygen in water can promote the regeneration of brain tissue in newts.
Pubmed 

 

Zhang P, Kratz AS, Salama M, Elabd S, Heinrich T, Wittbrodt J, Blattner C, Davidson G. (2015). Expression screening using a Medaka cDNA library identifies evolutionarily conserved regulators of the p53/Mdm2 pathway. BMC Biotechnol. 15(1):92.

Abstract
BACKGROUND: The p53 tumor suppressor protein is mainly regulated by alterations in the half-life of the protein, resulting in significant differences in p53 protein levels in cells. The major regulator of this process is Mdm2, which ubiquitinates p53 and targets it for proteasomal degradation. This process can be enhanced or reduced by proteins that associate with p53 or Mdm2 and several proteins have been identified with such an activity. Furthermore, additional ubiquitin ligases for p53 have been identified in recent years. Nevertheless, our understanding of how p53 abundance and Mdm2 activity are regulated remains incomplete. Here we describe a cell culture based overexpression screen to identify evolutionarily conserved regulators of the p53/Mdm2 circuit. The results from this large-scale screening method will contribute to a better understanding of the regulation of these important proteins. METHODS: Expression screening was based on co-transfection of H1299 cells with pools of cDNA's from a Medaka library together with p53, Mdm2 and, as internal control, Ror2. After cell lysis, SDS-PAGE/WB analysis was used to detect alterations in these proteins. RESULTS: More than one hundred hits that altered the abundance of either p53, Mdm2, or both were identified in the primary screen. Subscreening of the library pools that were identified in the primary screen identified several potential novel regulators of p53 and/or Mdm2. We also tested whether the human orthologues of the Medaka genes regulate p53 and/or Mdm2 abundance. All human orthologues regulated p53 and/or Mdm2 abundance in the same manner as the proteins from Medaka, which underscores the suitability of this screening methodology for the identification of new modifiers of p53 and Mdm2. CONCLUSIONS: Despite enormous efforts in the last two decades, many unknown regulators for p53 and Mdm2 abundance are predicted to exist. This cross-species approach to identify evolutionarily conserved regulators demonstrates that our Medaka unigene cDNA library represents a powerful tool to screen for these novel regulators of the p53/Mdm2 pathway.

 

Beccari L, Marco-Ferreres R, Tabanera N, Manfredi A, Souren M, Wittbrodt B, Conte I, Wittbrodt J, Bovolenta P. (2015). A trans-regulatory code for the forebrain expression of Six3.2 in the medaka fish. J Biol Chem. 2015 Sep 16. pii: jbc.M115.681254. [

 
Pubmed 

Bajoghli B, Kuri P, Inoue DAghaallaei N, Hanelt M, Thumberger T, Rauzi M, Wittbrodt J, Leptin M. (2015). Noninvasive In Toto Imaging of the Thymus Reveals Heterogeneous Migratory Behavior of Developing T Cells. J Immunol. 195(5):2177-86.

 
Pubmed 
 

Auer TO, Xiao T, Bercier V, Gebhardt V, Duroure K, Concordet J-P, Wyart C, Suster M, Kawakami K, Wittbrodt J, Baier H, Del Bene F. (2015). Deletion of a kinesin I motor unmasks a mechanism of homeostatic branching control by neurotrophin-3. eLIFE. 4:e05061.

 

Stemmer M, Thumberger T, Del Sol Keyer M, Wittbrodt JMateo JL. (2015). CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool. PLoS One. 10(4):e0124633.

 

Reinhardt R, Centanin L, Tavhelidse T, Inoue D, Wittbrodt B, Concordet JP, Martinez-Morales JR, Wittbrodt J. (2015). Sox2, Tlx, Gli3, and Her9 converge on Rx2 to define retinal stem cells in vivo. EMBO J. 2015 Apr 23. pii: e201490706.

Europe PMC 

 

Kirchmaier S, Naruse K, Wittbrodt J, Loosli F. (2015). The Genomic and Genetic Toolbox of the Teleost Medaka (Oryzias latipes). Genetics. 199(4):905-918.
 
Pubmed 
 

Zhang P, Elabd S, Hammer S, Solozobova V, Yan H, Bartel F, Inoue S, Henrich TWittbrodt J, Loosli F, Davidson G, Blattner C. (2015). TRIM25 has a dual function in the p53/Mdm2 circuit. 1. Oncogene. 2015 Mar 2.

 
 

Heermann S, Schütz L, Lemke S, Krieglstein K, Wittbrodt J. (2015). Eye morphogenesis driven by epithelial flow into the optic cup facilitated by modulation of bone morphogenetic protein. eLIFE. 4:e05216

 

Europe PMC 

 

Stemmer M, Schuhmacher LN, Foulkes NS, Bertolucci C, Wittbrodt J. (2015). Cavefish eye loss in response to an early block in retinal differentiation progression. Development142:743-752. 

 

 

 

 

 


 

2014

 

Chen Q, Su Y, Wesslowski J, Hagemann AI, Ramialison M, Wittbrodt J, Scholpp S, Davidson G. (2014). Tyrosine phosphorylation of LRP6 by Src and Fer inhibits Wnt/β-catenin signalling. 1. EMBO Rep. 2014 Nov 12. pii: e201439644.

Abstract
Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) function as transmembrane receptors to transduce Wnt signals. A key mechanism for signalling is Wnt-induced serine/threonine phosphorylation at conserved PPPSPxS motifs in the LRP6 cytoplasmic domain, which promotes pathway activation. Conserved tyrosine residues are positioned close to all PPPSPxS motifs, which suggests they have a functional significance. Using a cell culture-based cDNA expression screen, we identified the non-receptor tyrosine kinases Src and Fer as novel LRP6 modifiers. Both Src and Fer associate with LRP6 and phosphorylate LRP6 directly. In contrast to the known PPPSPxS Ser/Thr kinases, tyrosine phosphorylation by Src and Fer negatively regulates LRP6-Wnt signalling. Epistatically, they function upstream of β-catenin to inhibit signalling and in agreement with a negative role in regulating LRP6, MEF cells lacking these kinases show enhanced Wnt signalling. Wnt3a treatment of cells enhances tyrosine phosphorylation of endogenous LRP6 and, mechanistically, Src reduces cell surface LRP6 levels and disrupts LRP6 signalosome formation. Interestingly, CK1γ inhibits Fer-induced LRP6 phosphorylation, suggesting a mechanism whereby CK1γ acts to de-represses inhibitory LRP6 tyrosine phosphorylation. We propose that LRP6 tyrosine phosphorylation by Src and Fer serves a negative regulatory function to prevent over-activation of Wnt signalling at the level of the Wnt receptor, LRP6.
Pubmed 
Mateo JL, van den Berg DL, Haeussler M, Drechsel D, Gaber ZB, Castro DS, Robson P, Crawford GE, Flicek P, Ettwiller L, Wittbrodt J, Guillemot F, Martynoga B. (2014). Characterization of the neural stem cell gene regulatory network identifies OLIG2 as a multi-functional regulator of self-renewal. 2. Genome Res. 2014 Oct 7. pii: gr.173435.114.

 

Abstract
The gene regulatory network (GRN) that supports neural stem cell (NS cell) self-renewal has so far been poorly characterised. Knowledge of the central transcription factors (TFs), the non-coding gene regulatory regions that they bind to and the genes whose expression they modulate will be crucial in unlocking the full therapeutic potential of these cells. Here, we use DNase-seq in combination with analysis of histone modifications to identify multiple classes of epigenetically and functionally distinct cis-regulatory elements (CREs). Through motif analysis and ChIP-seq we identify several of the crucial TF regulators of NS cells. At the core of the network are TFs of the basic helix-loop-helix (bHLH), nuclear factor I (NFI), SOX and FOX families, with CREs often densely bound by several of these different TFs. We use machine learning to highlight several crucial regulatory features of the network that underpin NS cell self-renewal and multipotency. We validate our predictions by functional analysis of the bHLH TF OLIG2. This TF makes an important contribution to NS cell self-renewal by concurrently activating pro-proliferation genes and preventing the untimely activation of genes promoting neuronal differentiation and stem cell quiescence.
Pubmed 

Crespo CL, Vernieri C, Keller PJ, Garrè M, Bender JR, Wittbrodt J, Pardi R. (2014). The PAR complex controls the spatiotemporal dynamics of F-actin and the MTOC in directionally migrating leukocytes.  J Cell Sci. 127(20): 4381-4395.

Abstract

Inflammatory cells acquire a polarized phenotype to migrate toward sites of infection or injury. A conserved polarity complex comprising PAR-3, PAR-6, and atypical protein kinase C (aPKC) relays extracellular polarizing cues to control cytoskeletal and signaling networks affecting morphological and functional polarization. Yet, there is no evidence that myeloid cells use PAR signaling to migrate vectorially in 3D environments in vivo. Using genetically-encoded bioprobes and high-resolution live imaging we revealed the existence of F-actin oscillations in the trailing edge and constant MTOC repositioning to direct leukocyte migration in wounded medaka fish larvae. Genetic manipulation in live myeloid cells demonstrated that the catalytic activity of aPKC and the regulated interaction with PAR-3/PAR-6 are required for consistent F- actin oscillations, MTOC perinuclear mobility, aPKC repositioning and wound-directed migration upstream of Rho-kinase/ROCK/ROK activation. We propose that the PAR complex coordinately controls cytoskeletal changes affecting both traction force generation and directionality of leukocyte migration to sites of injury

Sinn R, Peravali R, Heermann S., Wittbrodt J. (2014). Differential responsiveness of distinct retinal domains to Atoh7. 2. Mech Dev. 2014 Aug 21. pii: S0925-4773(14)00038-0.

Abstract

During vertebrate eye development retinal progenitor cells (RPCs) differentiate into all neural cell types of the retina. Retinal ganglion cells (RGCs) represent the first cell type to be generated. For their development, Atoh7, a basic Helix Loop Helix (bHLH) transcription factor is crucial. Atoh7 loss of function results in a massive reduction or even a total loss of RGCs. However, inconsistent results have been obtained in atoh7 gain of function experiments with respect to ganglion cell genesis, implying that the effect of Atoh7 is likely to be dependent on the competence state of the RPC. In this study we addressed the differential susceptibilities of early RPCs to Atoh7 in vivo, using medaka. Unexpectedly, we observed a largely normal development of the dorsal retina, although atoh7 was precociously expressed. However, the development of the retina close to the optic nerve head (part of the ventral retina) was disturbed severely. Photoreceptors were largely absent and the Müller glia cell number was reduced significantly. The majority of cells in this domain were ganglion cells and the abnormal development of this area affected the closure of the optic fissure resulting in coloboma.

Pubmed 

Centanin L, Ander JJ, Hoeckendorf B, Lust K, Kellner T, Kraemer I, Urbany C, Hasel E, Harris WA, Simons BD, Wittbrodt J. (2014). Exclusive multipotency and preferential asymmetric divisions in post-embryonic neural stem cells of the fish retina. Development141(18):3472-82.

Abstract

The potency of post-embryonic stem cells can only be addressed in the living organism, by labeling single cells after embryonic development and following their descendants. Recently, transplantation experiments involving permanently labeled cells revealed multipotent neural stem cells (NSCs) of embryonic origin in the medaka retina. To analyze whether NSC potency is affected by developmental progression, as reported for the mammalian brain, we developed an inducible toolkit for clonal labeling and non-invasive fate tracking. We used this toolkit to address post-embryonic stem cells in different tissues and to functionally differentiate transient progenitor cells from permanent, bona fide stem cells in the retina. Using temporally controlled clonal induction, we showed that post-embryonic retinal NSCs are exclusively multipotent and give rise to the complete spectrum of cell types in the neural retina. Intriguingly, and in contrast to any other vertebrate stem cell system described so far, long-term analysis of clones indicates a preferential mode of asymmetric cell division. Moreover, following the behavior of clones before and after external stimuli, such as injuries, shows that NSCs in the retina maintained the preference for asymmetric cell division during regenerative responses. We present a comprehensive analysis of individual post-embryonic NSCs in their physiological environment and establish the teleost retina as an ideal model for studying adult stem cell biology at single cell resolution.

Pubmed 

 

Mazaheri F, Breus O, Durdu S, Haas PWittbrodt J, Gilmour D, Peri F. (2014). Distinct roles for BAI1 and TIM-4 in the engulfment of dying neurons by microglia. Nat Commun. 5:4046.

Abstract

The removal of dying neurons by microglia has a key role during both development and in several diseases. To date, little is known about the cellular and molecular processes underlying neuronal engulfment in the brain. Here we took a live imaging approach to quantify neuronal cell death progression in embryonic zebrafish brains and studied the response of microglia. We show that microglia engulf dying neurons by extending cellular branches that form phagosomes at their tips. At the molecular level we found that microglia lacking the phosphatidylserine receptors BAI1 and TIM-4, are able to recognize the apoptotic targets but display distinct clearance defects. Indeed, BAI1 controls the formation of phagosomes around dying neurons and cargo transport, whereas TIM-4 is required for phagosome stabilization. Using this single-cell resolution approach we established that it is the combined activity of BAI1 and TIM-4 that allows microglia to remove dying neurons.

Tena JJ, González-Aguilera C, Fernández-Miñán A, Vázquez-Marín J, Parra-Acero H, Cross JW, Rigby PW, Carvajal JJ, Wittbrodt J, Gómez-Skarmeta JL, Martínez-Morales JR. (2014). Comparative epigenomics in distantly related teleost species identifies conserved cis-regulatory nodes active during the vertebrate phylotypic period. Genome Res. 24(7):1075-85.

 

Abstract
The complex relationship between ontogeny and phylogeny has been the subject of attention and controversy since von Baer's formulations in the 19th century. The classic concept that embryogenesis progresses from clade general features to species-specific characters has often been revisited. It has become accepted that embryos from a clade show maximum morphological similarity at the so-called phylotypic period (i.e., during mid-embryogenesis). According to the hourglass model, body plan conservation would depend on constrained molecular mechanisms operating at this period. More recently, comparative transcriptomic analyses have provided conclusive evidence that such molecular constraints exist. Examining cis-regulatory architecture during the phylotypic period is essential to understand the evolutionary source of body plan stability. Here we compare transcriptomes and key epigenetic marks (H3K4me3 and H3K27ac) from medaka (Oryzias latipes) and zebrafish (Danio rerio), two distantly related teleosts separated by an evolutionary distance of 115-200 Myr. We show that comparison of transcriptome profiles correlates with anatomical similarities and heterochronies observed at the phylotypic stage. Through comparative epigenomics, we uncover a pool of conserved regulatory regions (≈700), which are active during the vertebrate phylotypic period in both species. Moreover, we show that their neighboring genes encode mainly transcription factors with fundamental roles in tissue specification. We postulate that these regulatory regions, active in both teleost genomes, represent key constrained nodes of the gene networks that sustain the vertebrate body plan.

Spivakov M, Auer TO, Peravali R, Dunham I, Dolle D, Fujiyama A, Toyoda A, Aizu T, Minakuchi Y, Loosli F, Naruse K, Birney E, Wittbrodt J. (2014). Genomic and Phenotypic Characterization of a Wild Medaka Population: Towards the Establishment of an Isogenic Population Genetic Resource in Fish. 1. G3 (Bethesda). 2014 Jan 9. pii: g3.113.008722v1.

Abstract
Oryzias latipes (medaka) has been established as a vertebrate genetic model for over a century, and has recently been rediscovered outside its native Japan. The power of new sequencing methods now makes it possible to reinvigorate medaka genetics, in particular by establishing a near-isogenic panel derived from a single wild population. Here we characterize the genomes of wild medaka catches obtained from a single Southern Japanese population in Kiyosu as a precursor for the establishment of a near isogenic panel of wild lines. The population is free of significant detrimental population structure, and has advantageous linkage disequilibrium properties suitable for establishment of the proposed panel. Analysis of morphometric traits in five representative inbred strains suggests phenotypic mapping will be feasible in the panel. In addition high throughput genome sequencing of these medaka strains confirms their evolutionary relationships on lines of geographic separation and provides further evidence that there has been little significant interbreeding between the Southern and Northern medaka population since the Southern/Northern population split. The sequence data suggest that the Southern Japanese medaka existed as a larger older population which went through a relatively recent bottleneck around 10,000 years ago. In addition we detect patterns of recent positive selection in the Southern population. These data indicate that the genetic structure of the Kiyosu medaka samples is suitable for the establishment of a vertebrate near isogenic panel and therefore inbreeding of 200 lines based on this population has commenced. Progress of this project can be tracked at http://www.ebi.ac.uk/birney-srv/medaka-ref-panel.

 

Centanin L, Wittbrodt J. (2014). Retinal neurogenesis. Development. 141(2):241-4.

Abstract
A unique feature of humans is the complexity of our central nervous system. A fully functional brain requires that billions of neurons make specific contacts in a highly coordinated way, an issue that is still not well understood. The neural retina constitutes an excellent system with which to analyze key aspects of neurogenesis and circuit formation in the central nervous system. Here, we provide an overview of retinal neurogenesis in vertebrates and discuss implications of the developmental mechanisms involved for regenerative therapy approaches.

 


2013

 

Müller C, Maeso I, Wittbrodt J, Martínez-Morales JR. (2013). The medaka mutation tintachina sheds light on the evolution of V-ATPase B subunits in vertebrates. Sci Rep. 3:3217.

 

Abstract

Vacuolar-type H(+) ATPases (V-ATPases) are multimeric protein complexes that play a universal role in the acidification of intracellular compartments in eukaryotic cells. We have isolated the recessive medaka mutation tintachina (tch), which carries an inactivating modification of the conserved glycine residue (G75R) of the proton pump subunit atp6v1Ba/vatB1. Mutant embryos show penetrant pigmentation defects, massive brain apoptosis and lethality before hatching. Strikingly, an equivalent mutation in atp6v1B1 (G78R) has been reported in a family of patients suffering from distal renal tubular acidosis (dRTA), a hereditary disease that causes metabolic acidosis due to impaired kidney function. This poses the question as to how molecularly identical mutations result in markedly different phenotypes in two vertebrate species. Our work offers an explanation for this phenomenon. We propose that, after successive rounds of whole-genome duplication, the emergence of paralogous copies allowed the divergence of the atp6v1B cis-regulatory control in different vertebrate groups.

Kirchmaier S, Lust K, Wittbrodt J. (2013). Golden GATEway Cloning - A Combinatorial Approach to Generate Fusion and Recombination Constructs. PLoS One. 8(10):e76117.

 

Abstract

The design and generation of DNA constructs is among the necessary but generally tedious tasks for molecular biologists and, typically, the cloning strategy is restricted by available restriction sites. However, increasingly sophisticated experiments require increasingly complex DNA constructs, with an intricacy that exceeds what is achievable using standard cloning procedures. Many transgenes such as inducible gene cassettes or recombination elements consist of multiple components that often require precise in-frame fusions. Here, we present an efficient protocol that facilitates the generation of these complex constructs. The golden GATEway cloning approach presented here combines two established cloning methods, namely golden Gate cloning and Multisite Gateway(TM) cloning. This allows efficient and seamless assembly as well as reuse of predefined DNA elements. The golden Gate cloning procedure follows clear and simple design rules and allows the assembly of multiple fragments with different sizes into one open reading frame. The final product can be directly integrated into the widely used Multisite Gateway(TM) cloning system, granting more flexibility when using a transgene in the context of multiple species. This adaptable and streamlined cloning procedure overcomes restrictions of "classical construct generation" and allows focusing on construct design.

Kirchmaier S, Höckendorf B, Möller EK, Bornhorst D, Spitz F, Wittbrodt J. (2013). Efficient site-specific transgenesis and enhancer activity tests in medaka using PhiC31 integrase. Development. 140(20):4287-95.

 

Abstract

Established transgenesis methods for fish model systems allow efficient genomic integration of transgenes. However, thus far a way of controlling copy number and integration sites has not been available, leading to variable transgene expression caused by position effects. The integration of transgenes at predefined genomic positions enables the direct comparison of different transgenes, thereby improving time and cost efficiency. Here, we report an efficient PhiC31-based site-specific transgenesis system for medaka. This system includes features that allow the pre-selection of successfully targeted integrations early on in the injected generation. Pre-selected embryos transmit the correctly integrated transgene through the germline with high efficiency. The landing site design enables a variety of applications, such as reporter and enhancer switch, in addition to the integration of any insert. Importantly, this allows assaying of enhancer activity in a site-specific manner without requiring germline transmission, thus speeding up large-scale analyses of regulatory elements.

Martynoga B, Mateo JL, Zhou B, Andersen J, Achimastou A, Urbán N, van den Berg D, Georgopoulou D, Hadjur S, Wittbrodt J, Ettwiller L, Piper M, Gronostajski RM, Guillemot F. (2013). Epigenomic enhancer annotation reveals a key role for NFIX in neural stem cell quiescence. Genes Dev. 27(16):1769-86.

Abstract

The majority of neural stem cells (NSCs) in the adult brain are quiescent, and this fraction increases with aging. Although signaling pathways that promote NSC quiescence have been identified, the transcriptional mechanisms involved are mostly unknown, largely due to lack of a cell culture model. In this study, we first demonstrate that NSC cultures (NS cells) exposed to BMP4 acquire cellular and transcriptional characteristics of quiescent cells. We then use epigenomic profiling to identify enhancers associated with the quiescent NS cell state. Motif enrichment analysis of these enhancers predicts a major role for the nuclear factor one (NFI) family in the gene regulatory network controlling NS cell quiescence. Interestingly, we found that the family member NFIX is robustly induced when NS cells enter quiescence. Using genome-wide location analysis and overexpression and silencing experiments, we demonstrate that NFIX has a major role in the induction of quiescence in cultured NSCs. Transcript profiling of NS cells overexpressing or silenced for Nfix and the phenotypic analysis of the hippocampus of Nfix mutant mice suggest that NFIX controls the quiescent state by regulating the interactions of NSCs with their microenvironment.

Schaafhausen MK, Yang WJ, Centanin L, Wittbrodt J, Bosserhoff A, Fischer A, Schartl M, Meierjohann S. (2013). Tumor angiogenesis is caused by single melanoma cells in a manner dependent on reactive oxygen species and NF-κB. J Cell Sci. 126(Pt 17):3862-72.

Abstract

Melanomas have a high angiogenic potential, but respond poorly to medical treatment and metastasize very early. To understand the early events in tumor angiogenesis, animal models with high tumor resolution and blood vessel resolution are required, which provide the opportunity to test the ability of small molecule inhibitors to modulate the angiogenic tumor program. We have established a transgenic melanoma angiogenesis model in the small laboratory fish species Japanese medaka. Here, pigment cells are transformed by an oncogenic receptor tyrosine kinase in fish expressing GFP throughout their vasculature. We show that angiogenesis occurs in a reactive oxygen species (ROS)- and NF-κB-dependent, but hypoxia-independent manner. Intriguingly, we observed that blood vessel sprouting is induced even by single transformed pigment cells. The oncogenic receptor as well as human melanoma cells harboring other oncogenes caused the production of pro-angiogenic factors, most prominently angiogenin, through NF-κB signaling. Inhibiting NF-κB prevented tumor angiogenesis and led to the regression of existing tumor blood vessels. In conclusion, our high-resolution medaka melanoma model discloses that ROS and NF-κB signaling from single tumor cells causes hypoxia-independent angiogenesis, thus, demonstrating that the intrinsic malignant tumor cell features are sufficient to initiate and maintain a pro-angiogenic signaling threshold.

Bärenz F, Inoue D, Yokoyama H, Tegha-Dunghu J, Freiss S, Draeger S, Mayilo D, Cado I, Merker S, Klinger M, Hoeckendorf B, Pilz S, Hupfeld K, Steinbeisser H, Lorenz H, Ruppert T, Wittbrodt J, Gruss OJ. (2013). The centriolar satellite protein SSX2IP promotes centrosome maturation. J Cell Biol. 202(1):81-95.

Abstract

Meiotic maturation in vertebrate oocytes is an excellent model system for microtubule reorganization during M-phase spindle assembly. Here, we surveyed changes in the pattern of microtubule-interacting proteins upon Xenopus laevis oocyte maturation by quantitative proteomics. We identified the synovial sarcoma X breakpoint protein (SSX2IP) as a novel spindle protein. Using X. laevis egg extracts, we show that SSX2IP accumulated at spindle poles in a Dynein-dependent manner and interacted with the γ-tubulin ring complex (γ-TuRC) and the centriolar satellite protein PCM-1. Immunodepletion of SSX2IP impeded γ-TuRC loading onto centrosomes. This led to reduced microtubule nucleation and spindle assembly failure. In rapidly dividing blastomeres of medaka (Oryzias latipes) and in somatic cells, SSX2IP knockdown caused fragmentation of pericentriolar material and chromosome segregation errors. We characterize SSX2IP as a novel centrosome maturation and maintenance factor that is expressed at the onset of vertebrate development. It preserves centrosome integrity and faithful mitosis during the rapid cleavage division of blastomeres and in somatic cells.

Herder C, Swiercz JM, Müller C, Peravali R, Quiring R, Offermanns S, Wittbrodt J, Loosli F. (2013). ArhGEF18 regulates RhoA-Rock2 signaling to maintain neuro-epithelial apico-basal polarity and proliferation. Development. 140(13):2787-97.

Abstract

The vertebrate central nervous system develops from an epithelium where cells are polarized along the apicobasal axis. Loss of this polarity results in abnormal organ architecture, morphology and proliferation. We found that mutations of the guanine nucleotide exchange factor ArhGEF18 affect apicobasal polarity of the retinal neuroepithelium in medaka fish. We show that ArhGEF18-mediated activation of the small GTPase RhoA is required to maintain apicobasal polarity at the onset of retinal differentiation and to control the ratio of neurogenic to proliferative cell divisions. RhoA signals through Rock2 to regulate apicobasal polarity, tight junction localization and the cortical actin cytoskeleton. The human ArhGEF18 homologue can rescue the mutant phenotype, suggesting a conserved function in vertebrate neuroepithelia. Our analysis identifies ArhGEF18 as a key regulator of tissue architecture and function, controlling apicobasal polarity and proliferation through RhoA activation. We thus identify the control of neuroepithelial apicobasal polarity as a novel role for RhoA signaling in vertebrate development.

Sinn R, Wittbrodt J. (2013). An eye on eye development. Mech Dev. 130(6-8):347-58.

Abstract

The vertebrate eye is composed of both surface ectodermal and neuroectodermal derivatives that evaginate laterally from an epithelial anlage of the forming diencephalon. The retina is composed of a limited number of neuronal and non-neuronal cell types and is seen as a model for the brain with reduced complexity. The eye develops in a stereotypic manner building on evolutionarily conserved molecular networks. Eye formation is initiated at the onset of gastrulation by the determination of the eye field in the anterior neuroectoderm. Homeobox transcription factors, in particular Six3 are crucially involved in the establishment and maintenance of retinal identity. The eye field expands by proliferation as gastrulation proceeds and is initially confined to a single retinal primordium by the differential activity of specifying transcription factors. This central field is subsequently split in response to secreted factors emanating from the ventral midline. Concomitant with medio-lateral patterning at the onset of neurulation, morphogenesis sets in and laterally evaginates the optic vesicle. Strikingly during this process the neuroectoderm in the eye field transiently loses epithelial features and cells migrate individually. In a second morphogenetic event, the vesicle is transformed into the optic cup, concomitant with onset and progression of retinal differentiation. Accompanying optic cup morphogenesis, neural differentiation is initiated from a retinal signalling centre in a stereotypic and species specific manner by secreted signalling factors. Here we will give an overview of key events during vertebrate eye formation and highlight key players in the respective processes.

Pubmed 


2012

Höckendorf B, Thumberger T, Wittbrodt J. (2012). Quantitative analysis of embryogenesis: a perspective for light sheet microscopy. Dev Cell. 23(6):1111-20.

Abstract

It is a challenge in developmental biology to understand how an embryo's genes, proteins, and cells function and interact to govern morphogenesis, cell fate specification, and patterning. These processes span very different spatial and temporal scales. Despite much progress, simultaneous observation of such vastly differing scales has been beyond the scope of conventional microscopy. Light sheet microscopy fills this gap and is increasingly used for long-term, high-speed recordings of large specimens with high contrast and up to subcellular spatial resolution. We provide an overview of applications of light sheet microscopy in developmental biology and discuss future perspectives in this field.

Pubmed 

Alten L, Schuster-Gossler K, Eichenlaub MPWittbrodt BWittbrodt J, Gossler A. (2012). A novel mammal-specific three partite enhancer element regulates node and notochord-specific Noto expression. PLoS One. 2012;7(10):e47785.

Abstract

The vertebrate organizer and notochord have conserved, essential functions for embryonic development and patterning. The restricted expression of developmental regulators in these tissues is directed by specific cis-regulatory modules (CRMs) whose sequence conservation varies considerably. Some CRMs have been conserved throughout vertebrates and likely represent ancestral regulatory networks, while others have diverged beyond recognition but still function over a wide evolutionary range. Here we identify and characterize a mammalian-specific CRM required for node and notochord specific (NNC) expression of NOTO, a transcription factor essential for node morphogenesis, nodal cilia movement and establishment of laterality in mouse. A 523 bp enhancer region (NOCE) upstream the Noto promoter was necessary and sufficient for NNC expression from the endogenous Noto locus. Three subregions in NOCE together mediated full activity in vivo. Binding sites for known transcription factors in NOCE were functional in vitro but dispensable for NOCE activity in vivo. A FOXA2 site in combination with a novel motif was necessary for NOCE activity in vivo. Strikingly, syntenic regions in non-mammalian vertebrates showed no recognizable sequence similarities. In contrast to its activity in mouse NOCE did not drive NNC expression in transgenic fish. NOCE represents a novel, mammal-specific CRM required for the highly restricted Noto expression in the node and nascent notochord and thus regulates normal node development and function.

Bogdanovic O, Delfino-Machín M, Nicolás-Pérez M, Gavilán MP, Gago-Rodrigues I, Fernández-Miñán A, Lillo C, Ríos RM, Wittbrodt J, Martínez-Morales JR. (2012). Numb/Numbl-Opo antagonism controls retinal epithelium morphogenesis by regulating integrin endocytosis. Dev Cell. 23(4):782-95.

Abstract

Polarized trafficking of adhesion receptors plays a pivotal role in controlling cellular behavior during morphogenesis. Particularly, clathrin-dependent endocytosis of integrins has long been acknowledged as essential for cell migration. However, little is known about the contribution of integrin trafficking to epithelial tissue morphogenesis. Here we show how the transmembrane protein Opo, previously described for its essential role during optic cup folding, plays a fundamental role in this process. Through interaction with the PTB domain of the clathrin adaptors Numb and Numbl via an integrin-like NPxF motif, Opo antagonizes Numb/Numbl function and acts as a negative regulator of integrin endocytosis in vivo. Accordingly, numb/numbl gain-of-function experiments in teleost embryos mimic the retinal malformations observed in opo mutants. We propose that developmental regulator Opo enables polarized integrin localization by modulating Numb/Numbl, thus directing the basal constriction that shapes the vertebrate retina epithelium.

Pubmed 

ENCODE Project Consortium, Dunham I, Kundaje A, Aldred SF, Collins PJ, Davis CA, Doyle F, Epstein CB, Frietze S, Harrow J, Kaul R, Khatun J, Lajoie BR, Landt SG, Lee BK, Pauli F, Rosenbloom KR, Sabo P, Safi A, Sanyal A, Shoresh N, Simon JM, Song L, Trinklein ND, Altshuler RC, Birney E, Brown JB, Cheng C, Djebali S, Dong X, Dunham I, Ernst J, Furey TS, Gerstein M, Giardine B, Greven M, Hardison RC, Harris RS, Herrero J, Hoffman MM, Iyer S, Kelllis M, Khatun J, Kheradpour P, Kundaje A, Lassmann T, Li Q, Lin X, Marinov GK, Merkel A, Mortazavi A, Parker SC, Reddy TE, Rozowsky J, Schlesinger F, Thurman RE, Wang J, Ward LD, Whitfield TW, Wilder SP, Wu W, Xi HS, Yip KY, Zhuang J, Bernstein BE, Birney E, Dunham I, Green ED, Gunter C, Snyder M, Pazin MJ, Lowdon RF, Dillon LA, Adams LB, Kelly CJ, Zhang J, Wexler JR, Green ED, Good PJ, Feingold EA, Bernstein BE, Birney E, Crawford GE, Dekker J, Elinitski L, Farnham PJ, Gerstein M, Giddings MC, Gingeras TR, Green ED, Guigó R, Hardison RC, Hubbard TJ, Kellis M, Kent WJ, Lieb JD, Margulies EH, Myers RM, Snyder M, Starnatoyannopoulos JA, Tennebaum SA, Weng Z, White KP, Wold B, Khatun J, Yu Y, Wrobel J, Risk BA, Gunawardena HP, Kuiper HC, Maier CW, Xie L, Chen X, Giddings MC, Bernstein BE, Epstein CB, Shoresh N, Ernst J, Kheradpour P, Mikkelsen TS, Gillespie S, Goren A, Ram O, Zhang X, Wang L, Issner R, Coyne MJ, Durham T, Ku M, Truong T, Ward LD, Altshuler RC, Eaton ML, Kellis M, Djebali S, Davis CA, Merkel A, Dobin A, Lassmann T, Mortazavi A, Tanzer A, Lagarde J, Lin W, Schlesinger F, Xue C, Marinov GK, Khatun J, Williams BA, Zaleski C, Rozowsky J, Röder M, Kokocinski F, Abdelhamid RF, Alioto T, Antoshechkin I, Baer MT, Batut P, Bell I, Bell K, Chakrabortty S, Chen X, Chrast J, Curado J, Derrien T, Drenkow J, Dumais E, Dumais J, Duttagupta R, Fastuca M, Fejes-Toth K, Ferreira P, Foissac S, Fullwood MJ, Gao H, Gonzalez D, Gordon A, Gunawardena HP, Howald C, Jha S, Johnson R, Kapranov P, King B, Kingswood C, Li G, Luo OJ, Park E, Preall JB, Presaud K, Ribeca P, Risk BA, Robyr D, Ruan X, Sammeth M, Sandu KS, Schaeffer L, See LH, Shahab A, Skancke J, Suzuki AM, Takahashi H, Tilgner H, Trout D, Walters N, Wang H, Wrobel J, Yu Y, Hayashizaki Y, Harrow J, Gerstein M, Hubbard TJ, Reymond A, Antonarakis SE, Hannon GJ, Giddings MC, Ruan Y, Wold B, Carninci P, Guigó R, Gingeras TR, Rosenbloom KR, Sloan CA, Learned K, Malladi VS, Wong MC, Barber GP, Cline MS, Dreszer TR, Heitner SG, Karolchik D, Kent WJ, Kirkup VM, Meyer LR, Long JC, Maddren M, Raney BJ, Furey TS, Song L, Grasfeder LL, Giresi PG, Lee BK, Battenhouse A, Sheffield NC, Simon JM, Showers KA, Safi A, London D, Bhinge AA, Shestak C, Schaner MR, Kim SK, Zhang ZZ, Mieczkowski PA, Mieczkowska JO, Liu Z, McDaniell RM, Ni Y, Rashid NU, Kim MJ, Adar S, Zhang Z, Wang T, Winter D, Keefe D, Birney E, Iyer VR, Lieb JD, Crawford GE, Li G, Sandhu KS, Zheng M, Wang P, Luo OJ, Shahab A, Fullwood MJ, Ruan X, Ruan Y, Myers RM, Pauli F, Williams BA, Gertz J, Marinov GK, Reddy TE, Vielmetter J, Partridge EC, Trout D, Varley KE, Gasper C, Bansal A, Pepke S, Jain P, Amrhein H, Bowling KM, Anaya M, Cross MK, King B, Muratet MA, Antoshechkin I, Newberry KM, McCue K, Nesmith AS, Fisher-Aylor KI, Pusey B, DeSalvo G, Parker SL, Balasubramanian S, Davis NS, Meadows SK, Eggleston T, Gunter C, Newberry JS, Levy SE, Absher DM, Mortazavi A, Wong WH, Wold B, Blow MJ, Visel A, Pennachio LA, Elnitski L, Margulies EH, Parker SC, Petrykowska HM, Abyzov A, Aken B, Barrell D, Barson G, Berry A, Bignell A, Boychenko V, Bussotti G, Chrast J, Davidson C, Derrien T, Despacio-Reyes G, Diekhans M, Ezkurdia I, Frankish A, Gilbert J, Gonzalez JM, Griffiths E, Harte R, Hendrix DA, Howald C, Hunt T, Jungreis I, Kay M, Khurana E, Kokocinski F, Leng J, Lin MF, Loveland J, Lu Z, Manthravadi D, Mariotti M, Mudge J, Mukherjee G, Notredame C, Pei B, Rodriguez JM, Saunders G, Sboner A, Searle S, Sisu C, Snow C, Steward C, Tanzer A, Tapanari E, Tress ML, van Baren MJ, Walters N, Washieti S, Wilming L, Zadissa A, Zhengdong Z, Brent M, Haussler D, Kellis M, Valencia A, Gerstein M, Raymond A, Guigó R, Harrow J, Hubbard TJ, Landt SG, Frietze S, Abyzov A, Addleman N, Alexander RP, Auerbach RK, Balasubramanian S, Bettinger K, Bhardwaj N, Boyle AP, Cao AR, Cayting P, Charos A, Cheng Y, Cheng C, Eastman C, Euskirchen G, Fleming JD, Grubert F, Habegger L, Hariharan M, Harmanci A, Iyenger S, Jin VX, Karczewski KJ, Kasowski M, Lacroute P, Lam H, Larnarre-Vincent N, Leng J, Lian J, Lindahl-Allen M, Min R, Miotto B, Monahan H, Moqtaderi Z, Mu XJ, O'Geen H, Ouyang Z, Patacsil D, Pei B, Raha D, Ramirez L, Reed B, Rozowsky J, Sboner A, Shi M, Sisu C, Slifer T, Witt H, Wu L, Xu X, Yan KK, Yang X, Yip KY, Zhang Z, Struhl K, Weissman SM, Gerstein M, Farnham PJ, Snyder M, Tenebaum SA, Penalva LO, Doyle F, Karmakar S, Landt SG, Bhanvadia RR, Choudhury A, Domanus M, Ma L, Moran J, Patacsil D, Slifer T, Victorsen A, Yang X, Snyder M, White KP, Auer T, Centarin L, Eichenlaub M, Gruhl F, Heerman S, Hoeckendorf B, Inoue D, Kellner T, Kirchmaier S, Mueller C, Reinhardt R, Schertel L, Schneider S, Sinn R, Wittbrodt B, Wittbrodt J, Weng Z, Whitfield TW, Wang J, Collins PJ, Aldred SF, Trinklein ND, Partridge EC, Myers RM, Dekker J, Jain G, Lajoie BR, Sanyal A, Balasundaram G, Bates DL, Byron R, Canfield TK, Diegel MJ, Dunn D, Ebersol AK, Ebersol AK, Frum T, Garg K, Gist E, Hansen RS, Boatman L, Haugen E, Humbert R, Jain G, Johnson AK, Johnson EM, Kutyavin TM, Lajoie BR, Lee K, Lotakis D, Maurano MT, Neph SJ, Neri FV, Nguyen ED, Qu H, Reynolds AP, Roach V, Rynes E, Sabo P, Sanchez ME, Sandstrom RS, Sanyal A, Shafer AO, Stergachis AB, Thomas S, Thurman RE, Vernot B, Vierstra J, Vong S, Wang H, Weaver MA, Yan Y, Zhang M, Akey JA, Bender M, Dorschner MO, Groudine M, MacCoss MJ, Navas P, Stamatoyannopoulos G, Kaul R, Dekker J, Stamatoyannopoulos JA, Dunham I, Beal K, Brazma A, Flicek P, Herrero J, Johnson N, Keefe D, Lukk M, Luscombe NM, Sobral D, Vaquerizas JM, Wilder SP, Batzoglou S, Sidow A, Hussami N, Kyriazopoulou-Panagiotopoulou S, Libbrecht MW, Schaub MA, Kundaje A, Hardison RC, Miller W, Giardine B, Harris RS, Wu W, Bickel PJ, Banfai B, Boley NP, Brown JB, Huang H, Li Q, Li JJ, Noble WS, Bilmes JA, Buske OJ, Hoffman MM, Sahu AO, Kharchenko PV, Park PJ, Baker D, Taylor J, Weng Z, Iyer S, Dong X, Greven M, Lin X, Wang J, Xi HS, Zhuang J, Gerstein M, Alexander RP, Balasubramanian S, Cheng C, Harmanci A, Lochovsky L, Min R, Mu XJ, Rozowsky J, Yan KK, Yip KY, Birney E. (2012). An integrated encyclopedia of DNA elements in the human genome. Nature. 489(7414):57-74.

Abstract

Comment in Nat Rev Genet. 2012 Oct;13(10):678. Am J Transplant. 2013 Feb;13(2):245. Nat Methods. 2012 Nov;9(11):1046.

Pubmed 

Schulz S, Chachami G, Kozaczkiewicz L, Winter U, Stankovic-Valentin N, Haas P, Hofmann K, Urlaub H, Ovaa H, Wittbrodt J, Meulmeester E, Melchior F. (2012). Ubiquitin-specific protease-like 1 (USPL1) is a SUMO isopeptidase with essential, non-catalytic functions. EMBO Rep. 13(10):930-8.

Abstract

Isopeptidases are essential regulators of protein ubiquitination and sumoylation. However, only two families of SUMO isopeptidases are at present known. Here, we report an activity-based search with the suicide inhibitor haemagglutinin (HA)-SUMO-vinylmethylester that led to the identification of a surprising new SUMO protease, ubiquitin-specific protease-like 1 (USPL1). Indeed, USPL1 neither binds nor cleaves ubiquitin, but is a potent SUMO isopeptidase both in vitro and in cells. C13orf22l--an essential but distant zebrafish homologue of USPL1--also acts on SUMO, indicating functional conservation. We have identified invariant USPL1 residues required for SUMO binding and cleavage. USPL1 is a low-abundance protein that colocalizes with coilin in Cajal bodies. Its depletion does not affect global sumoylation, but causes striking coilin mislocalization and impairs cell proliferation, functions that are not dependent on USPL1 catalytic activity. Thus, USPL1 represents a third type of SUMO protease, with essential functions in Cajal body biology.

Harden MV, Pereiro L, Ramialison M, Wittbrodt J, Prasad MK, McCallion AS, Whitlock KE. (2012). Close association of olfactory placode precursors and cranial neural crest cells does not predestine cell mixing. Dev Dyn. 241(7):1143-54.

Abstract

Vertebrate sensory organs originate from both cranial neural crest cells (CNCCs) and placodes. Previously, we have shown that the olfactory placode (OP) forms from a large field of cells extending caudally to the premigratory neural crest domain, and that OPs form through cell movements and not cell division. Concurrent with OP formation, CNCCs migrate rostrally to populate the frontal mass. However, little is known about the interactions between CNCCs and the placodes that form the olfactory sensory system. Previous reports suggest that the OP can generate cell types more typical of neural crest lineages such as neuroendocrine cells and glia, thus marking the OP as an unusual sensory placode. One possible explanation for this exception is that the neural crest origin of glia and neurons has been overlooked due to the intimate association of these two fields during migration. Using molecular markers and live imaging, we followed the development of OP precursors and of dorsally migrating CNCCs in zebrafish embryos. We generated a six4b:mCherry line (OP precursors) that, with a sox10:EGFP line (CNCCs), was used to follow cell migration. Our analyses showed that CNCCs associate with and eventually surround the forming OP with limited cell mixing occurring during this process.

Pubmed 

Ramialison M, Reinhardt R, Henrich T, Wittbrodt B, Kellner T, Lowy CM, Wittbrodt J. (2012). Cis-regulatory properties of medaka synexpression groups. Development. 139(5):917-28.

Abstract

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.


2011

 

Centanin L, Hoeckendorf B, Wittbrodt J. (2011). Fate restriction and multipotency in retinal stem cells. Cell Stem Cell. 9(6):553-62.

Abstract

Stem cells have the capacity to both self-renew and generate postmitotic cells. Long-term tracking of individual clones in their natural environment constitutes the ultimate way to validate postembryonic stem cells. We identify retinal stem cells (RSCs) using the spatiotemporal organization of the fish retina and follow the complete offspring of a single cell during the postnatal life. RSCs generate two tissues of the adult fish retina, the neural retina (NR) and the retinal-pigmented epithelium (RPE). Despite their common embryonic origin and tight coordination during continuous organ growth, we prove that NR and RPE are maintained by dedicated RSCs that contribute in a fate-restricted manner to either one or the other tissue. We show that in the NR, RSCs are multipotent and generate all neuron types and glia. The clonal origin of these different cell types from a multipotent NSC has far-reaching implications for cell type and tissue homeostasis.

Pubmed 

Keller PJ, Schmidt AD, Wittbrodt J, Stelzer EH. (2011). Digital scanned laser light-sheet fluorescence microscopy (DSLM) of zebrafish and Drosophila embryonic development. Cold Spring Harb Protoc. 2011(10):1235-43.

Abstract

PMID: 21969622 [PubMed - indexed for MEDLINE]

Mongin E, Auer TO, Bourrat F, Gruhl F, Dewar K, Blanchette M, Wittbrodt J, Ettwiller L. (2011). Combining computational prediction of cis-regulatory elements with a new enhancer assay to efficiently label neuronal structures in the medaka fish. PLoS One. 2011;6(5):e19747.

Abstract

The developing vertebrate nervous system contains a remarkable array of neural cells organized into complex, evolutionarily conserved structures. The labeling of living cells in these structures is key for the understanding of brain development and function, yet the generation of stable lines expressing reporter genes in specific spatio-temporal patterns remains a limiting step. In this study we present a fast and reliable pipeline to efficiently generate a set of stable lines expressing a reporter gene in multiple neuronal structures in the developing nervous system in medaka. The pipeline combines both the accurate computational genome-wide prediction of neuronal specific cis-regulatory modules (CRMs) and a newly developed experimental setup to rapidly obtain transgenic lines in a cost-effective and highly reproducible manner. 95% of the CRMs tested in our experimental setup show enhancer activity in various and numerous neuronal structures belonging to all major brain subdivisions. This pipeline represents a significant step towards the dissection of embryonic neuronal development in vertebrates.

Inoue D, Wittbrodt J. (2011). One for all--a highly efficient and versatile method for fluorescent immunostaining in fish embryos. PLoS One. 2011;6(5):e19713.

Abstract

BACKGROUND: For the detection and sub-cellular (co)-localization of proteins in the context of the tissue or organism immunostaining in whole mount preparations or on sections is still the best approach. So far, each antibody required its own fixation and antigen retrieval protocol so that optimizing immunostaining turned out to be tedious and time consuming. METHODOLOGY/PRINCIPAL FINDING: Here we present a novel method to efficiently retrieve the antigen in a widely applicable standard protocol, facilitating fluorescent immunostaining of both cryosections and whole mount preparations in zebrafish (Danio rerio) and medaka (Oryzias latipes). CONCLUSIONS/SIGNIFICANCE: Our method overcomes the loss of sections and damage of tissue and cell morphology, and allows parallel immunostaining in multiple colors, co-immunostaining with fluorescent proteins in transgenic fish lines and in combination with whole mount in situ hybridization.

Maurya AK, Tan H, Souren M, Wang X, Wittbrodt J, Ingham PW. (2011). Integration of Hedgehog and BMP signalling by the engrailed2a gene in the zebrafish myotome. Development. 138(4):755-65.

Abstract

Erratum in Development. 2012 May;139(10):1885.

Pubmed 


2010


Keller PJ, Schmidt AD, Santella A, Khairy K, Bao Z, Wittbrodt J, Stelzer EH. (2010). Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy. Nat Methods. 7(8):637-42.

Abstract

Recording light-microscopy images of large, nontransparent specimens, such as developing multicellular organisms, is complicated by decreased contrast resulting from light scattering. Early zebrafish development can be captured by standard light-sheet microscopy, but new imaging strategies are required to obtain high-quality data of late development or of less transparent organisms. We combined digital scanned laser light-sheet fluorescence microscopy with incoherent structured-illumination microscopy (DSLM-SI) and created structured-illumination patterns with continuously adjustable frequencies. Our method discriminates the specimen-related scattered background from signal fluorescence, thereby removing out-of-focus light and optimizing the contrast of in-focus structures. DSLM-SI provides rapid control of the illumination pattern, exceptional imaging quality and high imaging speeds. We performed long-term imaging of zebrafish development for 58 h and fast multiple-view imaging of early Drosophila melanogaster development. We reconstructed cell positions over time from the Drosophila DSLM-SI data and created a fly digital embryo.

Pubmed 

Haudry Y, Ramialison M, Paten B, Wittbrodt J, Ettwiller L. (2010). Using Trawler_standalone to discover overrepresented motifs in DNA and RNA sequences derived from various experiments including chromatin immunoprecipitation. Nat Protoc. 5(2):323-34.

Abstract

Genome-wide location analysis has become a standard technology to unravel gene regulation networks. The accurate characterization of nucleotide signatures in sequences is key to uncovering the regulatory logic but remains a computational challenge. This protocol describes how to best characterize these signatures (motifs) using the new standalone version of Trawler, which was designed and optimized to analyze chromatin immunoprecipitation (ChIP) data sets. In particular, we describe the three main steps of Trawler_standalone (motif discovery, clustering and visualization) and discuss the appropriate parameters to be used in each step depending on the data set and the biological questions addressed. Compared to five other motif discovery programs, Trawler_standalone is in most cases the fastest algorithm to accurately predict the correct motifs especially for large data sets. Its running time ranges within few seconds to several minutes, depending on the size of the data set and the parameters used. This protocol is best suited for bioinformaticians seeking to use Trawler_standalone in a high-throughput manner.

Pubmed 


2009

Brown KE, Keller PJ, Ramialison M, Rembold M, Stelzer EH, Loosli F, Wittbrodt J. (2009). Nlcam modulates midline convergence during anterior neural plate morphogenesis. Dev Biol. 339(1):14-25.

Abstract

During development, different cell types must undergo distinct morphogenetic programs so that tissues develop the right dimensions in the appropriate place. In early eye morphogenesis, retinal progenitor cells (RPCs) move first towards the midline, before turning around to migrate out into the evaginating optic vesicles. Neighbouring forebrain cells, however, converge rapidly and then remain at the midline. These differential behaviours are regulated by the transcription factor Rx3. Here, we identify a downstream target of Rx3, the Ig-domain protein Nlcam, and characterise its role in regulating cell migration during the initial phase of optic vesicle morphogenesis. Through sophisticated live imaging and comprehensive cell tracking experiments in zebrafish, we show that ectopic expression of Nlcam in RPCs, as is observed in Rx3 mutants, causes enhanced convergence of these cells. Expression levels of Nlcam therefore regulate the migratory properties of RPCs. Our results provide evidence that the two phases of optic vesicle morphogenesis: slowed convergence and outward-directed migration, are under different genetic control. We propose that Nlcam forms part of the guidance machinery directing rapid midline migration of forebrain precursors, where it is normally expressed, and that its ectopic expression upon loss of Rx3 imparts these migratory characteristics upon RPCs.

Pubmed 

Martinez-Morales JR, Wittbrodt J. (2009). Shaping the vertebrate eye. Curr Opin Genet Dev. 19(5):511-7.

Abstract

For over a century, the vertebrate eye has served as a paradigm for organogenesis. It forms through a complex sequence of morphogenetic events, involving the lateral evagination of the optic vesicles and their subsequent folding into the optic cups. Through intensive studies by experimental embryologists, anatomical descriptions of the process were available since many decades. Recent genetic and molecular work has illuminated essential features of the stereotyped cellular behaviour driving eye morphogenesis. The first pieces of the molecular machinery operating in each individual progenitor cell have been identified. These studies now set the groundwork for a system-wide approach towards understanding the cellular and molecular mechanisms involved in shaping the vertebrate eye.

Pubmed 

Bajoghli B, Ramialison M, Aghaallaei N, Czerny T, Wittbrodt J. (2009). Identification of starmaker-like in medaka as a putative target gene of Pax2 in the otic vesicle. Dev Dyn.238(11):2860-6.

Abstract

Otoliths in bony fishes are involved in the function of the ear in the senses of balance and hearing. In a large-scale random in situ hybridization screen of genes expressed in the medaka developing ear, we identified starmaker-like (stm-l) gene, a novel homologue of zebrafish starmaker and human dentine sialo-phosphoprotein (dspp) gene. Despite the absence of sequence similarity between these genes, here we describe their similar genomic structure and expression patterns hinting for a conserved function. In medaka fry, stm-l is expressed in various organs such as otoliths, teeth, gills, and kidney. Additionally, our results provide evidence that stm-l is a putative downstream target gene of Pax2 transcription factor and Pax2 itself has a promoting function in otolith formation.

Pubmed 

Mertes F, Martinez-Morales JR, Nolden T, Spörle R, Wittbrodt J, Lehrach H, Himmelbauer H. (2009). Cloning of mouse ojoplano, a reticular cytoplasmic protein expressed during embryonic development. Gene Expr Patterns.9(8):562-7.

Abstract

Ojoplano (Opo) is a morphogenetic gene playing an important role during embryogenesis in medaka. This report focuses on the identification and characterization of the mouse Opo gene. We examined Opo expression by whole-mount in situ hybridization and in situ hybridization on sagittal sections during mouse embryogenesis. First expression in whole-mounts was detected at Theiler stages 15-17 (E 9.5-10.5dpc) as a spotted specific staining in migrating neural crest cells and in placodal structures. A complex expression pattern was observed in Theiler stage 22-23 (E 14.5dpc) in sagittal sections, including expression in skeletal structures (skull, vertebrae, ribs, bones of the locomotor system), in the nasal region, the heart and the eye. Fusion proteins revealed the localization of OPO within the cytoplasm with a reticular distribution that largely overlapped with the endoplasmic reticulum. Opo shows homology to human transcripts linked to a hereditary craniofacial malformation, orofacial cleft 1 (OFC1). The expression of mouse Opo in neural crest derivatives and skull elements further supports this link.

Pubmed 

Souren M, Martinez-Morales JR, Makri P, Wittbrodt B, Wittbrodt J. (2009). A global survey identifies novel upstream components of the Ath5 neurogenic network. Genome Biol. 2009;10(9):R92.

Abstract

BACKGROUND: Investigating the architecture of gene regulatory networks (GRNs) is essential to decipher the logic of developmental programs during embryogenesis. In this study we present an upstream survey approach, termed trans-regulation screen, to comprehensively identify the regulatory input converging on endogenous regulatory sequences. RESULTS: Our dual luciferase-based screen queries transcriptome-scale collections of cDNAs. Using this approach we study the regulation of Ath5, the central node in the GRN controlling retinal ganglion cell (RGC) specification in vertebrates. The Ath5 promoter integrates the input of upstream regulators to enable the transient activation of the gene, which is an essential step for RGC differentiation. We efficiently identified potential Ath5 regulators that were further filtered for true positives by an in situ hybridization screen. Their regulatory activity was validated in vivo by functional assays in medakafish embryos. CONCLUSIONS: Our analysis establishes functional groups of genes controlling different regulatory phases, including the onset of Ath5 expression at cell-cycle exit and its down-regulation prior to terminal RGC differentiation. These results extent the current model of the GRN controlling retinal neurogenesis in vertebrates.

Pubmed 

Martinez-Morales JR, Rembold M, Greger K, Simpson JC, Brown KE, Quiring R, Pepperkok R, Martin-Bermudo MD, Himmelbauer H, Wittbrodt J. (2009). ojoplano-mediated basal constriction is essential for optic cup morphogenesis. Development. 136(13):2165-75.

Abstract

Although the vertebrate retina is a well-studied paradigm for organogenesis, the morphogenetic mechanisms that carve the architecture of the vertebrate optic cup remain largely unknown. Understanding how the hemispheric shape of an eye is formed requires addressing the fundamental problem of how individual cell behaviour is coordinated to direct epithelial morphogenesis. Here, we analyze the role of ojoplano (opo), an uncharacterized gene whose human ortholog is associated with orofacial clefting syndrome, in the morphogenesis of epithelial tissues. Most notably, when opo is mutated in medaka fish, optic cup folding is impaired. We characterize optic cup morphogenesis in vivo and determine at the cellular level how opo affects this process. opo encodes a developmentally regulated transmembrane protein that localizes to compartments of the secretory pathway and to basal end-feet of the neuroepithelial precursors. We show that Opo regulates the polarized localization of focal adhesion components to the basal cell surface. Furthermore, tissue-specific interference with integrin-adhesive function impairs optic cup folding, resembling the ocular phenotype observed in opo mutants. We propose a model of retinal morphogenesis whereby opo-mediated formation of focal contacts is required to transmit the mechanical tensions that drive the macroscopic folding of the vertebrate optic cup.

Pubmed 

Sano S, Takashima S, Niwa H, Yokoi H, Shimada A, Arenz A, Wittbrodt J, Takeda H. (2009). Characterization of teleost Mdga1 using a gene-trap approach in medaka (Oryzias latipes). Genesis. 47(8):505-13.

Abstract

MAM domain containing glycosilphosphatidilinositol anchor 1 (MDGA1) is an IgCAM protein present in many vertebrate species including humans. In mammals, MDGA1 is expressed by a subset of neurons in the developing brain and thought to function in neural cell migration. We identified a fish ortholog of mdga1 by a gene-trap screen utilizing the Frog Prince transposon in medaka (Japanese killifish, Oryzias latipes). The gene-trap vector was inserted into an intronic region of mdga1 to form a chimeric protein with green fluorescent protein, allowing us to monitor mdga1 expression in vivo. Expression of medaka mdga1 was seen in various types of embryonic brain neurons, and specifically in neurons migrating toward their target sites, supporting the proposed function of MDGA1. We also isolated the closely related mdga2 gene, whose expression partially overlapped with that of mdga1. Despite the fact that the gene-trap event eliminated most of the functional domains of the Mdga1 protein, homozygous embryos developed normally without any morphological abnormality, suggesting a functional redundancy of Mdga1 with other related proteins. High sequential homology of MDGA proteins between medaka and other vertebrate species suggests an essential role of the MDGA gene family in brain development among the vertebrate phylum.

Pubmed 

Signore IA, Guerrero N, Loosli F, Colombo A, Villalón A, Wittbrodt J, Concha ML. (2009). Zebrafish and medaka: model organisms for a comparative developmental approach of brain asymmetry. Philos Trans R Soc Lond B Biol Sci. 364(1519):991-1003.

Abstract

Pubmed 


2008

Ettwiller L, Budd A, Spitz F, Wittbrodt J. (2008). Analysis of mammalian gene batteries reveals both stable ancestral cores and highly dynamic regulatory sequences. Genome Biol. 9(12):R172.

Abstract

Pubmed 

Grabher C, Wittbrodt J.(2008). Recent advances in meganuclease-and transposon-mediated transgenesis of medaka and zebrafish. Methods Mol Biol. 461:521-39.

Abstract

PMID: 19030821 [PubMed - indexed for MEDLINE]

Keller PJ*, Schmidt AD, Wittbrodt J*, Stelzer EH. (2008). Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science. 322(5904):1065-9. * corresponding authors 

Abstract

A long-standing goal of biology is to map the behavior of all cells during vertebrate embryogenesis. We developed digital scanned laser light sheet fluorescence microscopy and recorded nuclei localization and movement in entire wild-type and mutant zebrafish embryos over the first 24 hours of development. Multiview in vivo imaging at 1.5 billion voxels per minute provides "digital embryos," that is, comprehensive databases of cell positions, divisions, and migratory tracks. Our analysis of global cell division patterns reveals a maternally defined initial morphodynamic symmetry break, which identifies the embryonic body axis. We further derive a model of germ layer formation and show that the mesendoderm forms from one-third of the embryo's cells in a single event. Our digital embryos, with 55 million nucleus entries, are provided as a resource.

Pubmed 

Ramialison M, Bajoghli B, Aghaallaei N, Ettwiller L, Gaudan S, Wittbrodt B, Czerny T, Wittbrodt J. (2008). Rapid identification of PAX2/5/8 direct downstream targets in the otic vesicle by combinatorial use of bioinformatics tools. Genome Biol. 9(10):R145.

Abstract

BACKGROUND: The pax2/5/8 genes belonging to the PAX family of transcription factors are key developmental regulators that are involved in the patterning of various embryonic tissues. More particularly, their function in inner ear specification has been widely described. However, little is known about the direct downstream targets and, so far, no global approaches have been performed to identify these target genes in this particular tissue. RESULTS: Here we present an original bioinformatics pipeline composed of comparative genomics, database querying and text mining tools, which is designed to rapidly and specifically discover PAX2/5/8 direct downstream targets involved in inner ear development. We provide evidence supported by experimental validation in medaka fish that brain 2 (POU domain, class 3, transcription factor 2), claudin-7, secretory pathway component sec31-like and meteorin-like precursor are novel direct downstream targets of PAX2/5/8. CONCLUSIONS: This study illustrates the power of extensive mining of public data repositories using bioinformatics methods to provide answers for a specific biological question. It furthermore demonstrates how the usage of such a combinatorial approach is advantageous for the biologist in terms of experimentation time and costs.

Pubmed 


2007

Grabher C, Wittbrodt J.  (2007). Meganuclease and transposon mediated transgenesis in medaka. Genome Biol. 8 Suppl 1:S10.

Abstract

From among a plethora of various gene delivery methods, the researcher must choose the right one according to availability for a given species and the precise application the transgenic animal is intended for. Here we review the progress in meganuclease and Sleeping Beauty transposon mediated transgenesis over recent years with a focus on medaka and zebrafish. We present a side-by-side comparison of these two approaches based on their biologic properties and provide interesting perspectives for future experiments and applications, which are different for the two techniques because of their distinct modes of action.

Pubmed 

Haudry Y, Berube H, Letunic I, Weeber PD, Gagneur J, Girardot C, Kapushesky M, Arendt D, Bork P, Brazma A, Furlong EE, Wittbrodt J, Henrich T. (2007). 4DXpress: a database for cross-species expression pattern comparisons. Nucleic Acids Res.36(Database issue):D847-53.

Abstract

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.

Pubmed 

Del Bene F, Ettwiller L, Skowronska-Krawczyk D, Baier H, Matter JM, Birney E, Wittbrodt J. (2007). In vivo validation of a computationally predicted conserved Ath5 target gene set. PLoS Genet. 3(9):1661-71.

Abstract

So far, the computational identification of transcription factor binding sites is hampered by the complexity of vertebrate genomes. Here we present an in silico procedure to predict target sites of a transcription factor in complex genomes using its binding site. In a first step sequence, comparison of closely related genomes identifies the binding sites in conserved cis-regulatory regions (phylogenetic footprinting). Subsequently, more remote genomes are introduced into the comparison to identify highly conserved and therefore putatively functional binding sites (phylogenetic filtering). When applied to the binding site of atonal homolog 5 (Ath5 or ATOH7), this procedure efficiently filters evolutionarily conserved binding sites out of more than 300,000 instances in a vertebrate genome. We validate a selection of the linked target genes by showing coexpression with and transcriptional regulation by Ath5. Finally, chromatin immunoprecipitation demonstrates the occupancy of the target gene promoters by Ath5. Thus, our procedure, applied to whole genomes, is a fast and predictive tool to in silico filter the target genes of a given transcription factor with defined binding site.

Pubmed 

Ettwiller L, Paten B, Ramialison M, Birney E, Wittbrodt J. (2007). Trawler: de novo regulatory motif discovery pipeline for chromatin immunoprecipitation. Nat Methods. 4(7):563-5.

Abstract

We developed Trawler, the fastest computational pipeline to date, to efficiently discover over-represented motifs in chromatin immunoprecipitation (ChIP) experiments and to predict their functional instances. When we applied Trawler to data from yeast and mammals, 83% of the known binding sites were accurately called, often with other additional binding sites, providing hints of combinatorial input. Newly discovered motifs and their features (identity, conservation, position in sequence) are displayed on a web interface.

Pubmed 

Rembold M, Lahiri K, Foulkes NS, Wittbrodt J. (2006). Transgenesis in fish: efficient selection of transgenic fish by co-injection with a fluorescent reporter construct. Nat Protoc. 1(3):1133-9.

Abstract

Small fish are a popular laboratory model for studying gene expression and function by transgenesis. If, however, the transgenes are not readily detectable by visual inspection, a large number of embryos must be injected, raised and screened to identify positive founder fish. Here, we describe a strategy to efficiently generate and preselect transgenic lines harbouring any transgene of interest. Co-injection of a selectable reporter construct (e.g., GFP), together with the transgene of interest on a separate plasmid using the I-SceI meganuclease approach, results in co-distribution of the two plasmids. The quality of GFP expression within the F0 generation therefore reflects the quality of injection and allows efficient and reliable selection of founder fish that are also positive for the second transgene of interest. In our experience, a large fraction (up to 50%) of GFP-positive fish will also be transgenic for the second transgene, thus providing a rapid (within 3-4 months) and efficient way to establish transgenic lines for any gene of interest in medaka and zebrafish.

Pubmed 

Martinez-Morales JR, Henrich T, Ramialison M, Wittbrodt J. (2007). New genes in the evolution of the neural crest differentiation program. Genome Biol. 8(3):R36.

Abstract

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.

Pubmed 

Steinmetz PR, Zelada-Gonzáles F, Burgtorf C, Wittbrodt J, Arendt D. (2007). Polychaete trunk neuroectoderm converges and extends by mediolateral cell intercalation. Proc Natl Acad Sci U S A. 104(8):2727-32.

Abstract

During frog and fish development, convergent extension movements transform the spherical gastrula into an elongated neurula. Such transformation of a ball- into a worm-shaped embryo is an ancestral and fundamental feature of bilaterian development, yet this is modified or absent in the protostome model organisms Caenorhabditis or Drosophila. In the polychaete annelid Platynereis dumerilii, early embryonic and larval stages resemble a sphere that subsequently elongates into worm shape. Cellular and molecular mechanisms of polychaete body elongation are yet unknown. Our in vivo time-lapse analysis of Platynereis axis elongation reveals that the polychaete neuroectoderm converges and extends by mediolateral cell intercalation. This occurs on both sides of the neural midline, the line of fusion of the slit-like blastopore. Convergent extension moves apart mouth and anus that are both derived from the blastopore. Tissue elongation is actin-dependent but microtubule-independent. Dependence on JNK activity and spatially restricted expression of strabismus indicates involvement of the noncanonical Wnt pathway. We detect a morphogenetic boundary between the converging and extending trunk neuroectoderm and the anterior otx-expressing head neuroectoderm that does not elongate. Our comparative analysis uncovers striking similarities but also differences between convergent extension in the polychaete and in the frog (the classical vertebrate model for convergent extension). Based on these findings, we propose that convergent extension movements of the trunk neuroectoderm represent an ancestral feature of bilaterian development that triggered the separation of mouth and anus along the elongating trunk.

Pubmed 


2006

Yokoi H, Shimada A, Carl M, Takashima S, Kobayashi D, Narita T, Jindo T, Kimura T, Kitagawa T, Kage T, Sawada A, Naruse K, Asakawa S, Shimizu N, Mitani H, Shima A, Tsutsumi M, Hori H, Wittbrodt J, Saga Y, Ishikawa Y, Araki K, Takeda H. (2006). Mutant analyses reveal different functions of fgfr1 in medaka and zebrafish despite conserved ligand-receptor relationships. Dev Biol.304(1):326-37.

Abstract

Medaka (Oryzias latipes) is a small freshwater teleost that provides an excellent developmental genetic model complementary to zebrafish. Our recent mutagenesis screening using medaka identified headfish (hdf) which is characterized by the absence of trunk and tail structures with nearly normal head including the midbrain-hindbrain boundary (MHB). Positional-candidate cloning revealed that the hdf mutation causes a functionally null form of Fgfr1. The fgfr1hdf is thus the first fgf receptor mutant in fish. Although FGF signaling has been implicated in mesoderm induction, mesoderm is induced normally in the fgfr1hdf mutant, but subsequently, mutant embryos fail to maintain the mesoderm, leading to defects in mesoderm derivatives, especially in trunk and tail. Furthermore, we found that morpholino knockdown of medaka fgf8 resulted in a phenotype identical to the fgfr1hdf mutant, suggesting that like its mouse counterpart, Fgf8 is a major ligand for Fgfr1 in medaka early embryogenesis. Intriguingly, Fgf8 and Fgfr1 in zebrafish are also suggested to form a major ligand-receptor pair, but their function is much diverged, as the zebrafish fgfr1 morphant and zebrafish fgf8 mutant acerebellar (ace) only fail to develop the MHB, but develop nearly unaffected trunk and tail. These results provide evidence that teleost fish have evolved divergent functions of Fgf8-Fgfr1 while maintaining the ligand-receptor relationships. Comparative analysis using different fish is thus invaluable for shedding light on evolutionary diversification of gene function.

Pubmed 

Grabher C, Cliffe A, Miura K, Hayflick J, Pepperkok R, Rørth P, Wittbrodt J. (2007). Birth and life of tissue macrophages and their migration in embryogenesis and inflammation in medaka. J Leukoc Biol.81(1):263-71.

Abstract

Macrophages detecting and migrating toward sites of injury and infection represent one of the first steps in an immune response. Here we directly image macrophage birth and migration in vivo in transgenic medaka fish. Macrophages are born as frequently dividing, immotile cells with spherical morphology that differentiate into flat, highly motile cells. They retain mitotic activity while spreading over the entire body. Cells follow restricted paths not only in directed migration, but also during patrolling. Along those paths the macrophages rapidly patrol the tissue and respond to wounding and bacterial infection from long distances. Upon injury they increase their speed and migratory persistence. Specifically targeting PI3-kinase isoforms efficiently blocks the wounding response and results in a distinct inhibition of cell motility and chemotaxis. Our study provides in situ insights into the properties of immature and migratory macrophages and presents a unique model to further test modulating compounds in vivo.

Pubmed 

Ason B, Darnell DK, Wittbrodt B, Berezikov E, Kloosterman WP, Wittbrodt J, Antin PB, Plasterk RH. (2006). Differences in vertebrate microRNA expression. Proc Natl Acad Sci U S A.103(39):14385-9.

Abstract

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.

Pubmed 

Rembold M, Loosli F, Adams RJ, Wittbrodt J. (2006). Individual cell migration serves as the driving force for optic vesicle evagination. Science. 313(5790):1130-4.

Abstract

The cellular mechanisms underlying organ formation are largely unknown. We visualized early vertebrate eye morphogenesis at single-cell resolution by in vivo imaging in medaka (Oryzias latipes). Before optic vesicle evagination, retinal progenitor cells (RPCs) modulate their convergence in a fate-specific manner. Presumptive forebrain cells converge toward the midline, whereas medial RPCs remain stationary, predetermining the site of evagination. Subsequent optic vesicle evagination is driven by the active migration of individual RPCs. The analysis of mutants demonstrated that the retina-specific transcription factor rx3 determines the convergence and migration behaviors of RPCs. Hence, the migration of individual cells mediates essential steps of organ morphogenesis.

Pubmed 

Swindell EC, Bailey TJ, Loosli F, Liu C, Amaya-Manzanares F, Mahon KA, Wittbrodt J, Jamrich M. (2006). Rx-Cre, a tool for inactivation of gene expression in the developing retina. Genesis.44(8):361-3.

Abstract

Nakamura S, Kobayashi D, Aoki Y, Yokoi H, Ebe Y, Wittbrodt J, Tanaka M. (2006). Identification and lineage tracing of two populations of somatic gonadal precursors in medaka embryos. Dev Biol.295(2):678-88.

Abstract

The gonad contains two major cell lineages, germline and somatic cells. Little is known, however, about the somatic gonadal cell lineage in vertebrates. Using fate mapping studies and ablation experiments in medaka fish (Oryzias latipes), we determined that somatic gonadal precursors arise from the most posterior part of the sdf-1a expression domain in the lateral plate mesoderm at the early segmentation stage; this region has the properties of a gonadal field. Somatic gonadal precursors in this field, which continuously express sdf-1a, move anteriorly and medially to the prospective gonadal area by convergent movement. By the stage at which these somatic gonadal precursors have become located adjacent to the embryonic body, the precursors no longer replace the surrounding lateral plate mesoderm, becoming spatially organized into two distinct populations. We further show that, prior to reaching the prospective gonadal area, these populations can be distinguished by expression of either ftz-f1 or sox9b. These results clearly indicate that different populations of gonadal precursors are present before the formation of a single gonadal primordium, shedding new light on the developmental processes of somatic gonadal cell and subsequent sex differentiation.

Pubmed 


publications before 2006

Ettwiller L, Paten B, Souren M, Loosli F, Wittbrodt J, Birney E. (2005). The discovery, positioning and verification of a set of transcription-associated motifs in vertebrates. Genome Biol. 6(12):R104.

Abstract

We have developed several new methods to investigate transcriptional motifs in vertebrates. We developed a specific alignment tool appropriate for regions involved in transcription control, and exhaustively enumerated all possible 12-mers for involvement in transcription by virtue of their mammalian conservation. We then used deeper comparative analysis across vertebrates to identify the active instances of these motifs. We have shown experimentally in Medaka fish that a subset of these predictions is involved in transcription.

Pubmed 

Wichmann O, Wittbrodt J, Schultz C. (2006). A small-molecule FRET probe to monitor phospholipase A2 activity in cells and organisms. Angew Chem Int Ed Engl.45(3):508-12.

Abstract

PMID: 16323228 [PubMed - indexed for MEDLINE]

Fritzsch B, Piatigorsky J, Tessmar-Raible K, Jékely G, Guy K, Raible F, Wittbrodt J, Arendt D. (2005). Ancestry of Photic and Mechanic Sensation? Science. 308(5725):1113-1114.

Abstract

Henrich T, Ramialison M, Wittbrodt B, Assouline B, Bourrat F, Berger A, Himmelbauer H, Sasaki T, Shimizu N, Westerfield M, Kondoh H, Wittbrodt J. (2005). MEPD: a resource for medaka gene expression patterns. Bioinformatics.21(14):3195-7.

Abstract

The Medaka Expression Pattern Database (MEPD) is a database for gene expression patterns determined by in situ hybridization in the small freshwater fish medaka (Oryzias latipes). Data have been collected from various research groups and MEPD is developing into a central expression pattern depository within the medaka community. Gene expression patterns are described by images and terms of a detailed medaka anatomy ontology of over 4000 terms, which we have developed for this purpose and submitted to Open Biological Ontologies. Sequences have been annotated via BLAST match results and using Gene Ontology terms. These new features will facilitate data analyses using bioinformatics approaches and allow cross-species comparisons of gene expression patterns. Presently, MEPD has 19,757 entries, for 1024 of them the expression pattern has been determined.

Pubmed 

Del Bene F, Wittbrodt J. (2005). Cell cycle control by homeobox genes in development and disease. Semin Cell Dev Biol. 16(3):449-60.

Abstract

Homeobox-containing transcription factors are among the most studied players during normal embryonic development. In the last few years, they have also been shown to act as modulators of cell proliferation. In the present review, we highlight recent studies that demonstrated the influences of homeobox genes on crucial components of the cell cycle machinery. Homeobox genes may have direct or indirect influence on their transcription levels but can also interact with those components via protein-protein interaction. In many cases, these studies were done in the context of pathological states like cancer as well as during vertebrate development.

Pubmed 

Schäfer M, Rembold M, Wittbrodt J, Schartl M, Winkler C. (2005). Medial floor plate formation in zebrafish consists of two phases and requires trunk-derived Midkine-a. Genes Dev. 19(8):897-902.

Abstract

The medial floor plate (MFP) organizes the specification of neurons and outgrowth of axons in the ventral spinal cord of vertebrates. We show that the growth factor Midkine-a, expressed in the paraxial mesoderm, is required for formation of the MFP in zebrafish. Our epistatic analyses demonstrate that development of MFP comprises two independent sequential phases. Following initial MFP induction in the gastrula organizer, Midkine-a regulates allocation of MFP cells during subsequent development. Thus in zebrafish, trunk-derived signals are required for complete MFP formation from a common pool of organizer-derived midline precursor cells.

Pubmed 

Martinez-Morales JR, Del Bene F, Nica G, Hammerschmidt M, Bovolenta P, Wittbrodt J. (2005). Differentiation of the vertebrate retina is coordinated by an FGF signaling center. Dev Cell. 8(4):565-74.

Abstract

In vertebrates, midline-derived sonic hedgehog and nodal are crucial for the initial proximal-distal patterning of the eye. The establishment of the distal optic stalk is in turn a prerequisite to initiate retinogenesis. However, the signal that activates this process is unknown. Here, we demonstrate that in both chick and fish, the initiation of retinal differentiation is triggered by a species-specific localized Fgf signaling center that acts as mediator of the midline signals. The concerted activity of Fgf8 and Fgf3 is both necessary and sufficient to coordinate retinal differentiation independent of the connecting optic stalk.

Pubmed 

Grabher C(1), Joly JS, Wittbrodt J. ( 65. Methods Cell Biol. 2004;77:381-401. ). Highly efficient zebrafish transgenesis mediated by the meganuclease I-SceI. 65. Methods Cell Biol. 2004;77:381-401.
Abstract
PMID: 15602923 [PubMed - indexed for MEDLINE]
Schartl M(1), Nanda I, Kondo M, Schmid M, Asakawa S, Sasaki T, Shimizu N, Henrich T, Wittbrodt J, Furutani-Seiki M, Kondoh H, Himmelbauer H, Hong Y, Koga A, Nonaka M, Mitani H, Shima A. ( 66. Methods Cell Biol. 2004;77:173-99. ). Current status of medaka genetics and genomics. The Medaka Genome Initiative (MGI). 66. Methods Cell Biol. 2004;77:173-99.
Abstract
PMID: 15602912 [PubMed - indexed for MEDLINE]
Arendt D(1), Tessmar-Raible K, Snyman H, Dorresteijn AW, Wittbrodt J. (2004). Ciliary photoreceptors with a vertebrate-type opsin in an invertebrate brain. Science. 306(5697):869-71.
Abstract
Comment in Science. 2005 May 20;308(5725):1113-4; author reply 1113-4. Science. 2004 Oct 29;306(5697):796-7.
Pubmed 
Grabher C(1), Wittbrodt J. (2004). Efficient activation of gene expression using a heat-shock inducible Gal4/Vp16-UAS system in medaka. BMC Biotechnol. 4:26.
Abstract
BACKGROUND: Genetic interference by DNA, mRNA or morpholino injection is a widely used approach to study gene function in developmental biology. However, the lack of temporal control over the activity of interfering molecules often hampers investigation of gene function required during later stages of embryogenesis. To elucidate the roles of genes during embryogenesis a precise temporal control of transgene expression levels in the developing organism is on demand. RESULTS: We have generated a transgenic Gal4/Vp16 activator line that is heat-shock inducible, thereby providing a tool to drive the expression of specific effector genes via Gal4/Vp16. Merging the Gal4/Vp16-UAS system with the I-SceI meganuclease and the Sleeping Beauty transposon system allows inducible gene expression in an entirely uniform manner without the need to generate transgenic effector lines. Combination of this system with fluorescent protein reporters furthermore facilitates the direct visualization of transgene expressing cells in live embryos. CONCLUSION: The combinatorial properties of this expression system provide a powerful tool for the analysis of gene function during embryonic and larval development in fish by ectopic expression of gene products.
Huisken J(1), Swoger J, Del Bene F, Wittbrodt J, Stelzer EH. (2004). Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science. 305(5686):1007-9.
Abstract
Large, living biological specimens present challenges to existing optical imaging techniques because of their absorptive and scattering properties. We developed selective plane illumination microscopy (SPIM) to generate multidimensional images of samples up to a few millimeters in size. The system combines two-dimensional illumination with orthogonal camera-based detection to achieve high-resolution, optically sectioned imaging throughout the sample, with minimal photodamage and at speeds capable of capturing transient biological phenomena. We used SPIM to visualize all muscles in vivo in the transgenic Medaka line Arnie, which expresses green fluorescent protein in muscle tissue. We also demonstrate that SPIM can be applied to visualize the embryogenesis of the relatively opaque Drosophila melanogaster in vivo.
Quiring R(1), Wittbrodt B, Henrich T, Ramialison M, Burgtorf C, Lehrach H, Wittbrodt J. (2004). Large-scale expression screening by automated whole-mount in situ hybridization. Mech Dev. 121(7-8):971-6.
Abstract
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.
Rembold M(1), Wittbrodt J. (2004). In vivo time-lapse imaging in medaka--n-heptanol blocks contractile rhythmical movements. Mech Dev. 121(7-8):965-70.
Abstract
Medaka is an ideal model system for developmental studies as it combines the advantages of powerful genetics and classical embryology. Due to the accessibility, transparency and fast development, embryogenesis and morphogenesis can be followed in vivo. Microscopic time-lapse imaging, however, requires the immobilization of the object to be observed. In medaka rhythmical contractile movements of the blastoderm during early development hampered time-lapse studies, as they cause the embryo to rotate vividly. Here we show that the contractile movements can be reduced by continuous treatment with the gap-junction uncoupling agent n-heptanol up to the 12-somite stage (stage 23) without interfering with development. This allows for the first time to perform high-resolution time-lapse studies in medaka.
Henrich T(1), Ramialison M, Segerdell E, Westerfield M, Furutani-Seiki M, Wittbrodt J, Kondoh H. (2004). GSD: a genetic screen database. Mech Dev. 121(7-8):959-63.
Abstract
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.
Khorasani MZ(1), Hennig S, Imre G, Asakawa S, Palczewski S, Berger A, Hori H, Naruse K, Mitani H, Shima A, Lehrach H, Wittbrodt J, Kondoh H, Shimizu N, Himmelbauer H. (2004). A first generation physical map of the medaka genome in BACs essential for positional cloning and clone-by-clone based genomic sequencing. Mech Dev. 121(7-8):903-13.
Abstract
In order to realize the full potential of the medaka as a model system for developmental biology and genetics, characterized genomic resources need to be established, culminating in the sequence of the medaka genome. To facilitate the map-based cloning of genes underlying induced mutations and to provide templates for clone-based genomic sequencing, we have created a first-generation physical map of the medaka genome in bacterial artificial chromosome (BAC) clones. In particular, we exploited the synteny to the closely related genome of the pufferfish, Takifugu rubripes, by marker content mapping. As a first step, we clustered 103,144 public medaka EST sequences to obtain a set of 21,121 non-redundant sequence entities. Avoiding oversampling of gene-dense regions, 11,254 of EST clusters were successfully matched against the draft sequence of the fugu genome, and 2363 genes were selected for the BAC map project. We designed 35mer oligonucleotide probes from the selected genes and hybridized them against 64,500 BAC clones of strains Cab and Hd-rR, representing 14-fold coverage of the medaka genome. Our data set is further supplemented with 437 results generated from PCR-amplified inserts of medaka cDNA clones and BAC end-fragment markers. Our current, edited, first generation medaka BAC map consists of 902 map segments that cover about 74% of the medaka genome. The map contains 2721 markers. Of these, 2534 are from expressed sequences, equivalent to a non-redundant set of 2328 loci. The 934 markers (724 different) are anchored to the medaka genetic map. Thus, genetic map assignments provide immediate access to underlying clones and contigs, simplifying molecular access to candidate gene regions and their characterization.
Watanabe T(1), Asaka S, Kitagawa D, Saito K, Kurashige R, Sasado T, Morinaga C, Suwa H, Niwa K, Henrich T, Hirose Y, Yasuoka A, Yoda H, Deguchi T, Iwanami N, Kunimatsu S, Osakada M, Loosli F, Quiring R, Carl M, Grabher C, Winkler S, Del Bene F, Wittbrodt J, Abe K, Takahama Y, Takahashi K, Katada T, Nishina H, Kondoh H, Furutani-Seiki M. (2004). Mutations affecting liver development and function in Medaka, Oryzias latipes, screened by multiple criteria. Mech Dev. 121(7-8):791-802.
Abstract
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.
Yoda H(1), Hirose Y, Yasuoka A, Sasado T, Morinaga C, Deguchi T, Henrich T, Iwanami N, Watanabe T, Osakada M, Kunimatsu S, Wittbrodt J, Suwa H, Niwa K, Okamoto Y, Yamanaka T, Kondoh H, Furutani-Seiki M. (2004). Mutations affecting retinotectal axonal pathfinding in Medaka, Oryzias latipes. Mech Dev. 121(7-8):715-28.
Abstract
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.
Loosli F(1), Del Bene F, Quiring R, Rembold M, Martinez-Morales JR, Carl M, Grabher C, Iquel C, Krone A, Wittbrodt B, Winkler S, Sasado T, Morinaga C, Suwa H, Niwa K, Henrich T, Deguchi T, Hirose Y, Iwanami N, Kunimatsu S, Osakada M, Watanabe T, Yasuoka A, Yoda H, Winkler C, Elmasri H, Kondoh H, Furutani-Seiki M, Wittbrodt J. (2004). Mutations affecting retina development in Medaka. Mech Dev. 121(7-8):703-14.
Abstract
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.
Kitagawa D(1), Watanabe T, Saito K, Asaka S, Sasado T, Morinaga C, Suwa H, Niwa K, Yasuoka A, Deguchi T, Yoda H, Hirose Y, Henrich T, Iwanami N, Kunimatsu S, Osakada M, Winkler C, Elmasri H, Wittbrodt J, Loosli F, Quiring R, Carl M, Grabher C, Winkler S, Del Bene F, Momoi A, Katada T, Nishina H, Kondoh H, Furutani-Seiki M. (2004). Genetic dissection of the formation of the forebrain in Medaka, Oryzias latipes. Mech Dev. 121(7-8):673-85.
Abstract
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.
Elmasri H(1), Winkler C, Liedtke D, Sasado T, Morinaga C, Suwa H, Niwa K, Henrich T, Hirose Y, Yasuoka A, Yoda H, Watanabe T, Deguchi T, Iwanami N, Kunimatsu S, Osakada M, Loosli F, Quiring R, Carl M, Grabher C, Winkler S, Del Bene F, Wittbrodt J, Abe K, Takahama Y, Takahashi K, Katada T, Nishina H, Kondoh H, Furutani-Seiki M. (2004). Mutations affecting somite formation in the Medaka (Oryzias latipes). Mech Dev. 121(7-8):659-71.
Abstract
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.
Furutani-Seiki M(1), Sasado T, Morinaga C, Suwa H, Niwa K, Yoda H, Deguchi T, Hirose Y, Yasuoka A, Henrich T, Watanabe T, Iwanami N, Kitagawa D, Saito K, Asaka S, Osakada M, Kunimatsu S, Momoi A, Elmasri H, Winkler C, Ramialison M, Loosli F, Quiring R, Carl M, Grabher C, Winkler S, Del Bene F, Shinomiya A, Kota Y, Yamanaka T, Okamoto Y, Takahashi K, Todo T, Abe K, Takahama Y, Tanaka M, Mitani H, Katada T, Nishina H, Nakajima N, Wittbrodt J, Kondoh H. (2004). A systematic genome-wide screen for mutations affecting organogenesis in Medaka, Oryzias latipes. Mech Dev. 121(7-8):647-58.
Abstract
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.
Furutani-Seiki M(1), Wittbrodt J. (2004). Medaka and zebrafish, an evolutionary twin study. Mech Dev. 121(7-8):629-37.
Abstract
Comparison of two related species is one of the most successful approaches to decipher general genetic principles in eukaryotes. This is best illustrated in yeast, where the model systems Saccharomyyces. cervisiae and Schizosaccharomyces. pombe have been examined. Powerful forward genetics in both species, species-specific differences in biological features and the phylogenetic distance between the two species, make them well suited for a comparative approach. Recent whole genome sequencing has also facilitated comparative genomics of these simple eukaryotes. It is now possible to go a step further using higher eukaryotes. A duplication of the genome at the base of the teleost radiation, facilitated evolution of almost 25,000 fish species, more than half of all vertebrate species together. Two teleost genetic model systems have emerged in the past few decades: zebrafish, in which large-scale mutagenesis has been successfully performed, and Medaka, a Japanese killifish with a century of history in genetics and now, as reported in this issue, many induced mutations. In this review we will illustrate how comparison of these two model species, Medaka and zebrafish, can reveal conserved and species-specific genetic and molecular mechanisms underlying vertebrate development.
Poggi L(1), Vottari T, Barsacchi G, Wittbrodt J, Vignali R. (2004). The homeobox gene Xbh1 cooperates with proneural genes to specify ganglion cell fate within the Xenopus neural retina. Development. 131(10):2305-15.
Abstract
Recent studies on vertebrate eye development have focused on the molecular mechanisms of specification of different retinal cell types during development. Only a limited number of genes involved in this process has been identified. In Drosophila, BarH genes are necessary for the correct specification of R1/R6 eye photoreceptors. Vertebrate Bar homologues have been identified and are expressed in vertebrate retinal ganglion cells during differentiation; however, their retinal function has not yet been addressed. In this study, we report on the role of the Xenopus Bar homologue Xbh1 in retinal ganglion cell development and its interaction with the proneural genes Xath5 and Xath3, whose ability to promote ganglion cell fate has been demonstrated. We show that XHB1 plays a crucial role in retinal cell determination, acting as a switch towards ganglion cell fate. Detailed expression analysis, animal cap assays and in vivo lipofection assays, indicate that Xbh1 acts as a late transcriptional repressor downstream of the atonal genes Xath3 and Xath5. However, the action of Xbh1 on ganglion cell development is different and more specific than that of the Xath genes, and accounts for only a part of their activities during retinogenesis.
Martinez-Morales JR(1), Naruse K, Mitani H, Shima A, Wittbrodt J. (2004). Rapid chromosomal assignment of medaka mutants by bulked segregant analysis. Gene. 329:159-65.
Abstract
Genetic screens in medaka are leading to the identification of an increasing number of unique mutant phenotypes. However, so far only a few genes responsible for these phenotypes have been characterized. Furthermore, no protocols using a systematic positional cloning strategy have been developed to determine the implicated genes. The PCR-based bulked segregant analysis is a fast and reliable tool to accomplish the initial steps of the positional cloning of a mutation. Here we describe the selection of a panel of genetic markers that, evenly distributed over the 24 chromosomes of medaka, provide a full coverage of the compact medaka genome (800 Mb) when used in bulked segregant analysis. The reference panel, which consists of 48 EST-derived markers, is anchored to a collection of more than 2000 polymorphic markers, thus facilitating a rapid transition from chromosomal assignment to fine mapping of the mutants. More importantly, since most of the genetic screens have been performed in the inbred Cab strain (derived from the Southern population), the selection of markers included in this panel was intended to optimize the recognition of polymorphisms between Cab and the polymorphic inbred mapping strain Kaga. Here we present a reliable mapping panel, confirmed both by the assignment of the locus responsible for the medaka mutation eyeless/Rx3 to chromosome 12, and by the analysis of its resolution power using representative markers.
Del Bene F(1), Tessmar-Raible K, Wittbrodt J. (2004). Direct interaction of geminin and Six3 in eye development. Nature. 427(6976):745-9.
Abstract
Comment in Nature. 2004 Feb 19;427(6976):687-8.
Pubmed 
Grabher C(1), Henrich T, Sasado T, Arenz A, Wittbrodt J, Furutani-Seiki M. (2003). Transposon-mediated enhancer trapping in medaka. Gene. 322:57-66.
Abstract
Erratum in Gene. 2004 Mar 3;327(2):239.
Pubmed 
Prud'homme B(1), de Rosa R, Arendt D, Julien JF, Pajaziti R, Dorresteijn AW, Adoutte A, Wittbrodt J, Balavoine G. (2003). Arthropod-like expression patterns of engrailed and wingless in the annelid Platynereis dumerilii suggest a role in segment formation. Curr Biol. 13(21):1876-81.
Abstract
The origin of animal segmentation, the periodic repetition of anatomical structures along the anteroposterior axis, is a long-standing issue that has been recently revived by comparative developmental genetics. In particular, a similar extensive morphological segmentation (or metamerism) is commonly recognized in annelids and arthropods. Mostly based on this supposedly homologous segmentation, these phyla have been united for a long time into the clade Articulata. However, recent phylogenetic analysis dismissed the Articulata and thus challenged the segmentation homology hypothesis. Here, we report the expression patterns of genes orthologous to the arthropod segmentation genes engrailed and wingless in the annelid Platynereis dumerilii. In Platynereis, engrailed and wingless are expressed in continuous ectodermal stripes on either side of the segmental boundary before, during, and after its formation; this expression pattern suggests that these genes are involved in segment formation. The striking similarities of engrailed and wingless expressions in Platynereis and arthropods may be due to evolutionary convergence or common heritage. In agreement with similarities in segment ontogeny and morphological organization in arthropods and annelids, we interpret our results as molecular evidence of a segmented ancestor of protostomes.
Loosli F(1), Staub W, Finger-Baier KC, Ober EA, Verkade H, Wittbrodt J, Baier H. (2003). Loss of eyes in zebrafish caused by mutation of chokh/rx3. EMBO Rep. 4(9):894-9.
Abstract
The vertebrate eye forms by specification of the retina anlage and subsequent morphogenesis of the optic vesicles, from which the neural retina differentiates. chokh (chk) mutant zebrafish lack eyes from the earliest stages in development. Marker gene analysis indicates that retinal fate is specified normally, but optic vesicle evagination and neuronal differentiation are blocked. We show that the chk gene encodes the homeodomain-containing transcription factor, Rx3. Loss of Rx3 function in another teleost,medaka, has also been shown to result in an eyeless phenotype. The medaka rx3 locus can fully rescue the zebrafish mutant phenotype. We provide evidence that the regulation of rx3 is evolutionarily conserved, whereas the downstream cascade contains significant differences in gene regulation. Thus, these mutations in orthologous genes allow us to study the evolution of vertebrate eye development at the molecular level.
Hammerschmidt M(1), Kramer C, Nowak M, Herzog W, Wittbrodt J. (2003). Loss of maternal Smad5 in zebrafish embryos affects patterning and morphogenesis of optic primordia. Dev Dyn. 227(1):128-33.
Abstract
The mechanisms of patterning and morphogenesis of vertebrate eye primordia are heavily debated. Taking advantage of the maternal effect of a zebrafish smad5 null mutation (Mm169), we investigate the effect of early signaling by members of the bone morphogenetic proteins (Bmps) on eye field patterning and optic vesicle morphogenesis. In contrast to previous Xenopus and chick studies demonstrating a late dorsalizing effect of Bmp4 expressed in the dorsal neural retina itself, we show that patterning of the eye primordia already starts during blastula and early gastrula stages. At these stages, bmps expressed on the ventrolateral side of the embryo promote dorso-distal fates in the entire neuroectoderm, including the eye primordia. Despite a normal split of the eye field in the midline, the eye primordia of Mm169 embryos fail to evaginate laterally. They display a concentric pattern with retinal cells in the center and optic stalk cells in the periphery, representing a flattened version of the topologic relationships present in the mature wild-type eye. Different interpretations of these latter findings are presented. They can be best explained with a model according to which zebrafish eye morphogenesis occurs as a telescopic extension of disc-like, concentric primordia, similar to the development of appendages from imaginal discs in Drosophila.
Pubmed 
Shima A(1), Himmelbauer H, Mitani H, Furutani-Seiki M, Wittbrodt J, Schartl M. (2003). Fish genomes flying. Symposium on Medaka Genomics. EMBO Rep. 4(2):121-5.
Abstract
PMCID: PMC1315840 PMID: 12612598 [PubMed - indexed for MEDLINE]
Henrich T(1), Ramialison M, Quiring R, Wittbrodt B, Furutani-Seiki M, Wittbrodt J, Kondoh H; Medaka Expression Pattern Database. (2003). MEPD: a Medaka gene expression pattern database. Nucleic Acids Res. 31(1):72-4.
Abstract
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.
López-Ríos J(1), Tessmar K, Loosli F, Wittbrodt J, Bovolenta P. (2003). Six3 and Six6 activity is modulated by members of the groucho family. Development. 130(1):185-95.
Abstract
Six3 and Six6 are two genes required for the specification and proliferation of the eye field in vertebrate embryos, suggesting that they might be the functional counterparts of the Drosophila gene sine oculis (so). Phylogenetic and functional analysis have however challenged this idea, raising the possibility that the molecular network in which Six3 and Six6 act may be different from that described for SO. To address this, we have performed yeast two-hybrid screens, using either Six3 or Six6 as a bait. In this paper, we report the results of the latter screen that led to the identification of TLE1 (a transcriptional repressor of the groucho family) and AES (a potential dominant negative form of TLE proteins) as cofactors for both SIX6 and SIX3. Biochemical and mutational analysis shows that the Six domain of both SIX3 and SIX6 strongly interact with the QD domain of TLE1 and AES, but that SIX3 also interacts with TLE proteins via the WDR domain. Tle1 and Aes are expressed in the developing eye of medaka fish (Oryzias latipes) embryos, overlapping with the distribution of both Six3 and Six6. Gain-of-function studies in medaka show a clear synergistic activity between SIX3/SIX6 and TLE1, which, on its own, can expand the eye field. Conversely, AES alone decreases the eye size and abrogates the phenotypic consequences of SIX3/6 over-expression. These data indicate that both Tle1 and Aes participate in the molecular network that control eye development and are consistent with the view that both Six3 and Six6 act in combination with either Tle1 and/or Aes.
Thermes V(1), Grabher C, Ristoratore F, Bourrat F, Choulika A, Wittbrodt J, Joly JS. (2002). I-SceI meganuclease mediates highly efficient transgenesis in fish. Mech Dev. 118(1-2):91-8.
Abstract
Erratum in Mech Dev. 2003 Feb;120(2):267.
Pubmed 
Tessmar K(1), Loosli F, Wittbrodt J. (2002). A screen for co-factors of Six3. Mech Dev. 117(1-2):103-13.
Abstract
The vertebrate Six3 gene a homeobox gene of the Six-family, plays a crucial role in early eye and forebrain development. Here we report the isolation of candidate factors that interact with Six3 in a yeast two-hybrid screen. Among these are two basic helix loop helix (bHLH) domain containing proteins. Biochemical analysis reveals that the bHLH proteins ATH5, ATH3, NEUROD as well as ASH1 interact specifically with XSix3. By defining the interacting domains we show that the bHLH domain of NEUROD interacts with the SIX domain of XSix3. The co-expression of the interacting molecules during late retina determination/differentiation suggests a new role for Six3 and the respective interaction partner also in these late steps of eye development.
Pubmed 
Poggi L(1), Carl M, Vignali R, Barsacchi G, Wittbrodt J. (2002). Expression of a medaka (Oryzias latipes) Bar homologue in the differentiating central nervous system and retina. Mech Dev. 114(1-2):193-6.
Abstract
The Bar homeobox genes play an essential role during nervous system and eye development in Drosophila. We isolated a medaka Bar gene closely related to the Drosophila and mammalian Bar genes. In the medaka embryo, OlBar is expressed from gastrula stages onwards, in a region demarcating the presumptive prosencephalic-mesencephalic boundary. Later in development, OlBar transcripts are found in populations of differentiating neuronal cells of the brain and the retina.
Pubmed 
Carl M(1), Loosli F, Wittbrodt J. (2002). Six3 inactivation reveals its essential role for the formation and patterning of the vertebrate eye. Development. 129(17):4057-63.
Abstract
The establishment of retinal identity and the subsequent patterning of the optic vesicle are the key steps in early vertebrate eye development. To date little is known about the nature and interaction of the genes controlling these steps. So far few genes have been identified that, when over-expressed, can initiate ectopic eye formation. Of note is Six3, which is expressed exclusively in the anterior neural plate. However, 'loss of function' analysis has not been reported. Using medaka fish, we show that vertebrate Six3 is necessary for patterning of the anterior neuroectoderm including the retina anlage. Inactivation of Six3 function by morpholino knock-down results in the lack of forebrain and eyes. Corroborated by gain-of-function experiments, graded interference reveals an additional role of Six3 in the proximodistal patterning of the optic vesicle. During both processes of vertebrate eye formation, Six3 cooperates with Pax6.
Arendt D(1), Tessmar K, de Campos-Baptista MI, Dorresteijn A, Wittbrodt J. (2002). Development of pigment-cup eyes in the polychaete Platynereis dumerilii and evolutionary conservation of larval eyes in Bilateria. Development. 129(5):1143-54.
Abstract
The role of Pax6 in eye development in insects and vertebrates supports the view that their eyes evolved from simple pigment-cup ocelli present in their last common ancestors (Urbilateria). The cerebral eyes in errant polychaetes represent prototype invertebrate pigment-cup ocelli and thus resemble the presumed ancestral eyes. We have analysed expression of conserved eye specification genes in the early development of larval and adult pigment-cup eyes in Platynereis dumerilii (Polychaeta, Annelida, Lophotrochozoa). Both larval and adult eyes form in close vicinity of the optic anlagen on both sides of the developing brain ganglia. While pax6 is expressed in the larval, but not in the developing, adult eyes, expression of six1/2 from trochophora stages onwards specifically outlines the optic anlagen and thus covers both the developing larval and adult eyes. Using Platynereis rhabdomeric opsin as differentiation marker, we show that the first pair of adult eye photoreceptor cells is detected within bilateral clusters that transitorily express ath, the Platynereis atonal orthologue, thus resembling proneural sensory clusters. Our data indicate that--similar to insects, but different from the vertebrates--polychaete six1/2 expression outlines the entire visual system from early developmental stages onwards and ath-positive clusters generate the first photoreceptor cells to appear. We propose that pax6-, six1/2- and ath-positive larval eyes, as found in today's trochophora, were present already in Urbilateria.
Loosli F(1), Mardon G, Wittbrodt J. (2002). Cloning and expression of medaka Dachshund. Mech Dev. 112(1-2):203-6.
Abstract
The nuclear factor dachshund (dac) is a regulator of retinal cell fate determination in Drosophila. We have cloned a Dachshund homologue of a lower vertebrate, the teleost medaka (Oryzias latipes). Sequence comparison reveals high similarity of medaka Dachshund (OlDach) to the known homologues in higher vertebrates and Drosophila. OlDach is first expressed at early somitogenesis stages in the otic placode territory and forming somites. Subsequently, expression is detected in the retina, specific regions of the central nervous system, pancreas and the finbuds.
Wittbrodt J(1), Shima A, Schartl M. (2002). Medaka--a model organism from the far East. Nat Rev Genet. 3(1):53-64.
Abstract
Genome sequencing has yielded a plethora of new genes the function of which can be unravelled through comparative genomic approaches. Increasingly, developmental biologists are turning to fish as model genetic systems because they are amenable to studies of gene function. Zebrafish has already secured its place as a model vertebrate and now its Far Eastern cousin--medaka--is emerging as an important model fish, because of recent additions to the genetic toolkit available for this organism. Already, the popularity of medaka among developmental biologists has led to important insights into vertebrate development.
Loosli F(1), Winkler S, Burgtorf C, Wurmbach E, Ansorge W, Henrich T, Grabher C, Arendt D, Carl M, Krone A, Grzebisz E, Wittbrodt J. (2001). Medaka eyeless is the key factor linking retinal determination and eye growth. Development. 128(20):4035-44.
Abstract
The complete absence of eyes in the medaka fish mutation eyeless is the result of defective optic vesicle evagination. We show that the eyeless mutation is caused by an intronic insertion in the Rx3 homeobox gene resulting in a transcriptional repression of the locus that is rescued by injection of plasmid DNA containing the wild-type locus. Functional analysis reveals that Six3- and Pax6- dependent retina determination does not require Rx3. However, gain- and loss-of-function phenotypes show that Rx3 is indispensable to initiate optic vesicle evagination and to control vesicle proliferation, by that regulating organ size. Thus, Rx3 acts at a key position coupling the determination with subsequent morphogenesis and differentiation of the developing eye.
Arendt D(1), Wittbrodt J. (2001). Reconstructing the eyes of Urbilateria. Philos Trans R Soc Lond B Biol Sci. 356(1414):1545-63.
Abstract
The shared roles of Pax6 and Six homologues in the eye development of various bilaterians suggest that Urbilateria, the common ancestors of all Bilateria, already possessed some simple form of eyes. Here, we re-address the homology of bilaterian cerebral eyes at the level of eye anatomy, of eye-constituting cell types and of phototransductory molecules. The most widespread eye type found in Bilateria are the larval pigment-cup eyes located to the left and right of the apical organ in primary, ciliary larvae of Protostomia and Deuterostomia. They can be as simple as comprising a single pigment cell and a single photoreceptor cell in inverse orientation. Another more elaborate type of cerebral pigment-cup eyes with an everse arrangement of photoreceptor cells is found in adult Protostomia. Both inverse larval and everse adult eyes employ rhabdomeric photoreceptor cells and thus differ from the chordate cerebral eyes with ciliary photoreceptors. This is highly significant because on the molecular level we find that for phototransduction rhabdomeric versus ciliary photoreceptor cells employ divergent rhodopsins and non-orthologous G-proteins, rhodopsin kinases and arrestins. Our comparison supports homology of cerebral eyes in Protostomia; it challenges, however, homology of chordate and non-chordate cerebral eyes that employ photoreceptor cells with non-orthologous phototransductory cascades.
Adamska M(1), Wolff A, Kreusler M, Wittbrodt J, Braun T, Bober E. (2001). Five Nkx5 genes show differential expression patterns in anlagen of sensory organs in medaka: insight into the evolution of the gene family. Dev Genes Evol. 211(7):338-49.
Abstract
We report the identification and characterisation of five different Nkx5-related genes in medaka fish (Oryzias latipes). They constitute homologues of genes previously isolated in higher vertebrates, Nkx5--1, Nkx5--2, Hmx1/Nkx5--3 and SOHo-1, and were named accordingly: OlNkx5--1.1, OlNkx5--2, OlNkx5--3 and OlSOHo. For the Nkx5--1 gene a new, second homologue, OlNkx5--1.2, was isolated. In medaka, Nkx5 and SOHo genes are differentially expressed in three developing sensory organs: eye, ear and lateral line and later in defined brain regions. Phylogenetic analyses of the entire Nkx5 family revealed that four paralogous Nkx5 groups, Nkx5--1, Nkx5--2, Hmx1/Nkx5--3/GH6 and SOHo, are present in vertebrates. Only some of the Nkx5 family members have been identified in singular vertebrate species so far. Here we present, for the first time, the isolation of representatives of each Nkx5 subgroup in one species, the medaka fish. Based on similarities in sequence and expression patterns, and genomic organisation we propose a model of the evolutionary history of the Nkx5 family. The model predicts that the four vertebrate Nkx5 genes arose by a tandem duplication, followed by chromosomal duplication. The two Nkx5--1 genes identified so far exclusively in medaka most probably result from an additional genome duplication in the fish lineage.

Loosli F(1), Köster RW, Carl M, Kühnlein R, Henrich T, Mücke M, Krone A, Wittbrodt J. (2000). A genetic screen for mutations affecting embryonic development in medaka fish (Oryzias latipes). Mech Dev. 97(1-2):133-9.

Abstract
In a pilot screen, we assayed the efficiency of ethylnitrosourea (ENU) as a chemical mutagen to induce mutations that lead to early embryonic and larval lethal phenotypes in the Japanese medaka fish, Oryzias latipes. ENU acts as a very efficient mutagen inducing mutations at high rates in germ cells. Three repeated treatments of male fish in 3 mM ENU for 1 h results in locus specific mutation rates of 1.1-1.95 x10(-3). Mutagenized males were outcrossed to wild type females and the F1 offspring was used to establish F2 families. F2 siblings were intercrossed and the F3 progeny was scored 24, 48 and 72 h after fertilization for morphological alterations affecting eye development. The presented mutant phenotypes were identified using morphological criteria and occur during early developmental stages of medaka. They are stably inherited in a Mendelian fashion. The high efficiency of ENU to induce mutations in this pilot screen indicates that chemical mutagenesis and screening for morphologically visible phenotypes in medaka fish allows the genetic analysis of specific aspects of vertebrate development complementing the screens performed in other vertebrate model systems.
Köster RW(1), Kühnlein RP, Wittbrodt J. (2000). Ectopic Sox3 activity elicits sensory placode formation. Mech Dev. 95(1-2):175-87.
Abstract
The induction of sensory organ placodes, in particular the lens placode, represents the paradigm for induction. We show that medaka Sox3 is expressed in the neuroectoderm and in the placodes of all sensory organs prior to placode formation and subsequently in placode-derived tissues. Ectopic Sox3 expression leads to ectopic expression of Pax6 and Eya1 in embryonic ectoderm and causes ectopic lens and otic vesicle formation. The descendants of cells ectopically expressing Sox3-mRNA contribute to ectopic lens tissue. This suggests a permissive role for Sox3 in establishing a placodal competence. In addition, ectopic Sox3 expression leads to the dysgenesis of the endogenous sensory organs. Both effects of ectopic Sox3 expression can be separated by ectopic expression of a truncated Sox3 variant depending on its expression level. Our data suggests that Sox3 is a permissive factor for sensory placode formation and plays an important role in sensory organ development.
Henrich T(1), Wittbrodt J. (2000). An in situ hybridization screen for the rapid isolation of differentially expressed genes. Dev Genes Evol. 210(1):28-33.
Abstract
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 cells. Of the nonhybridizing clones 153 were chosen randomly and further analyzed by whole-mount in situ hybridization. There were 67 selected clones differentially expressed in the developing embryos, and 48 of these were expressed in the developing head. Differentially expressed genes were either of novel type or showed homology to known genes containing DNA binding motifs or to putative housekeeping genes.
Loosli F(1), Winkler S, Wittbrodt J. (1999). Six3 overexpression initiates the formation of ectopic retina. Genes Dev. 13(6):649-54.
Abstract
The homeobox gene sine oculis (so) is essential for visual system formation in Drosophila. A vertebrate member of the so/Six gene family, Six3, is expressed in the developing eye and forebrain. Injection of Six3 RNA into medaka fish embryos causes ectopic Pax6 and Rx2 expression in midbrain and cerebellum, resulting in the formation of ectopic retinal primordia. Injected mouse Six3 RNA initiates ectopic expression of endogenous medaka Six3, uncovering a feedback control of Six3 expression. Initiation of ectopic retina formation reveals a pivotal role for Six3 in vertebrate retina development and hints at a conserved regulatory network underlying vertebrate and invertebrate eye development.
Loosli F(1), Köster RW, Carl M, Krone A, Wittbrodt J. (1998). Six3, a medaka homologue of the Drosophila homeobox gene sine oculis is expressed in the anterior embryonic shield and the developing eye. Mech Dev. 74(1-2):159-64.
Abstract
The conserved transcription factor Pax6 is essential for eye development in Drosophila and mammals (Hill, R.E., Favor, J., Hogan, B.L.M., Ton, C.C.T., Saunders, G.F., Hanson, I.M., Prosser, J., Jordan, T., Hastie, N.D., van Heyningen, V., 1991. Mouse small eye results from mutations in a paired-like homeobox containing gene. Nature 354, 522-525; Ton, C., Hirvonen, H., Miwa, H., Weil, M., Monaghan, P., Jordan, T., van Heyningen, V., Hastie, N., Meijers-Heijboer, H., Drechsler, M., Royer-Pokora, B., Collins, F., Swaroop, A., Strong, L.C., Saunders, G.F., 1991. Positional cloning and characterization of a paired box- and homeobox-containing gene from the aniridia region. Cell 6, 1059-1074; Matsuo, T., Osumi-Yamashita, N., Noji, S., Ohuchi, H., Koyama, E., Myokai, F., Matsuo, N., Toniguchi, S., Dari, H., Jseki, S., Ninomiya, Y., Fujiwara, M., Watanabe, T., Eto, K., 1993. A mutation at the Pax-6 gene in rat small eye is associated with impaired migration of midbrain crest cells. Nature genet. 3, 299-304; Quiring, R., Walldorf, U., Kloter, U., Gehring, W.J., 1994. Homology of the eyeless gene of Drosophila to the small eye gene in mice and aniridia in humans. Science 265, 785-789). These findings led to the hypothesis that additional genes involved in invertebrate and vertebrate eye development are structurally and functionally conserved (Halder, G., Callaerts, P., Gehring, W.J., 1995. New perspectives on eye evolution. Curr. Opin. Gen. Dev. 5, 602-609; Quiring, R., Walldorf, U., Kloter, U., Gehring, W.J., 1994. Homology of the eyeless gene of Drosophila to the small eye gene in mice and aniridia in humans. Science 265, 785-789). Candidates for such conserved genes are the Drosophila homeobox gene sine oculis (Cheyette, B.N.R., Green, P.J., Martin, K., Garren, H., Hartenstein, V., Zipursky, S.L., 1994. The Drosophila sine oculis locus encodes a homeodomain-containing protein required for the development of the entire visual system. Neuron l2, 977-996) and its murine homologue Six3 (Oliver, G., Mailhos, A., Wehr, R., Copeland, N.G., Jenkins, N.A., Gruss, P., 1995. Six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development. Development 121, 4045-4055). sine oculis (so) is essential for the development of the larval and adult visual system (Cheyette, B.N.R., Green, P.J., Martin, K., Garren, H., Hartenstein, V., Zipursky, S.L., 1994. The Drosophila sine oculis locus encodes a homeodomain-containing protein required for the development of the entire visual system. Neuron l2, 977-996). Six3 is expressed in the anterior neural plate and optic vesicles, lens, olfactory placodes and ventral forebrain (Oliver, G., Mailhos, A., Wehr, R., Copeland, N.G., Jenkins, N.A., Gruss, P., 1995. Six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development. Development 121, 4045-4055). Overexpression of mouse Six3 gene in medaka fish embryos (Orvzias latipes) results in the formation of an ectopic lens, indicating that Six3 activity can trigger the genetic pathway leading to lens formation (Oliver, G., Loosli, F., Koster, R., Wittbrodt, J., Gruss, P., 1996. Ectopic lens induction in fish in response to the murine homeobox gene Six3. Mech. Dev. 60, 233-239). We isolated the medaka Six3 homologue and analyzed its expression pattern in the medaka embryo. It is expressed initially in the anterior embryonic shield and later in the developing eye and prosencephalon. The early localized expression of Six3 suggests a role in the regionalization of the rostral head.
Pubmed 
Koster R(1), Stick R, Loosli F, Wittbrodt J. (1997). Medaka spalt acts as a target gene of hedgehog signaling. Development. 124(16):3147-56.
Abstract
In vertebrates, pattern formation in the eye, central nervous system, somites, and limb depends on hedgehog activity, but a general target gene controlled by hedgehog in all these signaling centers has remained largely elusive. The medaka fish gene spalt encodes a zinc-finger transcription factor, which is expressed in all known hedgehog signaling centers of the embryo and in the organizer region at the midbrain-hindbrain boundary. We show that the spalt expression domains expand in response to ectopic hedgehog activity and narrow in the presence of protein kinase A activity, an antagonist of hedgehog signaling, indicating that spalt is a hedgehog target gene. Our results also suggest a signaling mechanism for anterior-posterior patterning of the vertebrate brain that controls spalt expression at the midbrain-hindbrain boundary in a protein kinase A dependent manner likely to involve an unknown member of the hedgehog family.
Oliver G(1), Loosli F, Köster R, Wittbrodt J, Gruss P. (1996). Ectopic lens induction in fish in response to the murine homeobox gene Six3. Mech Dev. 60(2):233-9.
Abstract
Recent findings show an unexpected conservation of genes involved in vertebrate and insect eye development. The Drosophila homeobox gene sine oculis is crucial for eye development. Its murine homologue, Six3 is expressed in the anterior neural plate, a region which is involved in lens induction in Xenopus. To examine whether Six3 participates in the process of eye formation, mouse Six3 was ectopically expressed in fish embryos. The results show that Six3 is sufficient to promote ectopic lens formation in the area of the otic vesicle and that retinal tissue is not a prerequisite for ectopic lens differentiation. Our findings suggest a conserved function for Six3 in metazoan eye development.
Rissi M(1), Wittbrodt J, Délot E, Naegeli M, Rosa FM. (1995). Zebrafish Radar: a new member of the TGF-beta superfamily defines dorsal regions of the neural plate and the embryonic retina. Mech Dev. 49(3):223-34.
Abstract
Proper development of metazoan embryos requires cell to cell communications. In many instances, these communications involve diffusible molecules, particularly members of the Transforming Growth Factor beta superfamily. In an effort to identify new members of this superfamily involved in the control of early zebrafish embryogenesis, we have isolated a gene, Radar, which appears to be conserved throughout vertebrate evolution and defines a new subfamily within the superfamily. Its pattern of expression suggests that Radar plays a role in the dorso-ventral polarity of the neural plate, blood islands formation, blood cells differentiation, the establishment of retinal dorso-ventral polarity and/or proper axonal retinotectal projections. Radar expression in ntl homozygous mutants indicates that notochord and hypochord development are intimately linked.
Wittbrodt J(1), Rosa FM. (1994). Disruption of mesoderm and axis formation in fish by ectopic expression of activin variants: the role of maternal activin. Genes Dev. 8(12):1448-62.
Abstract
Formation of mesoderm in Xenopus embryos is the result of an induction event in which peptides such as FGF or activins have been implicated. It was recently demonstrated, by the ectopic expression of a truncated activin receptor, that activin receptor signaling pathways are involved in the processes of mesoderm and axis formation in vivo. However, this approach does not directly address the role of activin itself nor the involvement of activins in the formation of mesoderm in embryos from other vertebrates. In addition, activins are expressed maternally as a protein component of the egg as well as transcribed zygotically, and it is not clear which of the two forms is involved in mesoderm formation. To address those three issues, we analyzed the role of activins in the development of fish embryos by generating two activin dominant-negative variants. One of the variants behaves as an inhibitor of activin protein. The second variant was found to deplete the activin pool when cotranslated with wild-type activin. Injection of RNA encoding these variants into the two-cell embryo of the small teleost fish Oryzias latipes (Japanese medaka) demonstrates that only the maternally provided activin protein is required for mesoderm and axis formation in fish in vivo.
Wittbrodt J(1), Lammers R, Malitschek B, Ullrich A, Schartl M. (1992). The Xmrk receptor tyrosine kinase is activated in Xiphophorus malignant melanoma. EMBO J. 11(11):4239-46.
Abstract
Xmrk encodes a putative transmembrane glycoprotein of the tyrosine kinase family and is a melanoma-inducing gene in Xiphophorus. We attempted to investigate the biological function of the putative Xmrk receptor by characterizing its signalling properties. Since a potential ligand for Xmrk has not yet been identified, it has been difficult to analyse the biochemical properties and biological function of this cell surface protein. In an approach towards such analyses, the Xmrk extracellular domain was replaced by the closely related ligand-binding domain sequences of the human epidermal growth factor receptor (HER) and the ligand-induced activity of the chimeric HER-Xmrk protein was examined. We show that the Xmrk protein is a functional receptor tyrosine kinase, is highly active in malignant melanoma and displays a constitutive autophosphorylation activity possibly due to an activating mutation in its extracellular or transmembrane domain. In the focus formation assay the HER-Xmrk chimera is a potent transforming protein equivalent to other tyrosine kinase oncoproteins.
Winkler C(1), Hong Y, Wittbrodt J, Schartl M. (1992). Analysis of heterologous and homologous promoters and enhancers in vitro and in vivo by gene transfer into Japanese medaka (Oryzias latipes) Xiphophorus. Mol Mar Biol Biotechnol. 1(4-5):326-37.
Abstract
Efficient expression systems are required for analysis of gene regulation and function in teleost fish. To develop such systems, a number of inducible or constitutive promoter and enhancer sequences of fish or higher vertebrate origin were tested for activity in a variety of fish cell lines and in embryos of the Japanese medaka fish (Oryzias latipes) and Xiphophorus. The activity of the different promoter-enhancer combinations were quantitated. Considerable differences were found for some constructs if tested in vitro or in vivo. Fro the data obtained, a set of expression vectors for basic research as well as for aquaculture purposes were established.
Förnzler D(1), Wittbrodt J, Schartl M. (1991). Analysis of an esterase linked to a locus involved in the regulation of the melanoma oncogene and isolation of polymorphic marker sequences in Xiphophorus. Biochem Genet. 29(11-12):509-24.
Abstract
Melanoma formation in Xiphophorus hybrids is mediated by a growth factor receptor tyrosine kinase oncogene encoded by the Tu locus. In the wild-type parental fish no tumors occur due to the activity of a locus that regulates the activity of the melanoma oncogene. Molecular identification of this regulatory locus (R) requires a precise physical map of the chromosomal region. Therefore we studied esterase isozymes in Xiphophorus, two of which have been previously reported to be linked to locus R. We confirm that ES1 is a distant marker for R (approx. 30cM), and contrary to earlier studies, we show that this isozyme is present in all species of the genus and at similar activity levels in all organs tested. ES4, which has also been reported to be linked to R, was found to be a misclassification of liver ES1. In an attempt to identify markers that bridge the large distance between ES1 and R, we have generated DNA probes which are highly polymorphic. They will be useful in finding landmarks on a physical map of the R-containing chromosomal region.
Wiesmüller L(1), Wittbrodt J, Noegel AA, Schleicher M. (1991). Purification and cDNA-derived sequence of adenylosuccinate synthetase from Dictyostelium discoideum. J Biol Chem. 266(4):2480-5.
Abstract
Adenylosuccinate synthetase (IMP:L-aspartate ligase (GDP), EC 6.3.4.4) plays an important role in purine biosynthesis catalyzing the GTP-dependent conversion of IMP to AMP. The enzyme was purified from the cytosol of Dictyostelium discoideum using GTP-agarose chromatography as the critical step. It has an apparent molecular mass of 44 kDa. Monoclonal antibodies identified several forms of the enzyme with pI values between 8.1 and 9.0. Michaelis-Menten constants (Km) were low for the nucleotide substrates IMP (Km = 30 microM) and GTP (Km = 35 microM) as compared with the value for aspartic acid (Km = 440 microM). These values are in good agreement with constants reported from other organisms. Immunological studies indicated that the protein is predominantly localized in the cytosol and only partially associated with particulate fractions. The enzyme is present throughout the developmental cycle of D. discoideum. Using monoclonal antibodies, the gene was cloned from a lambda gt11 expression library. The complete sequence represents the first reported primary structure of an eucaryotic adenylosuccinate synthetase. Southern blots hybridized with a cDNA probe demonstrate that adenylosuccinate synthetase is encoded by a single gene and contains at least one intron. The deduced amino acid sequence shows 43% identity to adenylosuccinate synthetase from Escherichia coli. Homologous regions include short sequence motifs, such as the glycine-rich loop which is typical for GTP-binding proteins.

Wittbrodt J, Erhardt W. (1989). An inexpensive and versatile computer-controlled PCR machine using a Peltier Element as a thermoelectric heat pump. Trends Genet. 5(7):202-3.

Abstract
Wittbrodt J(1), Adam D, Malitschek B, Mäueler W, Raulf F, Telling A, Robertson SM, Schartl M. (1989). Novel putative receptor tyrosine kinase encoded by the melanoma-inducing Tu locus in Xiphophorus. Nature. 341(6241):415-21.
Abstract
Malignant melanoma in Xiphophorus fish hybrids is caused by the activity of a dominant oncogene Tu. By combining genetic and molecular approaches, we have isolated the melanoma oncogene. We show that its level of expression correlates with the degree of malignancy of the tumour. The corresponding proto-oncogene is developmentally regulated. The Tu gene codes for a novel receptor tyrosine kinase which is closely related to the receptor for epidermal growth factor.
Adam D(1), Wittbrodt J, Telling A, Schartl M. (1988). RFLP for an EGF-receptor related gene associated with the melanoma oncogene locus of Xiphophorus maculatus. Nucleic Acids Res. 16(14B):7212.
Abstract
PMCID: PMC338392 PMID: 2900499 [PubMed - indexed for MEDLINE]

 


/var/www/cos/ / http://www.cos.uni-heidelberg.de/ Prof. Dr. Joachim Wittbrodt