Cadherin-4 plays a role in the development of zebrafish cranial ganglia and lateral line system.

We previously reported that cadherin-4 (also called R-cadherin) was expressed by the majority of the developing zebrafish cranial and lateral line ganglia. Cadherin-4 (Cdh4) function in the formation of these structures in zebrafish was studied using morpholino antisense technology. Differentiation of the cranial and lateral line ganglia and lateral line nerve and neuromasts of the cdh4 morphants was analyzed using multiple neural markers. We found that a subset of the morphant cranial and lateral line ganglia were disorganized, smaller, with reduced staining, and/or with altered shape compared to control embryos. Increased cell death in the morphant ganglia likely contributed to these defects. Moreover, cdh4 morphants had shorter lateral line nerves and a reduced number of neuromasts, which was likely caused by disrupted migration of the lateral line primordia. These results indicate that Cdh4 plays a role in the normal formation of the zebrafish lateral line system and a subset of the cranial ganglia.     

Cadherin-2 function in the cranial ganglia and lateral line system of developing zebrafish.

Cadherins are cell surface molecules that mediate cell-cell adhesion through homophilic interactions. Cadherin-2 (also called N-cadherin), a member of classic cadherin subfamily, has been shown to play important roles in development of a variety of tissues and organs, including the nervous system. We recently reported that cadherin-2 was strongly expressed by the majority of cranial ganglia and lateral line system of developing zebrafish. To gain insight into cadherin-2 role in the formation of these structures, we have used several markers to analyze zebrafish embryos injected with a specific cadherin-2 antisense morpholino oligonucleotide (cdh2MO). We find that development of several cranial ganglia, including the trigeminal, facial, and vagal ganglia, and the lateral line ganglia and neuromasts of the cdh2MO-injected embryos are severely disrupted. These phenotypes were confirmed by analyzing a cadherin-2 mutant, glass onion. Our results suggest that cadherin-2 function is crucial for the normal formation of the zebrafish lateral line system and a subset of cranial ganglia.     

Cell adhesion molecule cadherin‐6 function in zebrafish cranial and lateral line ganglia development

Cadherins regulate the vertebrate nervous system development. We previously showed that cadherin‐6 message (cdh6) was strongly expressed in the majority of the embryonic zebrafish cranial and lateral line ganglia during their development. Here, we present evidence that cdh6 has specific functions during cranial and lateral line ganglia and nerve development. We analyzed the consequences of cdh6 loss‐of‐function on cranial ganglion and nerve differentiation in zebrafish embryos. Embryos injected with zebrafish cdh6 specific antisense morpholino oligonucleotides (MOs, which suppress gene expression during development; cdh6 morphant embryos) displayed a specific phenotype, including (i) altered shape and reduced development of a subset of the cranial and lateral line ganglia (e.g., the statoacoustic ganglion and vagal ganglion) and (ii) cranial nerves were abnormally formed. These data illustrate an important role for cdh6 in the formation of cranial ganglia and their nerves. Developmental Dynamics 240:1716–1726, 2011. © 2011 Wiley‐Liss, Inc.     

Activation of canonical Wnt/β‐catenin signaling stimulates proliferation in neuromasts in the zebrafish posterior lateral line

Background: The posterior lateral line in zebrafish develops from a migrating primordium that deposits clusters of cells that differentiate into neuromasts at regular intervals along the trunk. The deposition of these neuromasts is known to be coordinated by Wnt and FGF signals that control the proliferation, migration, and organization of the primordium. However, little is known about the control of proliferation in the neuromasts following their deposition. Results: We show that pharmacological activation of the Wnt/β‐catenin signaling pathway with 1‐azakenpaullone upregulates proliferation in neuromasts post‐deposition. This results in increased size of the neuromasts and overproduction of sensory hair cells. We also show that activation of Wnt signaling returns already quiescent supporting cells to a proliferative state in mature neuromasts. Additionally, activation of Wnt signaling increases the number of supporting cells that return to the cell cycle in response to hair cell damage and the number of regenerated hair cells. Finally, we show that inhibition of Wnt signaling by overexpression of dkk1b suppresses proliferation during both differentiation and regeneration. Conclusions: These data suggest that Wnt/β‐catenin signaling is both necessary and sufficient for the control of proliferation of lateral line progenitors during development, ongoing growth of the neuromasts, and hair cell regeneration. Developmental Dynamics 242:832–846, 2013. © 2013 Wiley Periodicals, Inc.     

Cell proliferation in the developing lateral line system of zebrafish embryos.

The sensory organs of the embryonic lateral line system are deposited by migrating primordia that originate in the otic region. Here, we examine the pattern of cell proliferation in the posterior lateral line system. We conclude that three phases of cell proliferation are involved in the generation of this system, separated by two phases of mitotic quiescence. The first phase corresponds to generalized proliferation during gastrulation, followed by a first period of quiescence that may be related to the determination of the lateral line precursor cells. A second phase of proliferation takes place in the placode and migrating primordium. This region is organized in annuli that correspond to the expression of proneural/neurogenic genes. A second period of quiescence follows, corresponding to deposition and differentiation of the sensory organs. The third period of proliferation corresponds to continued renewal of hair cells by division of support cells within each sensory organ.     

Mitotic patterns in the migrating lateral line cells of zebrafish embryos

The sense organs of the posterior lateral line system (neuromasts) are formed by a migrating primordium. In zebrafish, the primordium comprises approximately 100 cells at the onset of migration, and has deposited approximately 300 cells by the end of the process. Here, we report localized phases of mitotic activity and of mitotic quiescence within the migrating primordium. Quiescence in the leading region seems associated to the formation of a new prospective neuromast, whereas quiescence in the trailing region follows a wave of mitoses that synchronize trailing cells in G0/G1 phase, anticipating neuromast differentiation. Manipulating the size of the primordium does not lead to changes in the rate of cell proliferation. We also show that two mitoses often take place nearly synchronously in adjacent cells, suggestive of a determinate lineage. We conclude that proliferation in the migrating primordium follows a stereotyped pattern that closely anticipates the normal development of the system. Developmental Dynamics 238:1042–1051, 2009. © 2009 Wiley‐Liss, Inc.     

The development of muscle fiber type identity in zebrafish cranial muscles

Cranial skeletal muscles underlie breathing, eating, and eye movements. In most animals, at least two types of muscle fibers underlie these critical functions: fast and slow muscle fibers. We describe here the anatomical distribution of slow and fast twitch muscle in the zebrafish (Danio rerio) head in the adult and at an early larval stage just after feeding has commenced. We found that all but one of the cranial muscles examined contain both slow and fast muscle fibers, but the relative proportion of slow muscle in each varies considerably. As in the trunk, slow muscle fibers are found only in an anatomically restricted zone of each muscle, usually on the periphery. The relative proportion of slow and fast muscle in each cranial muscle changes markedly with development, with a pronounced decrease in the proportion of slow muscle with ontogeny. We discuss our results in relation to the functional roles of each muscle in larval and adult life and compare findings among a variety of vertebrates.     

Frequency response properties of lateral line superficial neuromasts in a vocal fish,with evidence for acoustic sensitivity

Laryngeal adductor responses to afferent stimulation play a key role in airway protection. Although vital for protection during cough and swallow, these responses also must be centrally controlled to prevent airway obstruction by laryngospasm during prolonged stimulation. Our purpose was to determine the role of N-methyl-D-aspartate (NMDA) receptors in modulating early R1 responses (at 9 ms) and/or later more prolonged R2 responses (at 36 ms) during electrical stimulation of the laryngeal afferent fibers contained in the internal branch of the superior laryngeal nerve in the cat. The percent occurrence, amplitude, and conditioning of muscle responses to single superior laryngeal nerve (SLN) stimuli presented in pairs at interstimulus intervals of 250 ms were measured in three experiments: 1) animals that had ketamine as anesthetic premedication were compared with those who did not, when both were maintained under alpha-chloralose anesthesia. 2) The effects of administering ketamine in one group of animals were compared with increasing the depth of alpha-chloralose anesthesia without NMDA receptor blockade in another group of animals. 3) The effects of dextromethorphan (without anesthetic effects) were examined in another group of animals. In the first experiment, the occurrence of R2 responses were reduced from 95% in animals without ketamine premedication to 25% in animals with ketamine premedication (P = 0.015). No differences occurred in the occurrence, amplitude, latency, or conditioning effects on R1 responses between these groups. In the second experiment, the occurrence of R2 responses was reduced from 96 to 79% after an increase in the depth of anesthesia with alpha-chloralose in contrast with reductions in R2 occurrence from 98 to 19% following the administration of ketamine to induce NMDA receptor blockade along with increased anesthesia (P = 0.025). In the third experiment, R2 occurrence was reduced from 89 to 27% (P = 0.017) with administration of dextromethorphan while R1 response occurrence and amplitude did not change. In each of these experiments, NMDA receptor blockade did not have significant effects on cardiac or respiratory rates in any of the animals. The results demonstrate that NMDA receptors play an essential role in long latency R2 laryngeal responses to laryngeal afferent stimulation. On the other hand, early R1 laryngeal adductor responses are likely to involve non-NMDA receptor activation.     

Postembryonic neural proliferation in the zebrafish forebrain and its relationship to prosomeric domains

Large gaps of knowledge exist regarding postembryonic brain morphogenesis of the zebrafish Danio rerio (Cyprinidae, Teleostei). The zebrafish represents together with the frog (Xenopus), chick and mouse--one of four major models for the genetic study of early brain development. Here, we used normal silver-stained Bodian material and immunohistochemical material stained with a monoclonal antibody against the proliferating cell nuclear antigen (PCNA, cyclin) to study the morphogenetic appearance and location of proliferation zones of the zebrafish brain between day 1 and day 10, focussing on the forebrain at day 5 postfertilization. Our results directly demonstrate that the dorsal telencephalic proliferation zone (i.e. the pallium) extends--consistent with the process of eversion--some distance laterally on top of the telencephalon. The subpallial telencephalic proliferation consists of dorsal and ventral zones. The preoptic region also includes dorsal and ventral proliferation zones. In the diencephalon proper, separate proliferation zones are present in the habenula, and in the periventricular cell masses of the dorsal thalamus, the ventral thalamus, and the pretectum. More ventrocaudally, the latter three massive proliferation zones appear to be replaced each by thinner, but distinct proliferation zones. Two of them represent ventrocaudal continuations of the dorsal and ventral thalamus and lie in the region referred to as the posterior tubercular area in adult teleostean neuroanatomy. The third lies in the region of the nucleus of the medial longitudinal fascicle. In addition, several hypothalamic proliferation zones are present. The data for the diencephalon are largely in agreement with the neuromeric model of brain organization of Puelles and Rubenstein (1993), which is mostly based on amniote data. Generally, the understanding of the prosomeric origin of teleostean prosencephalic cell masses may be regarded as pivotal for their comparative interpretation.     

Single‐cell analysis of somatotopic map formation in the zebrafish lateral line system

The zebrafish lateral line is a simple sensory system comprising a small number of neurons in addition to their sensory organs, the neuromasts. We have adopted this system as a model for single‐cell level analyses of topographic map formation and examined when and how the lateral line topographic map is established. Single‐neuron labeling demonstrated that somatotopic organization of the ganglion emerges by 54 hr postfertilization, but also that this initial map is not as accurate as that observed at 6 days postfertilization. During this initial stage, individual neurons exhibit extensively diverse behavior and morphologies. We identified leader neurons, the axons of which are the first to reach the tail, and later‐appearing axons that contribute to the initial map. Our data suggest that lateral line neurons are heterogeneous from the beginning of lateral line development, and that some of them are intrinsically fate determined to contribute to the somatotopic map. Developmental Dynamics 239:2058–2065, 2010. © 2010 Wiley‐Liss, Inc.     

In vivo physiological recording from the lateral line of juvenile zebrafish

AbstractHair cells are sensory receptors responsible for transducing auditory and vestibular information into electrical signals, which are then transmitted with remarkable precision to afferent neurons. The zebrafish lateral line is emerging as an excellent in vivo model for genetic and physiological analysis of hair cells and neurons. However, research has been limited to larval stages because zebrafish become protected from the time of independent feeding under European law (from 5.2 days post‐fertilization (dpf) at 28.5°C). In larval zebrafish, the functional properties of most of hair cells, as well as those of other excitable cells, are still immature. We have developed an experimental protocol to record electrophysiological properties from hair cells of the lateral line in juvenile zebrafish. We found that the anaesthetic benzocaine at 50 mg l⁻¹ was an effective and safe anaesthetic to use on juvenile zebrafish. Concentrations up to 300 mg l⁻¹ did not affect the electrical properties or synaptic vesicle release of juvenile hair cells, unlike the commonly used anaesthetic MS‐222, which reduces the size of basolateral membrane K⁺ currents. Additionally, we implemented a method to maintain gill movement, and as such respiration and blood oxygenation, via the intubation of > 21 dpf zebrafish. The combination of benzocaine and intubation provides an experimental platform to investigate the physiology of mature hair cells from live zebrafish. More generally, this method would allow functional studies involving live imaging and electrophysiology from juvenile and adult zebrafish.

Abbreviations

dpf

days post‐fertilization

MET

mechanoelectrical transducer

OHC

outer hair cell

PLLg

posterior lateral line ganglion

wpf

weeks post fertilization

     

Somite development in zebrafish.

A full understanding of somite development requires knowledge of the molecular genetic pathways for cell determination as well as the cellular behaviors that underlie segmentation, somite epithelialization, and somite patterning. The zebrafish has long been recognized as an ideal organism for cellular and histological studies of somite patterning. In recent years, genetics has proven to be a very powerful complementary approach to these embryological studies, as genetic screens for zebrafish mutants defective in somitogenesis have identified over 50 genes that are necessary for normal somite development. Zebrafish is thus an ideal system in which to analyze the role of specific gene products in regulating the cell behaviors that underlie somite development. We review what is currently known about zebrafish somite development and compare it where appropriate to somite development in chick and mouse. We discuss the processes of segmentation and somite epithelialization, and then review the patterning of cell types within the somite. We show directly, for the first time, that muscle cell and sclerotome migrations occur at the same time. We end with a look at the many questions about somitogenesis that are still unanswered.     

Relationship between surrounding tissue morphology and directional collective migration of the posterior lateral line primordium in zebrafish

Collective cell migration, in which cells assemble and move together, is an essential process in embryonic development, wound healing and cancer metastasis. Chemokine signaling guides cell assemblies to their destinations. In zebrafish posterior lateral line primordium (PLLP), a model system for collective cell migration, it has been proposed that the chemokine ligand Cxcl12a secreted from muscle pioneer cells (MPs) and muscle fast fibers (MFFs), which are distributed along with the horizontal midline, binds to the receptor Cxcr4b in PLLP and that Cxcl12a–Cxcr4b signaling guides the anterior‐to‐posterior migration of PLLP along the horizontal midline. However, how the surrounding tissues affect PLLP migration remains to be elucidated. Here, we investigated the relationship between the PLLP and the surrounding tissues and found that a furrow between the dorsal and ventral myotomes is generated by Sonic hedgehog (Shh) signaling‐dependent MP and MFF differentiation and that the PLLP migrates in this furrow. When transient inhibition of Shh signaling impaired both the furrow formation and differentiation of cxcl12a‐expressing MPs/MFFs, directional PLLP migration was severely perturbed. Furthermore, when differentiated MPs and MFFs were ablated by femtosecond laser irradiations, the furrow remained and PLLP migration was relatively unaffected. These results suggest that the furrow formation between the dorsal and ventral myotomes is associated with the migratory behavior of PLLP.     

The transmembrane inner ear (tmie) gene contributes to vestibular and lateral line development and function in the zebrafish (Danio rerio)

The inner ear is a complex organ containing sensory tissue, including hair cells, the development of which is not well understood. Our long-term goal is to discover genes critical for the correct formation and function of the inner ear and its sensory tissue. A novel gene, transmembrane inner ear (Tmie), was found to cause hearing-related disorders when defective in mice and humans. A homologous tmie gene in zebrafish was cloned and its expression characterized between 24 and 51 hours post-fertilization. Embryos injected with morpholinos (MO) directed against tmie exhibited circling swimming behavior (approximately 37%), phenocopying mice with Tmie mutations; semicircular canal formation was disrupted, hair cell numbers were reduced, and maturation of electrically active lateral line neuromasts was delayed. As in the mouse, tmie appears to be required for inner ear development and function in the zebrafish and for hair cell maturation in the vestibular and lateral line systems as well.     

Physiology of afferent neurons in larval zebrafish provides a functional framework for lateral line somatotopy

Fishes rely on the neuromasts of their lateral line system to detect water flow during behaviors such as predator avoidance and prey localization. Although the pattern of neuromast development has been a topic of detailed research, we still do not understand the functional consequences of its organization. Previous work has demonstrated somatotopy in the posterior lateral line, whereby afferent neurons that contact more caudal neuromasts project more dorsally in the hindbrain than those that contact more rostral neuromasts (Gompel N, Dambly-Chaudiere C, Ghysen A. Development 128: 387-393, 2001). We performed patch-clamp recordings of afferent neurons that contact neuromasts in the posterior lateral line of anesthetized, transgenic larval zebrafish (Danio rerio) to show that larger cells are born earlier, have a lower input resistance, a lower spontaneous firing rate, and tend to contact multiple neuromasts located closer to the tail than smaller neurons, which are born later, have a higher input resistance, a higher spontaneous firing rate, and tend to contact single neuromasts. We suggest that early-born neurons are poised to detect large stimuli during the initial stages of development. Later-born neurons are more easily driven to fire and thus likely to be more sensitive to local, weaker flows. Afferent projections onto identified glutamatergic regions in the hindbrain lead us to hypothesize a novel mechanism for lateral line somatotopy. We show that afferent fibers associated with tail neuromasts respond to stronger stimuli and are wired to dorsal hindbrain regions associated with Mauthner-mediated escape responses and fast, avoidance swimming. The ability to process flow stimuli by circumventing higher-order brain centers would ease the task of processing where speed is of critical importance. Our work lays the groundwork to understand how the lateral line translates flow stimuli into appropriate behaviors at the single cell level.     

Maternal factors in zebrafish development.

All processes that occur before the activation of the zygotic genome at the midblastula transition are driven by maternal products, which are produced during oogenesis and stored in the mature oocyte. Upon egg activation and fertilization, these maternal factors initiate developmental cascades that carry out the embryonic developmental program. Even after the initiation of zygotic gene expression, perduring maternal products continue performing essential functions, either together with other maternal factors or through interactions with newly expressed zygotic products. Advances in zebrafish research have placed this organism in a unique position to contribute to a detailed understanding of the role of maternal factors in early vertebrate development. This review summarizes our knowledge on the processes involved in the production and redistribution of maternal factors during zebrafish oogenesis and early development, as well as our understanding of the function of these factors in axis formation, germ layer and germ cell specification, and other early embryonic processes.