Kohei Hatta

Role of the floor plate in axonal patterning in the zebrafish CNS

To determine the role of the floor plate (FP) in CNS development, I have used labeling techniques, including immunolabeling, to analyze cyclops mutant embryos, which lack the FP. Except for the anterior brain, the mutant phenotype is almost exclusively confined to the vicinity of the ventral CNS midline. In the midbrain, the number of ventral neurons is reduced and cell patterning is disturbed. In contrast, the neuronal arrangement in the spinal cord is almost normal, including in particular both primary and secondary motoneurons. Longitudinal axonal bundles are disorganized in both the brain and spinal cord. Laser ablating the FP in wild-type embryos locally phenocopies cyclops axonal disturbances, and transplanting wild-type FP precursor cells into mutants locally rescues the disturbances. These results demonstrate a significant role for the FP in pathfinding and fasciculation by axons in situ, especially during their longitudinal courses.

Planar polarization of node cells determines the rotational axis of node cilia

Rotational movement of the node cilia generates a leftward fluid flow in the mouse embryo because the cilia are posteriorly tilted. However, it is not known how anterior-posterior information is translated into the posterior tilt of the node cilia. Here, we show that the basal body of node cilia is initially positioned centrally but then gradually shifts toward the posterior side of the node cells. Positioning of the basal body and unidirectional flow were found to be impaired in compound mutant mice lacking Dvl genes. Whereas the basal body was normally positioned in the node cells of Wnt3a−/− embryos, inhibition of Rac1, a component of the noncanonical Wnt signalling pathway, impaired the polarized localization of the basal body in wild-type embryos. Dvl2 and Dvl3 proteins were found to be localized to the apical side of the node cells, and their location was polarized to the posterior side of the cells before the posterior positioning of the basal body. These results suggest that posterior positioning of the basal body, which provides the posterior tilt to node cilia, is determined by planar polarization mediated by noncanonical Wnt signalling.

Cell tracking using a photoconvertible fluorescent protein

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.

E-cadherin is required for gastrulation cell movements in zebrafish

E-cadherin is a member of the classical cadherin family and is known to be involved in cell-cell adhesion and the adhesion-dependent morphogenesis of various tissues. We isolated a zebrafish mutant (cdh1(rk3)) that has a mutation in the e-cadherin/cdh1 gene. The mutation rk3 is a hypomorphic allele, and the homozygous mutant embryos displayed variable phenotypes in gastrulation and tissue morphogenesis. The most severely affected embryos displayed epiboly delay, decreased convergence and extension movements, and the dissociation of cells from the embryos, resulting in early embryonic lethality. The less severely affected embryos survived through the pharyngula stage and showed flattened anterior neural tissue, abnormal positioning and morphology of the hatching gland, scattered trigeminal ganglia, and aberrant axon bundles from the trigeminal ganglia. Maternal-zygotic cdh1(rk3) embryos displayed epiboly arrest during gastrulation, in which the enveloping layer (EVL) and the yolk syncytial layer but not the deep cells (DC) completed epiboly. A similar phenotype was observed in embryos that received antisense morpholino oligonucleotides (cdh1MO) against E-cadherin, and in zebrafish epiboly mutants. Complementation analysis with the zebrafish epiboly mutant weg suggested that cdh1(rk3) is allelic to half baked/weg. Immunohistochemistry with an anti-beta-catenin antibody and electron microscopy revealed that adhesion between the DCs and the EVL was mostly disrupted but the adhesion between DCs was relatively unaffected in the MZcdh1(rk3) mutant and cdh1 morphant embryos. These data suggest that E-cadherin-mediated cell adhesion between the DC and EVL plays a role in the epiboly movement in zebrafish.

Early axonal contacts during development of an identified dendrite in the brain of the zebrafish.

We have identified the initial synaptic contacts made onto the Mauthner (M) cell, an identified neuron that arises during early development of the zebrafish hindbrain. The contacts are made by a small bundle of pioneering trigeminal sensory axons onto the M cell soma before it forms dendrites. The sensory bundle is then partially enveloped by the M cell. The lateral dendrite appears at about the site of the contact, and eventually the trigeminal inputs are shifted to its trunk. As the dendrite elongates, other sensory contacts are made on its distal regions, sequentially from the acoustico-vestibular nerve and the lateral line nerves. To learn whether the earliest inputs induce the initial outgrowth of the M cell dendrite, we ablated the trigeminal neurons by laser irradiation before they contacted the M cell. Morphogenesis of the M cell, including its dendrite, appeared normal.

Visualizing neurons one‐by‐one in vivo: Optical dissection and reconstruction of neural networks with reversible fluorescent proteins

A great many axons and dendrites intermingle to fasciculate, creating synapses as well as glomeruli. During live imaging in particular, it is often impossible to distinguish between individual neurons when they are contiguous spatially and labeled in the same fluorescent color. In an attempt to solve this problem, we have taken advantage of Dronpa, a green fluorescent protein whose fluorescence can be erased with strong blue light, and reversibly highlighted with violet or ultraviolet light. We first visualized a neural network with fluorescent Dronpa using the Gal4‐UAS system. During the time‐lapse imaging of axonal navigation, we erased the Dronpa fluorescence entirely; re‐highlighted it in a single neuron anterogradely from the soma or retrogradely from the axon; then repeated this procedure for other single neurons. After collecting images of several individual neurons, we then recombined them in multiple pseudo‐colors to reconstruct the network. We have also successfully re‐highlighted Dronpa using two‐photon excitation microscopy to label individual cells located inside of tissues and were able to demonstrate visualization of a Mauthner neuron extending an axon. These “optical dissection” techniques have the potential to be automated in the future and may provide an effective means to identify gene function in morphogenesis and network formation at the single cell level. Developmental Dynamics 235:2192–2199, 2006.

Physiological properties of the mauthner system in the adult zebrafish

We investigated the morphological and electrophysiological properties of the Mauthner (M‐) cell and its networks in the adult zebrafish (Danio rerio) in comparison with those in the goldfish (Carassius auratus). The zebrafish M‐cell has an axon cap, a high resistivity structure which surrounds the initial segment of the M‐axon, and accounts for an unusual amplification of the fields generated within and around it. Second, extra‐ and intracellular recordings were performed with microelectrodes. The resting potential was ∼−80 mV with an input resistance of ∼0.42 MΩ. The M‐cell extracellular field was large (10‐20 mV), close to the axon hillock, and the latency of antidromic spikes short (∼0.4 milliseconds), confirming a high conduction velocity in the M‐axon. The extrinsic hyperpolarizing potential (EHP), which signals firing of presynaptic cells and collateral inhibition, was markedly lower at frequencies of spinal stimulation > ∼5/second, suggesting an organization of the recurrent collateral network similar to that in the goldfish. Inhibitory postsynaptic potentials (IPSPs) were highly voltage‐dependent; their decay time constant was increased by depolarizations. The presynaptic neurons which are numerous could be identified by their passive hyperpolarizing potential (PHP) produced by the M‐spike current. Auditory responses, mediated via mixed synapses (electrical and chemical), had short delays and hence are well suited to trigger the escape reaction. The similarities of their properties indicate that the wealth of information generated over decades in the goldfish can be extrapolated to the zebrafish. J. Comp. Neurol. 395:493–509, 1998.

Infrared laser-mediated local gene induction in medaka, zebrafish and Arabidopsis thaliana

Heat shock promoters are powerful tools for the precise control of exogenous gene induction in living organisms. In addition to the temporal control of gene expression, the analysis of gene function can also require spatial restriction. Recently, we reported a new method for in vivo, single‐cell gene induction using an infrared laser‐evoked gene operator (IR‐LEGO) system in living nematodes (Caenorhabditis elegans). It was demonstrated that infrared (IR) irradiation could induce gene expression in single cells without incurring cellular damage. Here, we report the application of IR‐LEGO to the small fish, medaka (Japanese killifish; Oryzias latipes) and zebrafish (Danio rerio), and a higher plant (Arabidopsis thaliana). Using easily observable reporter genes, we successfully induced gene expression in various tissues in these living organisms. IR‐LEGO has the potential to be a useful tool in extensive research fields for cell/tissue marking or targeted gene expression in local tissues of small fish and plants.

Secondary axis induction by heterospecific organizers in zebrafish

To investigate the inductive activities of the vertebrate organizer, we transplanted the chicken organizer (Hensens node) into zebrafish gastrula and analyzed resulting secondary axes. Grafted Hensens node did not differentiate or participate in the secondary axis. It also did not induce a secondary notochord or expression of the genes normally expressed by the fish organizer including no tail, axial, goosecoid. Nevertheless, it recruited fish cells to organize a variety of tissues: the dorsal portion of the central nervous system including Rohon‐Beard sensory neurons, otic vesicles, dorsal pigment stripe, dorsal fin, somites, heart, and pronephric ducts. Enlarged neural plate induced by the organizer was shown by the expression pattern of dlx3 and msxB genes, which demarcates the early presumptive neural tissue. In addition, Hensens node of an earlier stage chicken embryo displayed differential movement in zebrafish from that of a later stage. This might reflect unknown differences in properties between the organizer at two different developmental stages related to its normal organizer activity. To create a model system to study the molecular mechanisms of the organizer, we next transplanted genetically modified mouse cells into zebrafish embryos. We found that Wnt3A‐transfected NIH3T3 cells are much more potent in inducing a secondary axis than NIH3T3 cells alone. These results suggest that formation of a variety of tissues are controlled by signalling from the organizer itself with no requirement of participation of the organizer‐derived tissues. Additionally, the activities of the organizer may involve a function of Wnt‐family genes.

Patterning of body segments of the zebrafish embryo

Publisher Summary This chapter discusses the patterning of body segments of the zebrafish embryo. Descriptive studies in zebrafish have provided exceptionally clear information, often at the level of individual cells, about the structure, extent in the body, and development of the segments. Experimental analyses, including the use of mutations, reveal cellular interactions required for segmentation. Information from zebrafish provides a useful background for understanding how genes make vertebrate segments, an issue for the future, and for which this species also holds some promise. The developmental studies of zebrafish strengthen the notion that vertebrates seem to be metameric creatures. Attempts to dispel this notion in the past have pointed up the limited extent of vertebrate body segmentation. Segmentation does indeed seem to be limited, but it is even limited in annelids that are certainly segmented animals and that show no hint of segmental organization in lineages that produce the gut or the skin, organs that also appear unsegmented in vertebrates. Accordingly, the presence of nonsegmented tissues within a segmented body plan does not seem particularly problematic for the common segmented ancestor hypothesis; a most interesting question for both development and evolution is how the apparently unsegmented axial sets of cells, the notochord and the floor plate, come to be insinuated into the metameres. These axial tissues have dominant developmental roles and certainly have also been extremely important in chordate evolution.