Publications in 2003

Bahadori, R., Huber, M., Rinner, O., Seeliger, M.W., Geiger-Rudolph, S., Geisler, R. and Neuhauss, S.C.F. (2003). Retinal function and morphology in two zebrafish models of oculo-renal syndromes. Eur. J. Neurosci. 18, 1377-1386.

We characterized visual system defects in two recessive zebrafish mutants oval and elipsa . These mutants share the syndromic phenotype of outer retinal dystrophy in conjunction with cystic renal disorder. We tested the function of the larval visual system in a behavioural assay, eliciting optokinetic eye movements by high-contrast motion stimulation while recording eye movements in parallel. Visual stimulation did not elicit eye movements in mutant larvae, while spontaneous eye movements could be observed. The retina proved to be unresponsive to light using electroretinography, indicative of a defect in the outer retina. Histological analysis of mutant retinas revealed progressive degeneration of photoreceptors, initiated in central retinal locations and spreading to more peripheral regions with increasing age. The inner retina remains unaffected by the mutation. Photoreceptors display cell type-specific immunoreactivity prior to apoptotic cell death, arguing for a dystrophic defect. Genomic mapping employing simple sequence-length polymorphisms located both mutations on different regions of zebrafish linkage group 9. These mutants may serve as accessible animal models of human outer retinal dystrophies, including oculo-renal diseases, and show the general usefulness of a behavioural genetic approach to study visual system development in the model vertebrate zebrafish.

Bink, R.J., Habuchi, H., Lele, Z., Dolk, E., Joore, J., Rauch, G.-J., Geisler, R., Wilson, S.W., den Hertog, J., Kimata, K. and Zivkovic, D. (2003). Heparan sulfate 6-o-sulfotransferase is essential for muscle development in zebrafish. J. Biol. Chem. 15, 31118-31127.

Heparan sulfate proteoglycans function in development and disease. They consist of a core protein with attached heparan sulfate chains that are altered by a series of carbohydrate-modifying enzymes and sulfotransferases. Here, we report on the identification and characterization of a gene encoding zebrafish heparan sulfate 6-O-sulfotransferase (hs6st) that shows high homology to other heparan sulfate 6-O-sulfotransferases. When expressed as a fusion protein in cultured cells, the protein shows specific 6-O-sulfotransferase activity and preferentially acts on the iduronosyl N-sulfoglycosamine. In the developing embryo, hs6st is expressed in the brain, the somites, and the fins; the same structures that were affected upon morpholino-mediated functional knockdown. Morpholino injections significantly inhibited 6-O- but not 2-O-sulfation as assessed by HPLC. Morphants display disturbed somite specification independent of the somite oscillator mechanism and have impaired muscle differentiation. In conclusion, our results show that transfer of sulfate to specific positions on glycosaminoglycans is essential for muscle development.

Charatsi, I., Luschnig, S., Bartoszewski, S., Nüsslein-Volhard, C. and Moussian, B. (2003) Krapfen/dMyd88 is required for the establishment of dorsoventral pattern in the Drosophila embryo. Mechanisms of Development. 120(2):219-226.

In Drosophila, the dorsoventral axis is set up by the action of the dorsal group of genes and cactus, which have been ordered genetically in a linear pathway. We have identified and characterised krapfen (kra) as a new member of the dorsal-group genes. kra encodes for the Drosophila homologue of MyD88, an adapter protein operating in the mammalian IL-1 pathway. Epistasis experiments reveal that kra acts between the receptor Toll and the cytoplasmic factor Tube. We show that there is a direct interaction between Kra and Tube presumably mediated by the death domains present in both proteins. Tube in turn interacts with its downstream effector Pelle through death domain association. We therefore suggest that upon Toll activation, Kra associates with Pelle and Tube, in an heterotrimeric complex.

Dekens, M., Pelegri, F., Maischein, H.-M. and Nüsslein-Volhard, C. (2003): The maternal-effect gene futile cycle is essential for congression and mitotic spindle assembly in the zebrafish zygote. Development 130, 3907-3916.

Knaut, H., Werz, C., Geisler, R., Nüsslein-Volhard, C. and The Tübingen 2000 Screen Consortium (2003). A zebrafish homologue of the chemokine receptor Cxcr4 is a germ-cell guidance receptor. Nature. 421(6920):279-282

Germ cells preserve an individual's genetic information and transmit it to the next generation. Early in development germ cells are set aside and undergo a specialized developmental programme, a hallmark of which is the migration from their site of origin to the future gonad(1). In Drosophila, several factors have been identified that control germ-cell migration to their target tissues(2-4); however, the germ-cell chemoattractant or its receptor have remained unknown. Here we apply genetics and in vivo imaging to show that odysseus, a zebrafish homologue of the G-protein-coupled chemokine receptor Cxcr4, is required specifically in germ cells for their chemotaxis. odysseus mutant germ cells are able to activate the migratory programme, but fail to undergo directed migration towards their target tissue, resulting in randomly dispersed germ cells. SDF-1, the presumptive cognate ligand for Cxcr4, shows a similar loss-of-function phenotype and can recruit germ cells to ectopic sites in the embryo, thus identifying a vertebrate ligand-receptor pair guiding migratory germ cells at all stages of migration towards their target.

Knight, R.D., Nair, S., Nelson, S.S., Afshar, A., Javidan, Y., Geisler, R., Rauch, G.J. and Schilling, T.F. (2003). lockjaw encodes a zebrafish tfap2a required for early neural crest development. Development 130, 5755-5768.

The neural crest is a uniquely vertebrate cell type that gives rise to much of the craniofacial skeleton, pigment cells and peripheral nervous system, yet its specification and diversification during embryogenesis are poorly understood. Zebrafish homozygous for the lockjaw (low) mutation show defects in all of these derivatives and we show that low (allelic with montblanc) encodes a zebrafish tfap2a, one of a small family of transcription factors implicated in epidermal and neural crest development. A point mutation in low truncates the DNA binding and dimerization domains of tfap2a, causing a loss of function. Consistent with this, injection of antisense morpholino oligonucleotides directed against splice sites in tfap2a into wild-type embryos produces a phenotype identical to low. Analysis of early ectodermal markers revealed that neural crest specification and migration are disrupted in low mutant embryos. TUNEL labeling of dying cells in mutants revealed a transient period of apoptosis in crest cells prior to and during their migration. In the cranial neural crest, gene expression in the mandibular arch is unaffected in low mutants, in contrast to the hyoid arch, which shows severe reductions in dlx2 and hoxa2 expression. Mosaic analysis, using cell transplantation, demonstrated that neural crest defects in low are cell autonomous and secondarily cause disruptions in surrounding mesoderm. These studies demonstrate that low is required for early steps in neural crest development and suggest that tfap2a is essential for the survival of a subset of neural crest derivatives.

Maderspacher, F. and Nüsslein-Volhard, C.: Formation of the adult pigment pattern in zebrafish requires leopard and obelix dependent cell interactions. Development. 130(15):3447-57.

Colour patterns are a prominent feature of many animals and are of high evolutionary relevance. In zebrafish, the adult pigment pattern comprises alternating stripes of two pigment cell types, melanophores and xanthophores. How the stripes are defined and a straight boundary is formed remains elusive. We find that mutants lacking one pigment cell type lack a striped pattern. Instead, cells of one type form characteristic patterns by homotypic interactions. Using mosaic analysis, we show that juxtaposition of melanophores and xanthophores suffices to restore stripe formation locally. Based on this, we have analysed the pigment pattern of two adult specific mutants: leopard and obelix. We demonstrate that obelix is required in melanophores to promote their aggregation and controls boundary integrity. By contrast, leopard regulates homotypic interaction within both melanophores and xanthophores, and interaction between the two, thus controlling boundary shape. These findings support a view in which cell-cell interactions among pigment cells are the major driving force for adult pigment pattern formation.

Piotrowski, T., Ahn, D.-G., Schilling, T.F., Nair, S., Ruvinsky, I., Geisler, R., Rauch, G.-J., Haffter, P., Zon, L.I., Zhou ,Y., Foott, H., Dawid, I.B. and Ho, R.K. (2003). The zebrafish van gogh mutation disrupts tbx1, which is involved in the DiGeorge deletion syndrome in humans. Development 130, 5043-5052.

The van gogh (vgo ) mutant in zebrafish is characterized by defects in the ear, pharyngeal arches and associated structures such as the thymus. We show that vgo is caused by a mutation in tbx1 ,a member of the large family of T-box genes. tbx1 has been recently suggested to be a major contributor to the cardiovascular defects in DiGeorge deletion syndrome (DGS) in humans, a syndrome in which several neural crest derivatives are affected in the pharyngeal arches. Using cell transplantation studies, we demonstrate that vgo/tbx1 acts cell autonomously in the pharyngeal mesendoderm and influences the development of neural crest-derived cartilages secondarily. Furthermore, we provide evidence for regulatory interactions between vgo/tbx1 and edn1 and hand2 , genes that are implicated in the control of pharyngeal arch development and in the etiology of DGS.

Schröder, S., Otto, G.W., Siebert, J., Ott, S., Wegener, S., Geisler, R. and Krehan, A. (2003). Development of a zebrafish oligo test set. MWG Application Note 37.

(no abstract)