Kohei Hatta

Bio: Kohei Hatta is an academic researcher from University of Hyogo. The author has contributed to research in topics: Zebrafish & Floor plate. The author has an hindex of 25, co-authored 41 publications receiving 3230 citations. Previous affiliations of Kohei Hatta include University of Oregon & Pasteur Institute.

The cyclops mutation blocks specification of the floor plate of the zebrafish central nervous system

Abstract: The floor plate is a set of epithelial cells present in the ventral midline of the neural tube in vertebrates that seems to have an important role in the developmental patterning of central nervous system fibre pathways, and arrangements of specific neurons. The floor plate arises from dorsal ectodermal cells closely associated with the mesoderm that forms notochord, and it may depend on interactions from the notochord for its specification. To learn the nature of these interactions we have analysed mutations in zebrafish (Brachydanio rerio). We report here that in wild-type embryos the floor plate develops as a simply organized single cell row, but that its development fails in embryos bearing the newly discovered zygotic lethal 'cyclops' mutation, cyc-1(b16). Mosaic analysis establishes that cyc-1 blocks floor plate development autonomously and reveals the presence of homeogenetic induction between floor plate cells.

Developmental regulation of islet-1 mRNA expression during neuronal differentiation in embryonic zebrafish.

Abstract: Islet-1 (Zsl-1) is a LIM domain/ homeodomain-type transcription regulator that has been originally identified as an insulin gene enhancer binding protein Id-1 is also expressed by subsets of neurons in the central nervous sys- tem of rat and chick embryos We have cloned the ZsZ-1 cDNA from zebrafish and examined its ex- pression pattern using in situ hybridization to whole-mount embryos 191-1 mRNA first appears immediately after gastrulation in the polster, the cranial ganglia, and in Rohon-Beard neurons and ventromedial cells of the spinal cord The expres- sion by the ventromedial cells is segmentally re- peated and becomes restricted to the one or two cells slightly anterior to the segment borders Double staining by in situ hybridization and an antibody which stains most axons suggested that these segmentally distributed cells may be either the rostra1 primary motoneuron (RoP) or middle primary motoneuron (MiP) This raises a possibil- ity that 191-1 may be involved during determina- tion of subtype identities of the primary motoneu- rons Furthermore, the specific Id-1 mRNA expression in the spinal cord is under the control of the somites, since mutant embryo with defec- tive somite failed to maintain this pattern

Expression of a type II collagen gene in the zebrafish embryonic axis

Abstract: To understand the hierarchy of developmental controls underlying axis specification in vertebrate embryos, it is helpful to identify relationships between regulatory molecules and the genes that given axial cells their differentiated phenotypes. This work reports the cloning and expression pattern of one of these differentiation genes, a type II collagen (col2a1) gene from the zebrafish Danio rerio. Along th8e embryonic axis, col2a1 is expressed dynamically in three rows that are each a single cell wide: the notochord and the rows of cells immediately dorsal and ventral to it—the floor plate of the central nervous system, and the hypochord. In addition, col2a1 is expressed in the pharyngeal arches, the epithelium of the otic capsule, and in the mesenchyme of the neurocranium. Experiments probed the expression pattern of col2a1 relative to that of known or potential regulators of axis development, including axial, sonic hedgehog, twist, and cyclops. The results showed that the spatial and temporal pattern of col2a1 expression in axial mesoderm follows the expression of twist closer than other genes tested. In cyclops embryos, which lack an intact floor plate, col2a1 expression was usually low, but not missing in cells in the ventral spinal cord. Because col2a1 expression reveals abnormalities in the notochord of cyclopsb16 embryos, and less col2a1-expressing mesenchyme accumulates rostral to the notochord in cyclops embryos, the effects of the cyclopsb16 mutation are not confined to the central nervous system. ©1995 Wiley-Liss, Inc.

Diversity of expression of engrailed-like antigens in zebrafish.

Abstract: We have studied developmental expression of zebrafish engrailed-like (Eng) antigens. Many cell types are reproducibly labeled by two antibodies that recognize the Eng homeodomain, but other cells are labeled by only one or the other, suggesting a hitherto unrecognized complexity of Eng proteins. Expression patterns vary remarkably according to cell type and location. In the undifferentiated primordia of the brain and of each myotome, expression by a stripe of cells spatially subdivides the primordium at a location where a morphological boundary forms later, suggesting expression may be required for development of the boundaries. Supporting this hypothesis, trunk myotomal cells that express Eng are absent in spt-1 mutant embryos, just where the myotomal boundaries fail to form. Another pattern is present in rhombomeres, pharyngeal arches, and the pectoral girdle. In each of these cases, cells (neuron, muscle, cartilage) generating a subset of a series of repeated elements selectively express Eng. These subsets then form specialized derivatives, suggesting Eng homeoproteins are involved in determining the specializations. Epidermal expression is present in the ventral half of the pectoral fin rudiment, precisely 'compartmentalizing' the fin. Neuronal cells at a certain dorsoventral level in each hindbrain and spinal cord segment selectively express Eng, suggesting segmental control of neuronal identity. Specific expression patterns are observed in taste buds, otic vesicles and teeth. Thus we propose that eng genes function in diverse cell types in zebrafish, but play selector roles that can be classified into a few basic types.

Cell tracking using a photoconvertible fluorescent protein

Abstract: The tracking of cell fate, shape and migration is an essential component in the study of the development of multicellular organisms. Here we report a protocol that uses the protein Kaede, which is fluorescent green after synthesis but can be photoconverted red by violet or UV light. We have used Kaede along with confocal laser scanning microscopy to track labeled cells in a pattern of interest in zebrafish embryos. This technique allows the visualization of cell movements and the tracing of neuronal shapes. We provide illustrative examples of expression by mRNA injection, mosaic expression by DNA injection, and the creation of permanent transgenic fish with the UAS-Gal4 system to visualize morphogenetic processes such as neurulation, placode formation and navigation of early commissural axons in the hindbrain. The procedure can be adapted to other photoconvertible and reversible fluorescent molecules, including KikGR and Dronpa; these molecules can be used in combination with two-photon confocal microscopy to specifically highlight cells buried in tissues. The total time needed to carry out the protocol involving transient expression of Kaede by injection of mRNA or DNA, photoconversion and imaging is 2-8 d.

Stages of embryonic development of the zebrafish.

Abstract: We describe a series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad periods of embryogenesis--the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the changing spectrum of major developmental processes that occur during the first 3 days after fertilization, and we review some of what is known about morphogenesis and other significant events that occur during each of the periods. Stages subdivide the periods. Stages are named, not numbered as in most other series, providing for flexibility and continued evolution of the staging series as we learn more about development in this species. The stages, and their names, are based on morphological features, generally readily identified by examination of the live embryo with the dissecting stereomicroscope. The descriptions also fully utilize the optical transparancy of the live embryo, which provides for visibility of even very deep structures when the embryo is examined with the compound microscope and Nomarski interference contrast illumination. Photomicrographs and composite camera lucida line drawings characterize the stages pictorially. Other figures chart the development of distinctive characters used as staging aid signposts.

Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function.

Abstract: Targeted gene disruption in the mouse shows that the Sonic hedgehog (Shh) gene plays a critical role in patterning of vertebrate embryonic tissues, including the brain and spinal cord, the axial skeleton and the limbs. Early defects are observed in the establishment or maintenance of midline structures, such as the notochord and the floorplate, and later defects include absence of distal limb structures, cyclopia, absence of ventral cell types within the neural tube, and absence of the spinal column and most of the ribs. Defects in all tissues extend beyond the normal sites of Shh transcription, confirming the proposed role of Shh proteins as an extracellular signal required for the tissue-organizing properties of several vertebrate patterning centres.

Effective targeted gene ‘knockdown’ in zebrafish

Abstract: The sequencing of the zebrafish genome should be completed by the end of 2002. Direct assignment of function on the basis of this information would be facilitated by the development of a rapid, targeted 'knockdown' technology in this model vertebrate. We show here that antisense, morpholino-modified oligonucleotides (morpholinos) are effective and specific translational inhibitors in zebrafish. We generated phenocopies of mutations of the genes no tail (ref. 2), chordin (ref. 3), one-eyed-pinhead (ref. 4), nacre (ref. 5) and sparse (ref. 6), removing gene function from maternal through post-segmentation and organogenesis developmental stages. We blocked expression from a ubiquitous green fluorescent protein (GFP) transgene, showing that, unlike tissue-restricted limitations found with RNA-based interference in the nematode, all zebrafish cells readily respond to this technique. We also developed also morpholino-based zebrafish models of human disease. Morpholinos targeted to the uroporphyrinogen decarboxylase gene result in embryos with hepatoerythropoietic porphyria. We also used morpholinos for the determination of new gene functions. We showed that embryos with reduced sonic hedgehog (ref. 9) signalling and reduced tiggy-winkle hedgehog (ref. 10) function exhibit partial cyclopia and other specific midline abnormalities, providing a zebrafish genetic model for the common human disorder holoprosencephaly. Conserved vertebrate processes and diseases are now amenable to a systematic, in vivo, reverse-genetic paradigm using zebrafish embryos.