Loss of kidney function underlies many renal diseases1. Mammals can partly repair their nephrons (the functional units of the kidney), but cannot form new ones2,3. By contrast, fish add nephrons throughout their lifespan and regenerate nephrons de novo after injury4,5, providing a model for understanding how mammalian renal regeneration may be therapeutically activated. Here we trace the source of new nephrons in the adult zebrafish to small cellular aggregates containing nephron progenitors. Transplantation of single aggregates comprising 10–30 cells is sufficient to engraft adults and generate multiple nephrons. Serial transplantation experiments to test self-renewal revealed that nephron progenitors are long-lived and possess significant replicative potential, consistent with stem-cell activity. Transplantation of mixed nephron progenitors tagged with either green or red fluorescent proteins yielded some mosaic nephrons, indicating that multiple nephron progenitors contribute to a single nephron. Consistent with this, live imaging of nephron formation in transparent larvae showed that nephrogenic aggregates form by the coalescence of multiple cells and then differentiate into nephrons. Taken together, these data demonstrate that the zebrafish kidney probably contains self-renewing nephron stem/progenitor cells. The identification of these cells paves the way to isolating or engineering the equivalent cells in mammals and developing novel renal regenerative therapies.

In teleosts, cerebellar efferent neurons, known as eurydendroid cells, are dispersed within the cerebellar cortex rather than coalescing into deep cerebellar nuclei. To clarify their morphology, eurydendroid cells were labeled retrogradely by biotinylated dextran amine injection into the base of the corpus cerebelli. Labeling allowed the cells to be classified into three types-fusiform, polygonal, and monopolar-depending on their somal shapes and numbers of primary dendrites. The fusiform and polygonal type cells were distributed not only in the Purkinje cell layer but also in the molecular and granule cell layers. The monopolar type cells were distributed predominantly in the Purkinje cell layer of the ventrocaudal portion of the corpus cerebelli. These results suggest that there are some functional differences between these eurydendroid cell types. The eurydendroid cells were double-labeled by retrograde labeling and immunohistochemistry using specific antibodies against GABA, aspartate, and zebrin II. No GABA-like immunoreactivity was detected in the retrogradely labeled eurydendroid cells. About half of retrogradely labeled cells were immunoreactive to the anti-aspartate antibody, suggesting that some eurydendroid cells utilize aspartate as a neurotransmitter. Zebrin II reacts with cerebellar Purkinje cells but left all retrogradely labeled neurons nonreactive, although some of these were surrounded by immunopositive fibers. This relationship between the eurydendroid and Purkinje cells is similar to that between the deep cerebellar nuclei and Purkinje cells in mammals.

In tetrapods, cerebellar efferent systems are mainly mediated via the cerebellar nuclei. In teleosts, the cerebellum lacks cerebellar nuclei. Instead, the cerebellar efferent neurons, termed eurydendroid cells, are arrayed within and below the ganglionic layer. Tracer injections outside of the cerebellum, which retrogradely label eurydendroid cells demonstrate that most eurydendroid cells possess two or more primary dendrites which extend broadly into the molecular layer. Some eurydendroid cells mostly situated in caudal portions of the cerebellum have only one primary dendrite. The eurydendroid cells receive inputs from the Purkinje cells and parallel fibers, but apparently do not receive inputs from the climbing fibers. Eurydendroid cells of the corpus cerebelli and medial valvula project to many brain regions, from the diencephalon to the caudal medulla. A few eurydendroid cells in the valvula project directly to the telencephalon. About half of the eurydendroid cells are aspartate immunopositive. Anti-GABA and anti-zebrin II antibodies that are known as markers for the Purkinje cells in mammals also recognize the Purkinje cells in the teleost cerebellum, but do not recognize the eurydendroid cells. These results suggest that the eurydendroid cells receive GABAergic inputs from the Purkinje cells. This relationship between the eurydendroid and Purkinje cells is similar to that between the cerebellar nuclei and Purkinje cells in mammals. The eurydendroid cells of teleost have both dissimilar as well as similar features compared to neurons of the cerebellar nuclei in tetrapods.

Efferent fiber connections of the corpus and valvula cerebelli in the goldfish, Carassius auratus, were studied using an anterograde neural fiber tracing technique. Efferent targets of the corpus cerebelli are the posterior parvocellular preoptic nucleus, the ventromedial and ventrolateral thalamic nucleus, dorsal posterior thalamic nucleus, periventricular nucleus of posterior tuberculum, dorsal periventricular pretectal nucleus, inferior lobe, optic tectum, torus semicircularis, nucleus of the medial longitudinal fascicle, nucleus ruber, dorsal tegmental nucleus, nucleus lateralis valvulae, reticular formation, torus longitudinalis, and the medial and lateral lobe of the valvula cerebelli. Projections to the posterior parvocellular preoptic nucleus and the periventricular nucleus of posterior tuberculum are not reported in previous studies. Efferent targets of the medial lobe of the valvula cerebelli are similar to that of the corpus cerebelli except for lacking a projection to the inferior lobe and torus longitudinalis, but showing one to the corpus cerebelli. On the other hand, the lateral lobe of the valvula cerebelli projects only to the dorsal zone of the periventricular hypothalamus, the diffuse nucleus of the inferior lobe, corpus mamillare, vagal lobe and the corpus cerebelli. There are topographical projections from the lateral valvula to the inferior lobe. These results suggest that the function of the corpus and medial lobe of the valvula cerebelli include not only motor control but also functions similar to the mammalian higher cerebellum. This study also suggests that there are obvious functional divisions between the medial and lateral lobes of the valvula cerebelli.

Primary vagal gustatory afferents utilize glutamate as a neurotransmitter acting on AMPA/kainate receptors of second-order neurons. Some forms of ionotropic glutamate receptors permit passage of Ca++ ions upon activation by appropriate ligands. Calcium-binding proteins (CaBPs) play a buffering role for regulating the concentration of intracellular calcium. In the present study, we used immunohistochemistry to examine the distribution and morphology of neurons with CaBPs, including calretinin, calbindin, and parvalbumin, and to compare this distribution with neurons exhibiting Ca++-permeable glutamate receptors as determined by kainate-stimulated uptake of Co++ in the vagal lobe of goldfish. Calretinin- and calbindin-positive neurons occurred throughout the sensory zone including round unipolar, horizontal; and perpendicular bipolar or multipolar somata. Parvalbumin neurons were mainly round monopolar neurons, especially common in the superficial layers of the sensory zone. In the motor zone, while parvalbumin labeled nearly all motoneurons, calretinin labeled only external motoneurons. In double labeling with calretinin and parvalbumin, few neurons in the sensory layer labeled with both antisera. Immunocytochemistry following kainate-stimulate uptake of Co++ showed that most calretinin, but few parvalbumin immunopositive neurons also were labeled by cobalt in the central and deep layers of the sensory zone. All motoneurons were labeled by Co++, including those immunoreactive for calretinin or parvalbumin. These results indicate that calretinin expression is strongly correlated with calcium-permeable ionotropic glutamate receptors in the neurons of the sensory zone of the goldfish vagal lobe, but even within this limited region, not all Ca++-permeable neurons possess any of the CaBPs examined.

In the formation of the spinal network, various transcription factors interact to develop specific cell types. Using a gene trap technique, we established a stable line of zebrafish in which the red fluorescent protein (RFP) was inserted in the pax8 gene. RFP insertion marked putative pax8-lineage cells with fluorescence and inhibited pax8 expression in homozygous embryos. Pax8 homozygous embryos displayed defects in the otic vesicle, as previously reported in studies using morpholinos. The pax8 homozygous embryos survived to adulthood in contrast to mammalian counterparts that die prematurely. RFP is expressed in the dorsal spinal cord. Examination of the axon morphology revealed that RFP (+) neurons include Commissural Bifurcating Longitudinal (CoBL) interneurons, but other inhibitory neurons such as Commissural Local (CoLo) interneurons and Circumferential Ascending (CiA) interneurons do not express RFP. We examined the effect of inhibiting pax2a/pax8 expression on interneuron development. In pax8 homozygous fish, the RFP (+) cells undergo differentiation similar to that of pax8 heterozygous fish, and the swimming behavior remained intact. In contrast, the RFP (+) cells of pax2a/pax8 double mutants displayed altered cell fates. CoBLs were not observed. Instead, RFP (+) cells exhibited axons descending ipsilaterally: a morphology resembling that of V2a/V2b interneurons.

The contraction of skeletal muscle is dependent on synaptic transmission through acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). The lack of an AChR subunit causes a fetal akinesia in humans, leading to death in the first trimester and characteristic features of Fetal Akinesia Deformation Sequences (FADS). A corresponding null mutation of the ␦-subunit in zebrafish (sofa potato; sop) leads to the death of embryos around 5 d postfertilization (dpf). In sop Ϫ/Ϫ mutants, we expressed modified ␦-subunits, with one (␦1YFP) or two yellow fluorescent protein (␦2YFP) molecules fused at the intracellular loop, under the control of an ␣-actin promoter. AChRs containing these fusion proteins are fluorescent, assemble on the plasma membrane, make clusters under motor neuron endings, and generate synaptic current. We screened for germ-line transmission of the transgene and established a line of sop Ϫ/Ϫ fish stably expressing the ␦2YFP. These ␦2YFP/sop Ϫ/Ϫ embryos can mount escape behavior close to that of their wild-type siblings. Synaptic currents in these embryos had a smaller amplitude, slower rise time, and slower decay when compared with wild-type fish. Remarkably, these embryos grow to adulthood and display complex behaviors such as feeding and breeding. To the best of our knowledge, this is the first case of a mutant animal corresponding to first trimester lethality in human that has been rescued by a transgene and survived to adulthood. In the rescued fish, a foreign promoter drove the transgene expression and the NMJ had altered synaptic strength. The survival of the transgenic animal delineates requirements for gene therapies of NMJ.Key words: zebrafish; neuromuscular junction; acetylcholine receptor; synapse; fetal akinesia deformation sequence; fluorescent protein IntroductionThe synapse between the motor nerve and skeletal muscle, commonly referred to as the neuromuscular junction (NMJ), is cholinergic in vertebrates and is a model system for the investigation of synapses (Sanes and Lichtman, 2001;Ono, 2008). On the postsynaptic face of the NMJ, the AChRs span the membrane, with each AChR forming a pentameric structure. The five subunits comprising AChRs are 2␣s, ␤, ␦ and ␥/. ␥ and are developmentally regulated and can substitute for each other. The embryonic-type ␥ is replaced by the adult-type as the synapse matures (Mishina et al., 1986).A group of genetic disorders have mutations in genes coding for components of the NMJ. Congenital myasthenic disorders (CMDs) result from mutations in genes coding for expression of proteins such as AChR, rapsyn, MuSK, and cholinesterase (Engel and Sine, 2005). CMD patients display weak muscle strength resulting from compromised synaptic currents. Functional nulls of AChRs were predicted to be lethal and, indeed, fetuses homozygous for these mutations die in the first trimester (Michalk et al., 2008). Affected fetuses display characteristic anatomical features that are collectively called Fetal Akinesia Deformation Sequences (FADS).Zebrafish (Danio rerio)...

Glycine is a major inhibitory neurotransmitter in the central nervous system of vertebrates. Here, we report the initial development of glycine-immunoreactive (Gly-ir) neurons and fibers in zebrafish. The earliest Gly-ir cells were found in the hindbrain and rostral spinal cord by 20 h post-fertilization (hpf). Gly-ir cells in rhombomeres 5 and 6 that also expressed glycine transporter 2 (glyt2) mRNA were highly stereotyped; they were bilaterally located and their axons ran across the midline and gradually turned caudally, joining the medial longitudinal fascicles in the spinal cord by 24 hpf. Gly-ir neurons in rhombomere 5 were uniquely identified, since there was one per hemisegment, whereas the number of Gly-ir neurons in rhombomere 6 were variable from one to three per hemisegment. Labeling of these neurons by single-cell electroporation and tracing them until the larval stage revealed that they became MiD2cm and MiD3cm, respectively. The retrograde labeling of reticulo-spinal neurons in Tg(glyt2:gfp) larva, which express GFP in Gly-ir cells, and a genetic mosaic analysis with glyt2:gfp DNA construct also supported this notion. Gly-ir cells were also distributed widely in the anterior brain by 27 hpf, whereas glyt2 was hardly expressed. Double staining with anti-glycine and anti-GABA antibodies demonstrated distinct distributions of Gly-ir and GABA-ir cells, as well as the presence of doubly immunoreactive cells in the brain and placodes. These results provide evidence of identifiable glycinergic (Gly-ir/glyt2-positive) neurons in vertebrate embryos, and they can be used in further studies of the neurons' development and function at the single-cell level.