Rohon-Beard Sensory Neurons Research Articles

Isolation and expression of a novel neuron-specific onecut homeobox gene in zebrafish

A complete cDNA of a novel zebrafish gene named onecut has been isolated; this gene encodes a protein of 446 amino acids with a Cut domain (73 amino acid residues) and a homeodomain. The Cut domain of zebrafish Onecut is highly similar to those in mammalian hepatocyte nuclear factor-6 and Drosophila Onecut, sharing 90 and 88% amino acid identity, respectively. The expression of zebrafish onecut is restricted to neuronal cells, being first detected in trigeminal ganglia neurons at the end of gastrulation. By the 1-somite stage, onecut expression has begun in primary neurons of the lateral stripes in the neural plate, and appeared in neuronal cells of the medial stripes at the 2-somite stage. By the 4-somite stage, onecut expression expanded to the intermediate stripes and to subsets of neuronal cells in the midbrain and hindbrain. Subsequently, onecut expression intensified in the lateral region of midbrain and hindbrain, yet no onecut-positive cells were seen in the telencephalon. By 24 hpf, onecut transcripts remained abundant in the spinal cord but were no longer detectable in differentiated Rohon–Beard sensory neurons. The expression of onecut was greatly increased in the neural mutant mindbomb, while being decreased in narrowminded.

Zebrafish deadly seven Functions in Neurogenesis

In a genetic screen, we isolated a mutation that perturbed motor axon outgrowth, neurogenesis, and somitogenesis. Complementation tests revealed that this mutation is an allele of deadly seven (des). By creating genetic mosaics, we demonstrate that the motor axon defect is non-cell autonomous. In addition, we show that the pattern of migration for some neural crest cell populations is aberrant and crest-derived dorsal root ganglion neurons are misplaced. Furthermore, our analysis reveals that des mutant embryos exhibit a neurogenic phenotype. We find an increase in the number of primary motoneurons and in the number of three hindbrain reticulospinal neurons: Mauthner cells, RoL2 cells, and MiD3cm cells. We also find that the number of Rohon–Beard sensory neurons is decreased whereas neural crest-derived dorsal root ganglion neurons are increased in number supporting a previous hypothesis that Rohon–Beard neurons and neural crest form an equivalence group during development. Mutations in genes involved in Notch–Delta signaling result in defects in somitogenesis and neurogenesis. We found that overexpressing an activated form of Notch decreased the number of Mauthner cells in des mutants indicating that des functions via the Notch–Delta signaling pathway to control the production of specific cell types within the central and peripheral nervous systems.

Islet Reveals Segmentation in the Amphioxus Hindbrain Homolog

The vertebrate embryonic hindbrain is segmented into rhombomeres. Gene expression studies suggest that amphioxus, the closest invertebrate relative of vertebrates, has a hindbrain homolog. However, this region is not overtly segmented in amphioxus, raising the question of how hindbrain segmentation arose in chordate evolution. Vertebrate hindbrain segmentation includes the patterning of cranial motor neurons, which can be identified by their expression of the LIM-homeodomain transcription factor islet1. To learn if the amphioxus hindbrain homolog is cryptically segmented, we cloned an amphioxus gene closely related to islet1, which we named simply islet. We report that amphioxus islet expression includes a domain of segmentally arranged cells in the ventral hindbrain homolog. We hypothesize that these cells are developing motor neurons and reveal a form of hindbrain segmentation in amphioxus. Hence, vertebrate rhombomeres may derive from a cryptically segmented brain present in the amphioxus/vertebrate ancestor. Other islet expression domains provide evidence for amphioxus homologs of the pineal gland, adenohypophysis, and endocrine pancreas. Surprisingly, homologs of vertebrate islet1-expressing spinal motor neurons and Rohon-Beard sensory neurons appear to be absent.

Zebrafish narrowminded suggests a genetic link between formation of neural crest and primary sensory neurons.

In the developing vertebrate nervous system, both neural crest and sensory neurons form at the boundary between non-neural ectoderm and the neural plate. From an in situ hybridization based expression analysis screen, we have identified a novel zebrafish mutation, narrowminded (nrd), which reduces the number of early neural crest cells and eliminates Rohon-Beard (RB) sensory neurons. Mosaic analysis has shown that the mutation acts cell autonomously suggesting that nrd is involved in either the reception or interpretation of signals at the lateral neural plate boundary. Characterization of the mutant phenotype indicates that nrd is required for a primary wave of neural crest cell formation during which progenitors generate both RB sensory neurons and neural crest cells. Moreover, the early deficit in neural crest cells in nrd homozygotes is compensated later in development. Thus, we propose that a later wave can compensate for the loss of early neural crest cells but, interestingly, not the RB sensory neurons. We discuss the implications of these findings for the possibility that RB sensory neurons and neural crest cells share a common evolutionary origin.

Hox11-family genes XHox11 and XHox11L2 in xenopus: XHox11L2 expression is restricted to a subset of the primary sensory neurons.

The mouse genome contains a small family of homeobox genes related to Hox11, but relatively little is known about the expression of these genes during early development. Hox11 itself is expressed in the embryonic spleen, among other tissues, and is required for its formation. No description of Hox11L2 expression has been presented previously. We have isolated the Xenopus orthologs of Hox11 and Hox11L2 and have carefully compared their expression patterns during embryogenesis. The localization of Xhox11 transcripts in the branchial arches, cranial sensory ganglia and spinal cord is similar, but not identical, to that of mouse Hox11. Xhox11 expression is not detected in the developing spleen. XHox11L2 is expressed exclusively in a portion of the primary sensory system in the frog embryo, including the cranial sensory ganglia and the Rohon-Beard sensory neurons. There is significant overlap in the patterns of Xhox11 and XHox11L2 expression in the spinal cord and cranial sensory ganglia during early development, suggesting that they may function redundantly in these tissues. The timing of Xhox11 and Xhox11L2 expression indicates that Hox11-family members may participate in the final stages of the differentiation process.

Hox11‐family genes XHox11 and XHox11L2 in Xenopus: XHox11L2 expression is restricted to a subset of the primary sensory neurons

The mouse genome contains a small family of homeobox genes related to Hox11, but relatively little is known about the expression of these genes during early development. Hox11 itself is expressed in the embryonic spleen, among other tissues, and is required for its formation. No description of Hox11L2 expression has been presented previously. We have isolated the Xenopus orthologs of Hox11 and Hox11L2 and have carefully compared their expression patterns during embryogenesis. The localization of Xhox11 transcripts in the branchial arches, cranial sensory ganglia and spinal cord is similar, but not identical, to that of mouse Hox11. Xhox11 expression is not detected in the developing spleen. XHox11L2 is expressed exclusively in a portion of the primary sensory system in the frog embryo, including the cranial sensory ganglia and the Rohon-Beard sensory neurons. There is significant overlap in the patterns of Xhox11 and XHox11L2 expression in the spinal cord and cranial sensory ganglia during early development, suggesting that they may function redundantly in these tissues. The timing of Xhox11 and Xhox11L2 expression indicates that Hox11-family members may participate in the final stages of the differentiation process. Dev Dyn 1999;214:34–43. © 1999 Wiley-Liss, Inc.

Properties of Ectopic Neurons Induced byXenopusNeurogenin1 Misexpression

We have examined cells cultured from ectoderm-misexpressing Neurogenin1 (Ngn1) to describe better the extent to which this gene can control aspects of neuronal phenotype including motility, morphology, excitability, and synaptic properties. Like primary spinal neurons which normally express Ngn1, cells in Ngn1-misexpressing cultures exhibit a motility-correlated behavior called circus movements prior to neuritogenesis. Misexpression of NeuroD also causes circus movements and later neuronal differentiation. GSK3β, which inhibits NeuroD functionin vivo,blocks both Ngn1-induced and NeuroD-induced neuronal differentiation, while Notch signaling inhibits only Ngn1-induced neuronal differentiation, confirming that NeuroD is downstream of Ngn1 and insensitive to Notch inhibition. While interfering with NeuroD function in ventral ectoderm inhibits both circus movements and neuronal differentiation, such inhibition in the neural plate inhibits only neuronal differentiation, suggesting that additional factors regulate circus movements in the neural ectoderm. Ngn1-misexpressing cells extend N-tubulin-positive neurites and exhibit tetrodotoxin-sensitive action potentials. Unlike the majority of cultured spinal neurons, however, Ngn1-misexpressing cells do not respond to glutamate and do not form functional synapses with myocytes, suggesting that these cells are either like Rohon-Beard sensory neurons or are not fully differentiated.

Interleukin-1beta and its type 1 receptor are expressed in developing neural circuits in the frog, Xenopus laevis.

The cytokine interleukin-1 beta (IL-1beta) is an evolutionarily conserved molecule that was originally identified in the immune system. In addition to regulating peripheral immune responses, IL-1beta plays an important role in mediating neural-immune interactions and regulating glial activities during healing and repair in the damaged nervous system. Active IL-1beta is produced by interleukin-converting enzyme (ICE), a caspase thought to be involved in the induction of apoptosis. We report that, in the developing frog, Xenopus laevis, IL-1beta and the IL-1 type 1 receptor proteins are coexpressed in specific neurons that comprise early sensory-motor circuits. IL-1beta and IL-1 type 1 receptor proteins are colocalized in specific midbrain and hindbrain reticular cells, including Mauthner's neuron; specific cells in the trigeminal (fifth), lateral line (seventh), and vestibular (eighth) cranial ganglia; oculomotor neurons; and the primordial Purkinje cells of the lateral cerebellar auricle. In the spinal cord, Rohon-Beard sensory neurons, dorsal root ganglion cells, and primary motoneurons are immunopositive. Anteriorly, the olfactory pits, olfactory nerves, and olfactory bulbs are labeled, as are retinal cells, especially photoreceptor inner segments. With regard to the function of IL-1beta during neural development, IL-1beta and its type 1 receptor are present throughout the course of neural development in identifiable, long-lived neurons, such as Mauthner's neuron. These and other data suggest that IL-1beta and its type 1 receptor may be involved in the maintenance of cell survival rather than induction of neuronal death.

Efficient mutagenesis of zebrafish by a DNA cross-linking agent

We developed a novel procedure for efficient mutagenesis of zebrafish using a DNA cross-linking agent 4,5′,8-trimethylpsoralen (TMP), which is known to frequently induce small deletions in Escherichia coli and Caenorhabditis elegans. A specific-locus test and pilot screenings indicated that the TMP mutagenesis procedure was efficient. To confirm the successful mutagenesis by TMP, we characterized mutants with selective impairments in the nervous system. The no tectal neuron mutation hindered the development of the tectal neurons, while the edawakare mutation resulted in the enhancement of the extension and branching of the peripheral axons of trigeminal ganglion and Rohon–Beard sensory neurons. These results suggest that the TMP mutagenesis will provide an efficient method to isolate and characterize zebrafish mutants at molecular level.

Efficient mutagenesis of zebrafish by a DNA cross-linking agent

We developed a novel procedure for efficient mutagenesis of zebrafish using a DNA cross-linking agent 4,5',8-trimethylpsoralen (TMP), which is known to frequently induce small deletions in Escherichia coli and Caenorhabditis elegans. A specific-locus test and pilot screenings indicated that the TMP mutagenesis procedure was efficient. To confirm the successful mutagenesis by TMP, we characterized mutants with selective impairments in the nervous system. The no tectal neuron mutation hindered the development of the tectal neurons, while the edawakare mutation resulted in the enhancement of the extension and branching of the peripheral axons of trigeminal ganglion and Rohon-Beard sensory neurons. These results suggest that the TMP mutagenesis will provide an efficient method to isolate and characterize zebrafish mutants at molecular level.

Secondary axis induction by heterospecific organizers in zebrafish.

To investigate the inductive activities of the vertebrate organizer, we transplanted the chicken organizer (Hensen's node) into zebrafish gastrula and analyzed resulting secondary axes. Grafted Hensen's 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, Hensen's 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.

Two distinct rhythmic motor patterns are driven by common premotor and motor neurons in a simple vertebrate spinal cord

Xenopus embryos show two distinct rhythmic motor patterns: swimming and struggling. Both can be generated by spinal cord circuitry and evoked by stimulation of a single skin sensory pathway (Soffe, 1991b). This presents a valuable opportunity to explore mechanisms for vertebrate motor pattern switching. Swimming and struggling have been compared using intracellular recording from spinal neurons in immobilized embryos. Underlying synaptic drive was similar; motoneurons and premotor interneurons were excited in phase with ipsilateral motor root discharge and inhibited in phase with contralateral motor root discharge. Excitation was stronger during struggling and associated with short bursts of impulses, contrasting with single spikes per cycle during swimming. Excitation was reduced in both patterns by local application of 1 mM kynurenic acid, indicating excitatory amino acid mediation. Inhibition was antagonized by 1 microM strychnine, indicating glycine mediation. Many motoneurons (76%) and premotor interneurons (68%) fired during both swimming and struggling, including examples of all three spinal premotor interneuron classes. Most of the remaining motoneurons (20%) and premotor interneurons (24%) fired only during struggling, providing roughly 30% more active neurons than during swimming. To investigate whether new neuronal classes become active during struggling, recordings were made from sensory neurons and sensory interneurons. Rohon-Beard sensory neurons did not fire during either swimming or struggling. Dorsolateral commissural sensory interneurons received rhythmic, strychnine-sensitive inhibition during both swimming and struggling and also did not fire. Neither of these neuronal classes is therefore recruited to the circuitry for struggling. Although behaviorally distinct, Xenopus embryo swimming and struggling motor patterns appear to employ similar synaptic drive. I propose that this reflects the common nature of much of the premotor circuitry that drives them. Extra neurons are recruited to this circuitry during struggling, but only from within classes that also participate in swimming.

Spinal cord neuron classes in embryos of the smooth newt Triturus vulgaris: a horseradish peroxidase and immunocytochemical study.

Spinal cord neurons were investigated in embryos of Triturus vulgaris, the smooth newt, just prior to hatching. These embryos can swim if freed from their egg membranes. Horseradish peroxidase (HRP) labelling, together with GABA and glycine immunocytochemistry (ICC), revealed nine distinct anatomical classes of neuron. 1. Ventrolateral motoneurons with mainly dorsal dendrites, sometimes a descending central axon and peripheral axon innervating the trunk muscles. 2. Dorsal primary sensory Rohon-Beard neurons innervating skin and with dorsal ascending and descending axons in spinal cord. 3. Commissural interneurons with mid-cord unipolar soma, glycine-like immunoreactivity, dendrites on initial segment of ventral axon which crosses cord to ascend or branch. 4. Dorsolateral commissural interneurons with multipolar soma in dorsolateral position with dorsal dendrites and ventral axon which crosses and ascends or branches. 5. Giant dorsolateral commissural interneurons with large dorsolateral somata widely spaced (130-250 microns spacing) with process projecting dorsally to other side, dorsolateral dendrites and ventral axon which crosses to ascend and branch. 6. Dorsolateral ascending interneurons in dorsolateral position with multipolar soma and ascending axon on same side. 7. Ascending interneurons with unipolar soma, GABA-like immunoreactivity and ascending axon on same side. 8. Descending interneurons with bi- or multi-polar soma, extensive dorsal and ventral dendrites, and descending axon on same side. They may also have ascending axons. 9. Kolmer-Agduhr cerebrospinal fluid contacting neurons with cilia and microvilli in lateral corners of neural canal. GABA-like immunoreactivity, no dendrites and ascending axon. Eight of the nine cells classes were found to bear a marked resemblance to neurons previously described in zebrafish and Xenopus embryos in terms of their anatomy, distribution and immunoreactivity to GABA and glycine. Homologies and possible functions are discussed. Giant dorsolateral commissural neurons, were not found in Xenopus or teleosts but were present in Ambystoma mexicanum and Neoceratodus. The regular, possibly segmental longitudinal distribution pattern of these cells within the cord is unusual among amphibian spinal neurons.

Triggering and gating of motor responses by sensory stimulation: behavioural selection inXenopus embryos

Neural mechanisms underlying selection of motor responses are largely unknown in vertebrates. This study shows that in immobilized Xenopus embryos, brief mechanical or electrical stimulation of the trunk skin can trigger sustained fictive swimming, whereas sustained pressure or repetitive electrical stimulation can evoke fictive struggling. These two rhythmic motor patterns are distinct: alternating single motor root spikes propagate from head to tail during swimming; alternating motor root bursts propagate from tail to head during struggling. As both motor patterns can be evoked in embryos with the CNS transected caudal to the cranial roots, the sensory pathway responsible must have direct access to the spinal cord. Rohon-Beard sensory neurons provide the only such pathway known. They respond appropriately to brief stimuli applied to the trunk skin, and also to repetitive electrical stimuli and sustained pressure. The results suggest that Rohon-Beard sensory neurons can both trigger sustained swimming and 'gate in' struggling motor patterns, and thus effect behavioural selection according to their pattern of activity.

Expression of a homeobox gene product in normal and mutant zebrafish embryos: evolution of the tetrapod body plan

An antibody was used to detect antigens in zebrafish that appear to be homologous to the frog homeodomain-containing protein XlHbox 1. These antigens show a restricted expression in the anteroposterior axis and an anteroposterior gradient in the pectoral fin bud, consistent with the distribution of XlHbox 1 protein in frog and mouse embryos. In the somitic mesoderm, a sharp anterior limit of expression coincides exactly with the boundary between somites 4 and 5, and the protein level fades out posteriorly. A similar, graded expression of the antigen is seen within the series of Rohon-Beard sensory neurons of the CNS. We also immunostained the mutant spt-1 ('spadetail'), in which the trunk mesoderm is greatly depleted and disorganized in the region of XlHbox 1 expression. The defects stem from misdirected cell movements during gastrulation, but nervertheless, newly recruited cells that partially refill the trunk mesoderm express the antigen within the normal span of the anteroposterior axis. This finding suggests that the mutation does not delete positional information required for activation of the XlHbox 1 gene.

A neural degeneration mutation that spares primary neurons in the zebrafish

We describe an embryonic lethal mutation in the zebrafish Brachydanio rerio that specifically affects the viability of most cells in the embryonic central nervous system (CNS). The mutation ned-1 (b39rl) was induced with γ-irradiation and segregates as a single recessive allele closely linked to its centromere. It produces massive cell death in the CNS but a small set of specific neurons, including Rohon-Beard sensory neurons, large hindbrain interneurons, and primary motoneurons, survive embryogenesis and are functional. Synaptic connections between embryonic motoneurons and muscle cells appear physiologically normal, and the normally observed spontaneous flexions are present. Correlated with the presence of sensory neurons and interneurons, mutant embryos display reflexive movements in response to mechanical stimulation. Together, the surviving neurons, called primary neurons, form a class of cells that are prominent in size and arise early during development. Thus, this mutation may define a function that is differentially required by developmentally distinguishable sets of cells in the embryonic CNS.