Biomedical subjects

R W Köster

Publications and source records attributed to R W Köster.

7 recordsLinked to original sources

PubMed2003

The teleost fish medaka (Oryzias latipes) as genetic model to study gravity dependent bone homeostasis in vivo.

Long-term space flight and microgravity result in bone loss that can be explained by reduced activity of bone-forming cells (osteoblasts) and/or an increase in activity of bone resorbing cells (osteoclasts). Osteoprotegerin (OPG) has been shown to regulate the balance between osteoblast and osteoclast cell numbers and is involved in maintaining constant bone mass under normal gravitational conditions. The small bony fish medaka (Oryzias latipes) has attracted increasing attention as a genetic model system to study normal embryonic developmental and pathological processes. To analyze the molecular mechanisms of bone formation in this small vertebrate, we have isolated two opg genes, opgl and opg2, from medaka. Our phylogenetic analysis reveals that both genes originated from a common ancestor by fish-specific gene duplication and represent the orthologs of the mammalian opg gene. Both opg genes are differentially expressed during embryonic and larval development, in adult tissues and in cultured primary osteoblast-like cells. Furthermore, we have characterized the opg2 promoter region and identified consensus binding sites for the transcription factor core-binding-factor-1A (CBFA1). In mammals, CBFA1 has been shown to be a regulator of opg expression and to be essential for several steps during osteoblast differentiation. Here we show that sequence and expression domains of opg, cbfal and a member of the dlx gene family are highly conserved between medaka and higher vertebrates. This suggests that not only single genes but entire genetic networks for bone formation are conserved between teleosts and mammals. These findings will open medaka fish as a genetic model to monitor bone formation under different gravity conditions in a living whole animal allowing the identification of novel factors involved in bone homeostasis.

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PubMed2001

Direct imaging of in vivo neuronal migration in the developing cerebellum.

The upper rhombic lip (URL), a germinal zone in the dorsoanterior hindbrain, has long been known to be a source for neurons of the vertebrate cerebellum. It was thought to give rise to dorsally migrating granule cell precursors (Figure 1e); however, recent fate mapping studies have questioned the exclusive contributions of the URL to granule cells. By taking advantage of the clarity of the zebrafish embryo during the stages of brain morphogenesis, we have followed the fate of neuronal precursor cells generated within the upper rhombic lip directly. Combining a novel GFP labeling strategy with in vivo time-lapse imaging, we find, contrary to the former view, that most URL-descendants migrate anterior toward the midhindbrain boundary (MHB) and then course ventrally along the MHB (Figure 1f). As the migrating neuronal precursors reach the MHB, they form ventrally extending projections, likely axons, and continue ventral migration to settle outside of the cerebellum, in the region of the ventral brainstem. Thus, we define a new pathway for URL-derived neuronal precursor cells consistent with the recent fate maps. In addition, our results strongly suggest that the MHB plays a crucial role, not only in induction and patterning of the cerebellar anlage, but also in organizing its later morphogenesis by influencing cell migration.

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PubMed2001

Tracing transgene expression in living zebrafish embryos.

Ectopic expression by injection of plasmid DNA is rarely used in zebrafish embryos due to a low frequency of cells expressing a transgene of interest at detectable levels. Furthermore, the mosaic nature of ectopic expression by plasmid injection requires the direct detection of transgene-expressing cells. We have used the transcriptional activator Gal4-VP16 to amplify transgene expression in living zebrafish embryos. In comparison to conventional expression vectors, Gal4-VP16-amplified expression results in a significant higher number of cells which express a transgene at detectable levels. The Gal4-VP16-activator and the Gal4-VP16-dependent transgene can be placed on a single expression vector. Using tissue-specific regulatory elements, we show that expression of a Gal4-VP16-dependent transgene can be reliably restricted to muscle, notochordal, or neuronal tissues. Furthermore, Gal4-VP16 can drive the expression of two or more transgenes from the same construct resulting in simultaneous coexpression of both genes in virtually all expressing cells. The reported expression system works effectively not only in zebrafish embryos but also in Xenopus embryos, chicken, mouse, and human cultured cells and is thus applicable to a broad variety of vertebrates. The high frequency of transgene expression together with the linked coexpression of more than one transgene opens the possibility of easily monitoring the behavior of individual transgene-expressing cells in real time by labeling them with the fluorescent reporter GFP. The combinatorial nature of the expression system greatly facilitates changing the tissue-specificity, the transgene expressed, or the cell compartment-specific GFP reporter, making it simpler to address a gene's function in different tissues as well as its cell biological consequences.

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PubMed2001

GAP-43 promoter elements in transgenic zebrafish reveal a difference in signals for axon growth during CNS development and regeneration.

A pivotal event in neural development is the point at which differentiating neurons become competent to extend long axons. Initiation of axon growth is equally critical for regeneration. Yet we have a limited understanding of the signaling pathways that regulate the capacity for axon growth during either development or regeneration. Expression of a number of genes encoding growth associated proteins (GAPs) accompanies both developmental and regenerative axon growth and has led to the suggestion that the same signaling pathways regulate both modes of axon growth. We have tested this possibility by asking whether a promoter fragment from a well characterized GAP gene, GAP-43, is sufficient to activate expression in both developing and regenerating neurons. We generated stable lines of transgenic zebrafish that express green fluorescent protein (GFP) under regulation of a 1 kb fragment of the rat GAP-43 gene, a fragment that contains a number of evolutionarily conserved elements. Analysis of GFP expression in these lines confirms that the rat 1 kb region can direct growth-associated expression of the transgene in differentiating neurons that extend long axons. Furthermore, this region supports developmental down-regulation of transgene expression which, like the endogenous gene, coincides with neuronal maturation. Strikingly, these same sequences are insufficient for directing expression in regenerating neurons. This finding suggests that signaling pathways regulating axon growth during development and regeneration are not the same. While these results do not exclude the possibility that pathways involved in developmental axon growth are also active in regenerative growth, they do indicate that signaling pathway(s) controlling activation of the GAP-43 gene after CNS injury differ in at least one key component from the signals controlling essential features of developmental axon growth.

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PubMed2000

Ectopic Sox3 activity elicits sensory placode formation.

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.

Amino Acid Sequence↗

PubMed2000

A genetic screen for mutations affecting embryonic development in medaka fish (Oryzias latipes).

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.

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PubMed1998

Six3, a medaka homologue of the Drosophila homeobox gene sine oculis is expressed in the anterior embryonic shield and the developing eye.

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.

Amino Acid Sequence↗