Laboratory fish as models for embryonic development and disease

Christoph Winkler, Department of Biological Sciences

central question in Developmental Biology is how patterns form in uniform fields of apparently equivalent cells. For many decades, biologists have been fascinated by the processes that control the coordinated development from a unilateral zygote with only one axis of polarity (animalvegetal) to a complex, multi-cellular embryo with several polarities (anteriorposterior, dorso-ventral, left-right) and hundreds of different cell types. The identification of signaling cascades and molecular key players involved in these processes gave important insight into the specification of distinct germ layers and different cell types. Small laboratory fish, like zebrafish (Danio rerio ) and medaka ( Oryzias latipes ) have considerably contributed to our understanding of fundamental developmental processes. They offer a combination of descriptive and analytical strategies in experimental embryology and the power of genetic manipulations. Zebrafish and medaka

Assoc Prof Christoph Winkler

Assoc Prof Christoph Winkler received his Diploma in Biology from the University of Munich in 1989 and his Dr. rer. nat. (summa cum laude) from the University of Wuerzburg, Germany in 1994. From 1996 to 1998, he worked as a Human-Frontier-Science-Program postdoctoral fellow at the University of Washington in Seattle. He then established his own laboratory at the University of Wuerzburg, received his Habilitation in Developmental Biology in 2005 and became a member of the Faculty for Biology at the University of Wuerzburg. He has been an Ad hoc reviewer for NSF (USA), ANR (France), the German-Israeli Research Foundation and other grant funding agencies and is a member of the Editorial Board of the Journal of Zoology. Dr. Winkler joined the National University of Singapore as Associate Professor in April 2007. Research Interests Formation and patterning of the vertebrate nervous system Molecular mechanisms of neural degeneration Development of somites and bones Fish models for human disorders

Contact Information National University of Singapore Department of Biological Sciences National University of Singapore 14 Science Drive 4, Block S2, Level 4 Singapore 117543 Phone: 0065-6516 7376 Fax: 0065-6779 2486 e-mail: dbswcw@nus.edu.sg

are oviparous and can be easily maintained in large numbers in the laborator y. Their embr yos are transparent (Fig. 1) and within a few days develop into free swimming larvae. Over the last years, efficient methods for generating transgenic lines and for gene knock-down have been developed. Most notably, several largescale mutagenesis screens resulted in thousands of different mutants affecting a wide range of developmental processes. The identification of mutated loci is facilitated by the recent completion of genome sequencing projects in both fish species. Fish mutants have provided significant insight into many developmental processes and are important tools to uncover the underlying molecular networks. Furthermore, many mutants are valuable models to study the pathogenesis of several genetic diseases. During the last years, especially the zebrafish has been developed as animal model for a variety of human disorders, including cardiovascular diseases, cancer, retinal disorders and many others (1). Their unique experimental features make fish especially suited for small molecule drug screenings that open the way for identification of relevant drug targets and therapeutic approaches (2). In our laborator y, we use fish models to investigate fundamental processes of nervous system and bone development and to approach the molecular basis of different human disorders.

Figure 1 The zebrafish as model for biomedical research. Fast development of transparent embryos, short generation time and the possibility to perform genetic and drug screens make zebrafish a valuable model for basic and applied research.
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Pattern formation in the embryonic spinal cord

The spinal cord of vertebrates consists of millions of different neurons that are interconnected in a highly coordinated

fashion to mediate proper function of our ner vous system. During embr yogenesis, these neurons are formed at specific positions in the developing neural tube. Motoneurons, which innervate our muscles, are formed in the ventral part, while sensory neurons that relay sensor y input are positioned in the dorsal part of the neural tube. The positioning of distinct neuron classes along the dorsoventral axis of the Figure 2 Midkine-a is a growth factor secreted from the paraxial neural tube is controlled by mesoderm and controls formation of the floor plate (I.). The floor plate in turn is an important organizer of the embryonic spinal two crucial signaling centers. cord and releases Shh. This acts as a morphogen and establishes These are the floor and the an activity gradient that controls specification of different neurons roof plate, situated at the at distinct positions of the spinal cord (II.). Shown are schematic cross sections of the trunk of zebrafish embryos at 16 hours post ventral and dorsal tips of the fertilization. (M. Schaefer) neural tube, respectively (Fig. 2). Both centers produce growth factors (3) and identified important structural (morphogens) that establish features of this organizing tissue (4). antagonistic activity gradients. The roof Notably, Midkine-a is derived from the plate secretes members of the Bone paraxial mesoderm surrounding the Morphogenetic Protein (BMP) and WNT spinal cord, establishing a novel families, while the floor plate releases mechanism for floor plate specification Sonic Hedgehog (Shh). Naive cells at (Fig. 2). Future projects aim to elucidate different positions in the early spinal cord the signalling cascades that are are able to interpret these gradients. activated by Midkine growth factors They respond to distinct threshold levels and the role of Midkine-b, a second by activating different sets of neural member of this family, in roof plate differentiation genes. Consequently, this formation. leads to regionalized gene expression patterns and cell differentiation Defects in RNA metabolism as according to the position in the neural cause of neurodegenerative tube. diseases: Spinal Muscular It still is a matter of intense research to find out how and when floor and roof plate are formed in the early embryo. We have characterized the family of Midkine growth factors, which have neurotrophic activities in cell culture assays. Their in vivo roles in mammals, however, remain unclear, as the corresponding knock-out mice show no morphological abnormalities. We could show that in zebrafish Midkine-a plays a crucial role in setting up the floor plate

Atrophy
Spinal muscular atrophy (SMA) is a common motoneuron disease that represents one of the most frequent genetic causes of infant death in humans. In SMA patients, motoneurons in the spinal cord are lost, which results in progressive paralysis of the trunk and limbs. SMA patients carry mutations in the ubiquitously expressed survival of motor neuron 1 (SMN1) gene.

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Figure 3 Motor axonal defects in the spinal cord of zebrafish embryos after knock-down of snRNP assembly components. Motor axons are truncated (arrow) or bifurcated (arrowheads) and do not reach their proper targets in the musculature (top). These defects can be rescued by supplementation of intact spliceosomal snRNPs into zebrafish embryos (bottom). (M. Giegerich)

embryos (5). These include truncations and ectopic branching of outgrowing motor axons (Fig. 3). Reduction of SMN and Gemin2 protein levels to approx. 10% results in significant paralysis and swimming deficiencies in zebrafish lar vae. Importantly, motor axonal defects in SMN deficient zebrafish can be rescued by injection of intact UsnRNPs. Thus, our findings suggest that motoneuron degeneration in SMA patients is a direct consequence of impaired production of UsnRNPs. Our working hypothesis proposes the presence of distinct pre-mRNAs in motoneurons that are particularly sensitive to inefficient splicing. The zebrafish offers many advantages to search for transcripts that are not correctly spliced in SMN deficient embryos and to gain more insight into the pathomolecular mechanisms leading to this common neurodegenerative disease in humans.

The SMN1 protein is implicated in the assembly of spliceosomal UsnRNPs, and possibly has additional functions in RNA transcription, splicing and localization. Knock-down studies in mouse and zebrafish have recently shown that reduced levels of SMN cause defects specifically in motor axonal growth. It however remains unclear, whether this reflects a particular sensitivity of motoneurons to general splicing deficiencies or a motoneuron specific function of the SMN protein, which is not related to assembly of spliceosomal UsnRNPs. Because of the transparency of its embryos and the relatively simple organization of motoneuronal organization in the spinal cord, the zebrafish is especially suited to study the cause of these defects. Using a Morpholino antisense approach, we have shown that knock-down not only of SMN, but also of two other essential factors in UsnRNP assembly, Gemin2 and pICln, result in similar motor axonal phenotypes in developing zebrafish

Laboratory fish as models for human bone disease

In an effort to establish fish models also for other relevant human diseases, we recently started to analyze bone formation and homeostasis in medaka. Osteoporosis and other related bone diseases are a major public health concern and have increasing impact onto our human society. Research during the last decade identified several key regulators controlling the activity of osteoblasts (bone forming cells) and osteoclasts (bone resorbing cells). We want to use medaka as experimental system to functionally characterize regulatory networks that have been implicated in human bone diseases. Teleost fish such as medaka grow during their entire life span. Constant body growth requires continuous reorganization of bony structures to ensure appropriate space for growing soft tissues. This process needs permanent resorption of bone material,

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as well as new bone formation to ensure structural stability. It has been shown that osteoblasts and osteoclasts of teleosts share many features with those of mammals. Recent findings suggest a high conservation also of the underlying genetics. As partner of the ENFORM (European Network using Fish as Osteoporosis Research Model) consortium, we have characterized several fish homologs of human genes, which play important regulatory roles during bone formation (6). Furthermore, we established stable transgenic fish that express fluorescent reporter genes in either early bone precursor cells or in differentiated bones (Fig. 4). These fish will be used to follow bone formation by real time imaging in control versus genetically altered individuals and, in addition, for high-throughput in vivo screens for bone anabolic compounds. This will generate targets for future therapeutic evaluation in humans. In summary, we hope that our work will give insight into the function and evolution of factors regulating osteoblast and osteoclast specification in vertebrates.

Literature
1. Rubinstein, A.L. (2003). Zebrafish: from disease modeling to drug discovery. Curr Opin Drug Discov Devel 6, 218223. Zon, L.I., and Peterson, R.T. (2005). In vivo drug discovery in the zebrafish. Nat Rev Drug Discov 4, 35-44. Schfer, M., Rembold, M., Wittbrodt, J., Schartl, M., and Winkler, C. (2005). Medial floor plate formation in zebrafish consists of two phases and requires trunk-derived Midkine-a. Genes & Development 19, 897-902. Schfer, M., Kinzel, D., and Winkler, C. (2007). Discontinuous organization and specification of the lateral floor plate in zebrafish. Dev. Biol. 301, 117129. Winkler, C., Eggert, C., Gradl, D., Meister, G., Giegerich, M., Wedlich, D., Laggerbauer, B. and Fischer, U. (2005). Reduced RNP assembly causes motor axon degeneration in an animal model for spinal muscular atrophy. Genes & Development 19, 2320-2330. Renn, J., Schaedel, M., Volff, J.N., Goerlich, R., Schartl, M., and Winkler, C. (2006). Dynamic expression of Sparc precedes formation of skeletal elements in the Medaka (Oryzias latipes). Gene 372, 208-218.

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Figure 4 Transgenic medaka fish expressing green fluorescent protein in osteoblasts of the larval head skeleton (ventral view, anterior to the top). The transparency of embryos allows monitoring of bone development in real time in vivo. (J. Renn)

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