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Publications in 1999

Barth, K. A., Kishimoto, Y., Rohr, K. B., Seydler, C., Schulte-Merker, S., Wilson, S. W. (1999). Bmp activity establishes a gradient of positional information throughout the mentire neural plate Development. 126, 4977-4987.

Bone morphogenetic proteins (Bmps) are key regulators of dorsoventral (DV) patterning. Within the ectoderm, Bmp activity has been shown to inhibit neural development, promote epidermal differentiation and influence the specification of dorsal neurons and neural crest. In this study, we examine the patterning of neural tissue in mutant zebrafish embryos with compromised Bmp signalling activity, We find that although Bmp activity does not influence anteroposterior (AP) patterning, it does affect DV patterning at all AP levels of the neural plate, Thus, we show that Bmp activity is required for specification of cell fates around the margin of the entire neural plate, including forebrain regions that do not form neural crest. Surprisingly, we find that Bmp activity is also required for patterning neurons at all DV levels of the CNS, In swirl/bmp2b(-) (swr(-)) embryos, laterally positioned sensory neurons are absent whereas more medial interneuron populations are hugely expanded, However, in somitabun(-)(sbn(-)) embryos, which probably retain higher residual Bmp activity, it is the sensory neurons and not the interneurons that are expanded, Conversely, in severely Bmp depleted embryos, both interneurons and sensory neurons are absent and it is the most medial neurons that are expanded, These results are consistent with there being a gradient of Bmp-dependent positional information extending throughout the entire neural and non-neural ectoderm.

Campione, M., Steinbeisser, H., Schweickert, A., Deissler, K., van Bebber, F., Lowe, L. A., Nowotschin, S., Viebahn, C., Haffter, P., Kuehn, M. R., and Blum, M. (1999). The homeobox gene Pitx2: mediator of asymmetric left-right signaling in vertebrate heart and gut looping. Development 126, 1225-34.

Left-right asymmetry in vertebrates is controlled by activities emanating from the left lateral plate. How these signals get transmitted to the forming organs is not known. A candidate mediator in mouse, frog and zebrafish embryos is the homeobox gene Pitx2. It is asymmetrically expressed in the left lateral plate mesoderm, tubular heart and early gut tube. Localized Pitx2 expression continues when these organs undergo asymmetric looping morphogenesis. Ectopic expression of Xnr1 in the right lateral plate induces Pitx2 transcription in Xenopus. Misexpression of Pitx2 affects situs and morphology of organs. These experiments suggest a role for Pitx2 in promoting looping of the linear heart and gut.

Chandrasekhar, A., Schauerte, H. E., Haffter, P., and Kuwada, J. Y. (1999). The zebrafish detour gene is essential for cranial but not spinal motor neuron induction. Development 126, 2727-37.

The zebrafish detour (dtr) mutation generates a novel neuronal phenotype. In dtr mutants, most cranial motor neurons, especially the branchiomotor, are missing. However, spinal motor neurons are generated normally. The loss of cranial motor neurons is not due to aberrant hindbrain patterning, failure of neurogenesis, increased cell death or absence of hh expression. Furthermore, activation of the Hh pathway, which normally induces branchiomotor neurons, fails to induce motor neurons in the dtr hindbrain. Despite this, not all Hh-mediated regulation of hindbrain development is abolished since the regulation of a neural gene by Hh is intact in the dtr hindbrain. Finally, dtr can function cell autonomously to induce branchiomotor neurons. These results suggest that detour encodes a component of the Hh signaling pathway that is essential for the induction of motor neurons in the hindbrain but not in the spinal cord and that dtr function is required for the induction of only a subset of Hh-mediated events in the hindbrain.

Dorsky, R., Cretekos, C., Grunwald, D., Geisler, R., Haffter, P., Moon, R. T., and Raible, D. (1999). Maternal and embryonic expression of zebrafish lef1. Mech Dev 86, 147-50.


Transcription factors of the TCF/LEF family interact with the Wnt signaling pathway to control transcription of downstream genes (Clevers, H., van de Wetering, M., 1997. TCF/LEF factor earn their wings. Trends Genet. 13, 485-489). We were interested in cloning family members which were expressed in zebrafish neural crest, because Wnt signaling modulates specification of neural crest fate (Dorsky, R.I., Moon, R.T., Raible, D.W., 1998. Control of neural crest cell fate by the Wnt signalling pathway. Nature 396, 370-373). We cloned a zebrafish homolog of lef1 and localized its chromosomal position by radiation hybrid mapping. lef1 is expressed in the neural crest as well as the tailbud and developing mesoderm, and is maternally expressed in zebrafish, unlike mouse and Xenopus homologs. In addition, we cloned two tcf3 genes and a homolog of tcf4, neither of which were strongly expressed in premigratory neural crest.

Fritsch, C., Brown, J. L., Kassis, J. A. and Müller , J. (1999). The DNA-binding Polycomb group protein Pleiohomeotic mediates silencing of a Drosophila homeotic gene. Development 126, 3905-13.

Polycomb group (PcG) proteins repress homeotic genes in cells where these genes must remain inactive during development. This repression requires cis-acting silencers, also called PcG response elements. Currently, these silencers are ill-defined sequences and it is not known how PcG proteins associate with DNA. Here, we show that the Drosophila PcG protein Pleiohomeotic binds to specific sites in a silencer of the homeotic gene Ultrabithorax. In an Ultrabithorax reporter gene, point mutations in these Pleiohomeotic binding sites abolish PcG repression in vivo. Hence, DNA-bound Pleiohomeotic protein may function in the recruitment of other non-DNA-binding PcG proteins to homeotic gene silencers.

Geisler, R., Rauch, G.-J., Baier, H., van Bebber, F., Broß, L., Davis, R. W., Dekens, M., Finger, K., Fricke, C., Gates, M. A., Geiger, H., Geiger-Rudolph, S., Gilmour, D., Glaser, S., Gnügge, L., Habeck, H., Hingst, K., Holley, S., Keenan, J., Kirn, A., Knaut, H., Lashkari, D., Maderspacher, F., Martyn, U., Neuhauss, S., Neumann, C., Nicolson, T., Pelegri, F., Ray, R., Rick, J., Roehl, H., Roeser, T., Schauerte, H. E., Schier, A. F., Schönberger, U., Schönthaler, H.-B., Schulte-Merker, S., Seydler, C., Talbot, W. S., Weiler, C., Nüsslein-Volhard, C. and Haffter, P. (1999). A radiation hybrid map of the zebrafish genome. Nature Genet 23, 86-9.

Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map in order to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning we have created a radiation hybrid (RH) map of the zebrafish genome. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat, and mouse. For our map of the zebrafish genome we used a RH panel generated by P.N. Goodfellow and 1451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. 1275 of these (87.9 %) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9 %. We found the average marker retention frequency to be 18.4 %. 1 cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb.

Großhans, J., Schnorrer, F., and Nüsslein-Volhard, C. (1999). Oligomerisation of Tube and Pelle leads to nuclear localisation of dorsal. Mech Dev 81, 127-38.

In the Drosophila embryo the nuclear localisation of Dorsal, a member of the Rel family, is regulated by an extracellular signal, which is transmitted to the interior of the egg cell by a cascade of proteins involving the novel protein Tube and the protein kinase Pelle. Here we analyse the activation mechanism of Tube and Pelle and the interaction between these two components. We show that both proteins, although having different biochemical activities, are activated by the same mechanism. Membrane association alone is not sufficient, but oligomerisation is required for full activation of Tube and Pelle. By deletion analysis we determined the domains of Tube and Pelle mediating the physical interaction and the signalling to downstream components. In order to investigate the link between Pelle and the target of the signalling cascade, the Dorsal/Cactus complex, we isolated and characterised the novel, but evolutionary conserved protein Pellino, which associates with the kinase domain of Pelle.

Hild, M., Dick, A., Rauch, G. J., Meier, A., Bouwmeester, T., Haffter, P., and Hammerschmidt, M. (1999). The smad5 mutation somitabun blocks Bmp2b signaling during early dorsoventral patterning of the zebrafish embryo. Development. 126, 2149-59.

Signaling by members of the TGF beta superfamily is thought to be transduced by Smad proteins. Here, we describe a zebrafish mutant in smad5, designated somitabun (sbn). The dominant maternal and zygotic effect of the sbn(tc24) mutation is caused by a change in a single amino acid in the L3 loop of Smad5 protein which transforms Smad5 into an antimorphic version, inhibiting wild-type Smad5 and related Smad proteins. sba mutant embryos are strongly dorsalized, similarly to mutants in Bmp2b, its putative upstream signal. Double mutant analyses and RNA injection experiments show that sbn and bmp2b interact and that sbn acts downstream of Bmp2b signaling to mediate Bmp2b autoregulation during early dorsoventral (D-V) pattern formation. Comparison of early marker gene expression patterns, chimera analyses and rescue experiments involving temporally controlled misexpression of bmp or smad in mutant embryos reveal three phases of D-V patterning: an early sbn- and bmp2b-independent phase when a coarse initial D-V pattern is set up, an intermediate sbn- and bmp2b-dependent phase during which the putative morphogenetic Bmp2/4 gradient is established, and a later sbn-independent phase during gastrulation when the Bmp2/4 gradient is interpreted and cell fates are specified.

Holley, S. A. and Nüsslein-Volhard, C. (1999). Somitogenesis in Zebrafish. Current Topics in Developmental Biology. 47, 248-272.

Munchberg, S. R., Ober, E. A., Steinbeisser, H. (1999). Expression of the Ets transcription factors erm and pea3 in early zebrafish development. Mechanisms of Development. 88, 233-236.

Here we report the cloning of zebrafish erm and pea3 cDNAs, which are members of the PEA3 subgroup of Ets transcription factors. The expression patterns of these two genes were examined during zebrafish embryogenesis. Maternal mRNAs of both genes are detectable and transcripts are found ubiquitously until the late blastula, in the marginal zone of gastrula stages and in the presumptive fore- and hindbrain and in the trunk region of early somite stages. Later, erm expression is observed in distinct regions of the forebrain, the mid-/hindbrain boundary, the differentiating rhombomeres, branchial arches, otic vesicles, the pectoral fins, the somites and the tailbud in a dynamic fashion. In contrast, pea3 is not expressed in the rhombomeres but in the Rohon-Beard neurons, the sensory lateral line placodes and the heart.

Neumann CJ. Grandel H. Gaffield W. Schulte-Merker S. Nüsslein-Volhard C. (1999) Transient establishment of anteroposterior polarity in the zebrafish pectoral fin bud in the absence of sonic hedgehog activity. Development 126 (21): 4817-4826.

Sonic hedgehog (Shh) is expressed in the posterior vertebrate limb bud mesenchyme and directs anteroposterior patterning and growth during limb development. Here we report an analysis of the pectoral fin phenotype of zebrafish sonic you mutants, which disrupt the shh gene, We show that Shh is required for the establishment of some aspects of anteroposterior polarity, while other aspects of anteroposterior polarity are established independently of Shh, and only later come to depend on Shh for their maintenance, We also demonstrate that Shh is required for the activation of posterior HoxD genes by retinoic acid, Finally, we show that Shh is required for normal development of the apical ectodermal fold, for growth of the fin bud, and for formation of the fin endoskeleton.

Ober EA. Schulte-Merker S. (1999) Signals from the yolk cell induce mesoderm, neuroectoderm, the trunk organizer, and the notochord in zebrafish. Developmental Biology 215 (2):167-181.

We have analyzed the role of the zebrafish yolk cell in the processes of mesoderm induction and establishment of the organizer. By recombining blastomere-free yolk cells and animal cap tissue we have shown that the yolk cell itself can induce mesoderm in neighboring blastomeres. We further demonstrate the competence of all blastomeres to form mesoderm, suggesting the endogenous mesoderm inducing signal to be locally restricted. Ablation of the vegetal third of the yolk cell during the first 20 min of development does not interfere with mesoderm formation in general, but results in completely ventralized embryos. These embryos lack the notochord, neuroectoderm, and the anterior-most 14-15 somites, demonstrating that the ablation affects the formation of the trunk-, but not the tail region of the embryo. This suggests the presence of a trunk organizer in fish. The dorsalized mutant swirl (zbmp-2b) shows expanded dorsal structures and missing ventral structures. In contrast to the phenotypes obtained upon the ablation treatment in wild-type embryos, removal of the vegetal-most yolk in swirl mutants results in embryos which do form neuroectoderm and anterior trunk somites. However, both wild-type and swirl mutants lack a notochord upon vegetal yolk removal. These ablation experiments in wild-type and swirl mutant embryos demonstrate that in zebrafish dorsal determining factors originate from the vegetal part of the yolk cell. These factors set up two independent activities: one induces the notochord and the other is involved in the formation of the neuroectoderm and the trunk region by counteracting the function of swirl. In addition, these experiments show that the establishment of the anteroposterior axis is independent of the dorsoventral axis.

Pelegri, F., Knaut, H., Maischein, H.-M., Schulte-Merker, S. and C. Nüsslein-Volhard (1999). A mutation in the zebrafish maternal gene nebel affects furrow formation and vasa RNA localization. Curr. Biol. 9, 1431-1440.


Background: In many animals, embryonic patterning depends on a careful interplay between cell division and the segregation of localized cellular components. Both of these processes in turn rely on cytoskeletal elements and motor proteins. A type of localized cellular component found in most animals is the germ plasm, a specialized region of cytoplasm that specifies the germ-cell fate. The gene vasa has been shown in Drosophila to encode an essential component of the germ plasm and is thought to have a similar function in other organisms. In the zebrafish embryo, the vasa RNA is localized to the furrows of the early cellular divisions. Results: We identified the gene nebel in a pilot screen for zebrafish maternal-effect mutations. Embryos from females homozygous for a mutation in nebel exhibit defects in cell adhesion. Our analysis provides genetic evidence for a function of the microtubule array that normally develops at the furrow in the deposition of adhesive membrane at the cleavage plane. In addition, nebel mutant embryos show defects in the early localization of vasa RNA. The vasa RNA localization phenotype could be mimicked with microtubule-inhibiting drugs, and confocal microscopy suggests an interaction between microtubules and vasa-RNA-containing aggregates. Conclusions: Our data support two functions for the microtubule reorganization at the furrow, one for the exocytosis of adhesive membrane, and another for the translocation of vasa RNA along the forming furrow.

Pelegri, F., and Schulte-Merker, S. (1999). A gynogenesis-based screen for maternal-effect genes in the zebrafish, Danio rerio. Methods Cell Biol 60, 1-20.

In many animals, embryonic patterning depends on a careful interplay between cell division and the segregation of localized cellular components. Both of these processes in turn rely on cytoskeletal elements and motor proteins. A type of localized cellular component found in most animals is the germ plasm, a specialized region of cytoplasm that specifies the germ-cell fate. The gene vasa has been shown in Drosophila to encode an essential component of the germ plasm and is thought to have a similar function in other organisms. In the zebrafish embryo, the vasa RNA is localized to the furrows of the early cellular divisions. RESULTS: We identified the gene nebel in a pilot screen for zebrafish maternal-effect mutations. Embryos from females homozygous for a mutation in nebel exhibit defects in cell adhesion. Our analysis provides genetic evidence for a function of the microtubule array that normally develops at the furrow in the deposition of adhesive membrane at the cleavage plane. In addition, nebel mutant embryos show defects in the early localization of vasa RNA. The vasa RNA localization phenotype could be mimicked with microtubule-inhibiting drugs, and confocal microscopy suggests an interaction between microtubules and vasa-RNA-containing aggregates. CONCLUSIONS: Our data support two functions for the microtubule reorganization at the furrow, one for the exocytosis of adhesive membrane, and another for the translocation of vasa RNA along the forming furrow.

Schvarzstein, M., Kirn, A., Haffter, P., and Cordes, S. P. (1999). Expression of Zkrml2, a homologue of the Krml1/val segmentation gene, during embryonic patterning of the zebrafish (Danio rerio). Mech Dev 80, 223-6.

We have identified Zkrml2, a novel homologue of the segmentation gene Krml/val in zebrafish (Danio rerio). Zkrml2 shows 72% and 92% identity in its basic leucine zipper domain with mouse Krml1 and zebrafish val, respectively. Zkrml2 is expressed coincident with MyoD throughout the somites starting at the three somite stage, becomes restricted to the dermomyotome, and subsequently disappears. Transient expression is also detected in the reticulospinal and oculomotor neurons. Zkrml2 maps to the Oregon linkage group 11 (Boston Linkage group 14) with no mapped zebrafish mutations nearby.

van Eeden, F. J., Granato, M., Odenthal, J., and Haffter, P. (1999). Developmental mutant screens in the zebrafish. Methods Cell Biol 60, 21-41.

Warga, R.M., and Nüsslein-Volhard, C. (1999). Origin and development of the zebrafish endoderm. Development 126, 827-38.

The segregation of cells into germ layers is one of the earliest events in the establishment of cell fate in the embryo. In the zebrafish, endoderm and mesoderm are derived from cells that involute into an internal layer, the hypoblast, whereas ectoderm is derived from cells that remain in the outer layer, the epiblast. In this study, we examine the origin of the zebrafish endoderm and its separation from the mesoderm. By labeling individual cells located at the margin of the blastula, we demonstrate that all structures that are endodermal in origin are derived predominantly from the more dorsal and lateral cells of the blastoderm margin. Frequently marginal cells give rise to both endodermal and mesodermal derivatives, demonstrating that these two lineages have not yet separated. Cells located farther than 4 cell diameters from the margin give rise exclusively to mesoderm, and not to endoderm. Following involution, we see a variety of cellular changes indicating the differentiation of the two germ layers. Endodermal cells gradually flatten and extend filopodial processes forming a noncontiguous inner layer of cells against the yolk. At this time, they also begin to express Forkhead-domain 2 protein. Mesodermal cells form a coherent layer of round cells separating the endoderm and ectoderm. In cyclops-mutant embryos that have reduced mesodermal anlage, we demonstrate that by late gastrulation not only mesodermal but also endodermal cells are fewer in number. This suggests that a common pathway initially specifies germ layers together before a progressive sequence of determinative events segregate endoderm and mesoderm into morphologically distinct germ layers.