Zebrafish, A Practical Approach. Christiane Nüsslein-Volhard and Ralf Dahm (eds.), Oxford University Press (2002). 303 pages.Chapters from the book by authors from Department 3: Introduction: Zebrafish as a system to study development and organogenesis
by Christiane Nüsslein-Volhard, Darren T. Gilmour and Ralf Dahm
Chapter 1: Keeping and raising zebrafish
by Michael Brand, Michael Granato and Christiane Nüsslein-Volhard
Chapter 5: Manipulating gene expression in the zebrafish
by Darren T. Gilmour, Jason R. Jessen and Shuo Lin
Chapter 7: Mapping and cloning
by Robert Geisler
Appendix 1: Atlas of embryonic stages of development in the zebrafish
by Ralf Dahm
Appendix 2: Table of zebrafish mutations
by Hans Georg FrohnhöferAberle, H., Haghighi, A. P., Fetter, R. D., McCabe, B. D., Magalhães, T. R. and Goodman, C. S. (2002). Wishful thinking encodes a BMP Type II receptor that regulates synaptic growth in Drosophila. Neuron, 33: 545-558
We conducted a large-scale screen for Drosophila mutants that have structural abnormalities of the larval neuromuscular junction (NMJ). We recovered mutations in wishful thinking (wit), a gene that positively regulates synaptic growth. wit encodes a BMP type II receptor. In wit mutant larvae, the size of the NMJs is greatly reduced relative to the size of the muscles. wit NMJs have reduced evoked excitatory junctional potentials, decreased levels of the synaptic cell adhesion molecule Fasciclin II, and synaptic membrane detachment at active zones. Wit is expressed by a subset of neurons, including motoneurons. The NMJ phenotype is specifically rescued by transgenic expression of Wit only in motoneurons. Thus, Wit appears to function as a presynaptic receptor that regulates synaptic size at the Drosophila NMJ.Ciruna, B., Weidinger, G., Knaut, H., Thisse, B., Thisse, C., Raz, E. and Schier, A.F., (2002) Production of maternal-zygotic mutant zebrafish by germ-line replacement. Proceedings of the National Academy of Sciences of the United States of America. 99(23): 14919-14924
We report a generally applicable strategy for transferring zygotic lethal mutations through the zebrafish germ line. By using a morpholino oligonucleotide that blocks primordial germ cell (PGC) development, we generate embryos devoid of endogenous PGCs to serve as hosts for the transplantation of germ cells derived from homozygous mutant donors. Successful transfers are identified by the localization of specifically labeled donor PGCs to the region of the developing gonad in chimeric embryos. This strategy, which results in the complete replacement of the host germ line with donor PGCs, was validated by the generation of maternal and maternal-zygotic mutants for the miles apart locus. This germ-line replacement technique provides a powerful tool for studying the maternal effects of zygotic lethal mutations. Furthermore, the ability to generate large clutches of purely mutant embryos will greatly facilitate embryological, genetic, genomic, and biochemical studies.Gilmour, D.T., Maischein, H.-M. and Nüsslein-Volhard, C. (2002). Migration and Function of a Glial Subtype in the Vertebrate Peripheral Nervous System. Neuron 34: 577-588.
Glia-axon interactions are essential for the development and function of the nervous system. We combine in vivo imaging and genetics to address the mechanism by which the migration of these cells is coordinated during embryonic development. Using stable transgenic lines, we have followed the migration of one subset of glial cells and their target axons in living zebrafish embryos. These cells coalesce at an early stage and remain coupled throughout migration, with axons apparently pathfinding for glia. Mutant analysis demonstrates that axons provide instructive cues that are sufficient to control glial guidance. Furthermore, mutations in the transcription factor Sox10/cls uncouple the migration of axons and glia. Finally, genetic ablation of this glial subtype reveals an essential role in nerve fasciculation.Grandel, H., Lun, K., Rauch, G.J., Rhinn, M., Piotrowski, T., Houart, C., Sordino, P., Kuchler, A.M., Schulte-Merker, S., Geisler, R., Holder, N., Wilson, S.W. and Brand, M. (2002). Retinoic acid signalling in the zebrafish embryo is necessary during pre-segmentation stages to pattern the anterior-posterior axis of the CNS and to induce a pectoral fin bud. Development, 129(12): 2851-65.
A number of studies have suggested that retinoic acid (RA) is an important signal for patterning the hindbrain, the branchial arches and the limb bud. Retinoic acid is thought to act on the posterior hindbrain and the limb buds at somitogenesis stages in chick and mouse embryos. Here we report a much earlier requirement for RA signalling during pre-segmentation stages for proper development of these structures in zebrafish. We present evidence that a RA signal is necessary during pre-segmentation stages for proper expression of the spinal cord markers hoxb5a and hoxb6b, suggesting an influence of RA on anteroposterior patterning of the neural plate posterior to the hindbrain. We report the identification and expression pattern of the zebrafish retinaldehyde dehydrogenase2 (raldh2/aldh1a2) gene. Raldh2 synthesises retinoic acid (RA) from its immediate precursor retinal. It is expressed in a highly ordered spatial and temporal fashion during gastrulation in the involuting mesoderm and during later embryogenesis in paraxial mesoderm, branchial arches, eyes and fin buds, suggesting the involvement of RA at different times of development in different functional contexts. Mapping of the raldh2 gene reveals close linkage to no-fin (nof), a newly discovered mutant lacking pectoral fins and cartilaginous gill arches. Cloning and functional tests of the wild-type and nof alleles of raldh2 reveal that nof is a raldh2 mutant. By treating nof mutants with RA during different time windows and by making use of a retinoic acid receptor antagonist, we show that RA signalling during pre-segmentation stages is necessary for anteroposterior patterning in the CNS and for fin induction to occur.Holley, S.A., Julich, D., Rauch, G.J., Geisler, R. and Nüsslein-Volhard, C. (2002). her1 and the notch pathway function within the oscillator mechanism that regulates zebrafish somitogenesis. Development,129(5):1175-83.
Somite formation is thought to be regulated by an unknown oscillator mechanism that causes the cells of the presomitic mesoderm to activate and then repress the transcription of specific genes in a cyclical fashion. These oscillations create stripes/waves of gene expression that repeatedly pass through the presomitic mesoderm in a posterior-to-anterior direction. In both the mouse and the zebrafish, it has been shown that the notch pathway is required to create the stripes/waves of gene expression. However, it is not clear if the notch pathway comprises part of the oscillator mechanism or if the notch pathway simply coordinates the activity of the oscillator among neighboring cells. In the zebrafish, oscillations in the expression of a hairy-related transcription factor, her1 and the notch ligand deltaC precede somite formation. Our study focuses on how the oscillations in the expression of these two genes is affected in the mutants aei/deltaD and des/notch1, in 'morpholino knockdowns' of deltaC and her1 and in double 'mutant' combinations. This analysis indicates that these oscillations in gene expression are created by a genetic circuit comprised of the notch pathway and the notch target gene her1. We also show that a later function of the notch pathway can create a segmental pattern even in the absence of prior oscillations in her1 and deltaC expression. Supplementary data available at www.eb.tuebingen.mpg.de/papers/holley_dev_2002.htmlKnaut, H., Steinbeisser, H., Schwarz, H. and Nüsslein-Volhard, C. (2002). An Evolutionary Conserved Region in the vasa 3'UTR Targets RNA Translation to the Germ Cells in the Zebrafish. Current Biology 12: 1-20.
In many animals, germ cells are set aside from somatic cells early during development to give rise to sperm in males and eggs in females. One strategy to achieve this separation is to localize special cytoplasmic granules to the precursors of the germline. In Drosophila, the vasa gene has been shown to encode an essential component of these granules. While Vasa protein is directly targeted to the forming germ cells of Drosophila, Vasa protein expression in the germline of Xenopus and zebrafish is thought to be achieved by RNA localization.Results: To analyze whether the machinery responsible for RNA localization is conserved among lower vertebrates, we tested different vasa homologs for their ability to localize in Xenopus oocytes. Reporter transcripts fused to the vasa 3'UTR of zebrafish are recruited to the germ plasm of injected Xenopus oocytes, although the 3'UTR shows no clear sequence similarity to the Xenopus vasa-like DEADsouth 3'UTR. However, isolation, expression pattern analysis, and sequence inspection of vasa genes from different teleosts indicate that RNA localization correlates with the presence of several conserved regions in the 3'UTR. Introduction of reporter transcripts fused to different vasa 3'UTR deletions into Xenopus and zebrafish demonstrates that one of these conserved regions is sufficient for RNA localization in either species. Moreover, these regions target GFP translation to the germline of transgenic fish.Conclusions: Our results suggest the existence of a common RNA localization machinery in lower vertebrates that uses a functionally conserved localization signal to target gene expression to the germline.Lele, Z., Folchert, A., Concha, M., Rauch, G.J., Geisler, R., Rosa, F., Wilson, S.W., Hammerschmidt, M. and Bally-Cuif, L. (2002). parachute/n-cadherin is required for morphogenesis and maintained integrity of the zebrafish neural tube. Development, 129(14): 3281-94.
N-cadherin (Ncad) is a classical cadherin that is implicated in several aspects of vertebrate embryonic development, including somitogenesis, heart morphogenesis, neural tube formation and establishment of left-right asymmetry. However, genetic in vivo analyses of its role during neural development have been rather limited. We report the isolation and characterization of the zebrafish parachute (pac) mutations. By mapping and candidate gene analysis, we demonstrate that pac corresponds to a zebrafish n-cadherin (ncad) homolog. Three mutant alleles were sequenced and each is likely to encode a non-functional Ncad protein. All result in a similar neural tube phenotype that is most prominent in the midbrain, hindbrain and the posterior spinal cord. Neuroectodermal cell adhesion is altered, and convergent cell movements during neurulation are severely compromised. In addition, many neurons become progressively displaced along the dorsoventral and the anteroposterior axes. At the cellular level, loss of Ncad affects beta-catenin stabilization/localization and causes mispositioned and increased mitoses in the dorsal midbrain and hindbrain, a phenotype later correlated with enhanced apoptosis and the appearance of ectopic neurons in these areas. Our results thus highlight novel and crucial in vivo roles for Ncad in the control of cell convergence, maintenance of neuronal positioning and dorsal cell proliferation during vertebrate neural tube development.Schnorrer, F., Luschnig, S., Koch, I. and Nüsslein-Volhard, C. (2002) gamma-Tubulin37C and gamma-tubulin ring complex protein 75 are essential for bicoid RNA localization during Drosophila oogenesis. Developmental Cell. 3(5): 685-696.
bicoid (bcd) RNA localization requires the activity of exuperantia and swallow at sequential steps of oogenesis and is microtubule dependent. In a genetic screen, we identified two novel genes essential for bcd RNA localization. They encode maternal gamma-Tubulin37C (gammaTub37C) and gamma-tubulin ring complex protein 75 (Dgrip75), both of which are gamma-tubulin ring complex components. Mutations in these genes specifically affect bcd RNA localization, whereas other microtubule-dependent processes during oogenesis are not impaired. This provides direct evidence that a subset of microtubules organized by the gamma-tubulin ring complex is essential for localization of bcd RNA. At stage 10b we find gammaTub37C and Dgrip75 anteriorly concentrated and propose the formation of a microtubule-organizing center at the anterior pole of the oocyte.