Anterior Lateral Line Research Articles

Expression patterns of prune2 is regulated by Notch and retinoic acid signaling pathways in the zebrafish embryogenesis

PRUNE2 has been identified as a susceptible gene for Alzheimer's disease and a marker for leiomyosarcomas. Isoforms of Prune2 regulate neuronal cell differentiation and synaptogenesis. Although expression pattern of Prune2 has been reported in the murine brain, its expression patterns and regulation along vertebrate embryogenesis remain to be further investigated. We thus defined the expression profiles and transcriptional regulation of prune2 in zebrafish embryos. prune2 exhibits maternal expression, but is increased in later embryonic stages, and expressed in the telencephalon, epiphysis cluster, nucleus of the tract of the post optic commissure, spinal cord, cerebellum, tegmentum, anterior lateral line ganglion, posterior lateral line ganglion and rhombomeres 2 through 5. Two color WISH with a post-mitotic neuron specific marker, huC defined that prune2 is expressed in the post mitotic neurons. The level of prune2 transcripts is upregulated in Notch signaling homozygous mutant, mib1−/−(mibta52b), indicating that Notch signaling regulates transcription of prune2. Interestingly, in silico analysis of prune2 promoter found retinoic acid (RA) response elements (AGGTCAcaTGACCA) located at −3 to −16 relative to the first exon. It turned out that RA signaling altered the expression pattern of prune2 in the hindbrain. We further propose that Prune2 might be a putative regulator for CNS development in zebrafish embryogenesis.

An optimized method to assess ototoxic effects in the lateral line of zebrafish (Danio rerio) embryos

In order to clarify the suitability of the lateral line of zebrafish (Danio rerio) embryos as a model for the screening of ototoxic (neurotoxic) effects, existing neuromast assays were adapted, improved and validated with a series of chemicals known or unknown for their ototoxic potential (caffeine copper sulfate, dichlorvos, 2.4-dinitrotoluene, neomycin, 4-nonylphenol, perfluorooctanesulfonic acid). Present methods were improved by (1) the introduction of a 4-step scoring system, (2) the selection of neuromasts from both the anterior and posterior lateral line systems, (3) a combined DASPEI/DAPI staining applied after both a continuous and pulse exposure scenario, and (4) an additional screening for nuclear fragmentation. Acute toxicities of the model substances were determined by means of the fish embryo test as specified in OECD TG 236, and EC10 concentrations were used as the highest test concentration in the neuromast assay. The enhanced neuromast assay identified known ototoxic substances such as neomycin and copper sulfate as ototoxic at sensitivities similar to those of established methods, with pulse exposure leading to stronger effects than continuous exposure. Except for caffeine, all substances tested (dichlorvos, 2.4-dinitrotoluene, 4-nonylphenol, perfluorooctanesulfonic acid) produced significant toxic effects in neuromasts at EC10 concentrations. Depending on the test substances and their location along the lateral line, specific neuromasts differed in sensitivity. Generally, neuromasts proved more sensitive in the pulse exposure scenario. Whereas for neomycin and copper sulfate neuromasts located along the anterior lateral line were more sensitive, posterior lateral line neuromasts proved more sensitive for the other test substances. Nuclear fragmentation could not only be associated with all test substances, but, albeit at lower frequencies, also with negative controls, and could, therefore, not be assigned specifically to chemical damage. The study thus documented that for a comprehensive evaluation of lateral line damage both neuromasts from the anterior and the posterior lateral line have to be considered. Given the apparently rapid regeneration of hair cells, pulse exposure seems more appropriate for the identification of lateral line neurotoxicity than continuous exposure.

The anterior talofibular ligament: A detailed morphological study

The anterior talofibular ligament (ATFL) is commonly injured and may result in ankle instability. Good results from ATFL reconstruction have been reported; however complications and movement restrictions have also been observed. ATFL differences have been reported; however details of its precise bony attachment are lacking. This study provides a detailed morphology of the ATFL with respect to surgical and clinical applications. ATFL morphology, number of bands and the exact insertion points were studied in 50 formaldehyde embalmed feet. ATFL length was measured in different joint positions to assess its functional role: ATFL length varied from 18.81mm in dorsiflexion to 21.06mm in plantarflexion: mid-length width and thickness were 4.97mm and 1.01mm respectively. The bony attachment lengths were also measured: mean proximal and distal bony attachment lengths were 4.68mm and 3.1mm respectively, while 13.04mm had no bony attachment. One (22.9%), two (56.3%) and three (20.8%) band morphologies were observed originating 10.37mm anterosuperior to the lateral malleolar tip and inserting 3.92mm anterior to the anterior lateral malleolar line (ALML). Detailed morphology of the ATFL may help in restoring injured ATFL function by appropriate ligament reconstruction, as well as aid the understanding of the mechanism of ligament injury.

Anterior lateral line nerve encoding to tones and play back vocalisations in free swimming oyster toadfish, Opsanus tau

In the underwater environment, sound propagates both as a pressure wave and as particle motion, with particle motion dominating close to the source. At the receptor level, the fish ear and the neuromast hair cells act as displacement detectors, and both are potentially stimulated by the particle motion component of sound. The encoding of the anterior lateral line nerve to acoustic stimuli in freely behaving oyster toadfish, Opsanus tau, was examined. Nerve sensitivity and directional responses were determined using spike rate and vector strength analysis, a measure of phase-locking of spike times to the stimulus waveform. All units showed greatest sensitivity to 100 Hz stimulus. While sensitivity was independent of stimuli orientation, the neuron's ability to phase-lock was correlated with stimuli origin. Two different types of units were classified, type 1 (tonic), and type 2 (phasic). The type 1 fibres were further classified into two sub-types based on their frequency response (type 1-1 and type 1-2), which was hypothesised to be related to canal (type 1-1) and superficial (type 1-2) neuromast innervation. Lateral line units also exhibited sensitivity and phase locking to boatwhistle vocalisations, with greatest spike rates exhibited at the onset of the call. These results provide direct evidence that oyster toadfish can use their lateral line to detect behaviourally relevant acoustic stimuli, which could provide a sensory pathway to aid in sound source localisation.

Time-Lapse Analysis of Primordium Migration during the Development of the Fish Lateral Line

The lateral line is a mechanosensory system that comprises a set of discrete sense organs called neuromasts, which are arranged in reproducible patterns on the surface of fish and amphibians. The system is used by these animals to extract information from the movements of water around their body, providing them with a sense of "touch-at-a-distance" that is involved in most aspects of fish behavior. Each neuromast has a core of mechanosensory hair cells, each of which is depolarized by water motion in one direction and hyperpolarized by motion in the other direction. Based on the position of their ganglion, two components of the lateral-line system can be distinguished: the anterior lateral-line (ALL) system comprises the neuromasts on the head and has its ganglion just anterior to the otic vesicle, the posterior lateral-line (PLL) system comprises the neuromasts on the body and tail and has its ganglion just posterior to the otic vesicle. The peripheral location of the PLL system makes it accessible and easily visualized by imaging methods. The PLL develops from the migrating primordium and so can be used to examine various aspects of neural development, including the control of long-range, collective cell migration and the mechanisms underlying the establishment of appropriate connectivity. Here we discuss imaging methods for exploring these processes.

Recurrent facial taste neurons of sea catfish Plotosusjaponicus: morphology and organization in the ganglion

This study investigated the morphology of the recurrent facial taste neurons and their organization in the recurrent ganglion of the sea catfish Plotosus japonicus. The recurrent ganglion is independent of the anterior ganglion, which consists of trigeminal, facial and anterior lateral line neurons that send peripheral fibres to the head region. The recurrent taste neurons are round or oval and bipolar, with thick peripheral and thin central fibres, and completely wrapped by membranous layers of satellite cells. Two peripheral nerve branches coursing to the trunk or pectoral fin originate from the recurrent ganglion. The results presented here show that the trunk and pectoral-fin neurons are independently distributed to form various sizes of groups, and the groups are intermingled throughout the ganglion. No distinct topographical relationship of the two nerve branches occurs in the ganglion. Centrally, the trunk and pectoral-fin branches project somatotopically in the anterolateral and intermediate medial regions of the trunk tail lobule of the facial lobe, respectively.

Zebrafish Foxi1 provides a neuronal ground state during inner ear induction preceding the Dlx3b/4b-regulated sensory lineage

Vertebrate inner ear development is a complex process that involves the induction of a common territory for otic and epibranchial precursors and their subsequent segregation into otic and epibranchial cell fates. In zebrafish, the otic-epibranchial progenitor domain (OEPD) is induced by Fgf signaling in a Foxi1- and Dlx3b/4b-dependent manner, but the functional differences of Foxi1 and Dlx3b/4b in subsequent cell fate specifications within the developing inner ear are poorly understood. Based on pioneer tracking (PioTrack), a novel Cre-dependent genetic lineage tracing method, and genetic data, we show that the competence to embark on a neuronal or sensory fate is provided sequentially and very early during otic placode induction. Loss of Foxi1 prevents neuronal precursor formation without affecting hair cell specification, whereas loss of Dlx3b/4b inhibits hair cell but not neuronal precursor formation. Consistently, in Dlx3b/4b- and Sox9a-deficient b380 mutants almost all otic epithelial fates are absent, including sensory hair cells, and the remaining otic cells adopt a neuronal fate. Furthermore, the progenitors of the anterior lateral line ganglia also arise from the OEPD in a Foxi1-dependent manner but are unaffected in the absence of Dlx3b/4b or in b380 mutants. Thus, in addition to otic fate Foxi1 provides neuronal competence during OEPD induction prior to and independently of the Dlx3b/4b-mediated sensory fate of the developing inner ear.

Developmental change in neuromast distribution in the anterior lateral line of muskellunge (Esox masquinongy)

The lateral line system of teleost fish is comprised of individual sensory elements, neuromasts, which together form a mechanosensory system. Because neuromasts have been studied in relatively few species, the biology of the lateral line in diverse taxa of fishes remains an open area of inquiry. Musky are predatory fish native to central North America; these prized game fish have been extensively propagated and introduced to many locations. This study investigated the development of neuromasts in the anterior lateral line system of muskellunge. I hypothesized that as juvenile muskellunge grow, the number of neuromasts in the anterior lateral line would increase. Live staining with DASPEI, fluorescence microscopy and image analysis were used to determine the number and spatial distribution of head neuromasts in developing muskellunge. Results indicate that the number of head neuromasts increases over early development; specifically, neuromast number increases in the pre‐orbital line, a sub‐region of the anterior lateral line. This change was further determined to occur via increased neuromast density, as opposed to elongation of the preorbital line. These findings contribute specifically to understanding of muskellunge sensory biology and generally to development of the lateral line system. This work was supported by Westminster College's Drinko Center for Experiential Learning.

Electroreception in Elasmobranchs: Sawfish as a Case Study

The ampullae of Lorenzini are the electroreceptors of elasmobranchs. Ampullary pores located in the elasmobranch skin are each connected to a gel-filled canal that ends in an ampullary bulb, in which the sensory epithelium is located. Each ampulla functions as an independent receptor that measures the potential difference between the ampullary pore opening and the body interior. In the elasmobranch head, the ampullary bulbs of different ampullae are aggregated in 3–6 bilaterally symmetric clusters, which can be surrounded by a connective tissue capsule. Each cluster is innervated by one branch of the anterior lateral line nerve (ALLN). Only the dorsal root of the ALLN carries electrosensory fibers, which terminate in the dorsal octavo-lateral nucleus (DON) of the medulla. Each ampullary cluster projects into a distinctive area in the central zone of the DON, where projection areas are somatotopically arranged. Sharks and rays can possess thousands of ampullae. Amongst other functions, the use of electroreception during prey localization is well documented. The distribution of ampullary pores in the skin of elasmobranchs is influenced by both the phylogeny and ecology of a species. Pores are grouped in distinct pore fields, which remain recognizable amongst related taxa. However, the density of pores within a pore field, which determines the electroreceptive resolution, is influenced by the ecology of a species. Here, I compare the pore counts per pore field between rhinobatids (shovelnose rays) and pristids (sawfish). In both groups, the number of ampullary pores on the ventral side of the rostrum is similar, even though the pristid rostrum can comprise about 20% of the total length. Ampullary pore numbers in pristids are increased on the upper side of the rostrum, which can be related to a feeding strategy that targets free-swimming prey in the water column. Shovelnose rays pin their prey onto the substrate with their disk, while repositioning their mouth for ingestion and thus possess large numbers of pores ventrally around the mouth and in the area between the gills.

Visualization and exploration of Tcf/Lef function using a highly responsive Wnt/β-catenin signaling-reporter transgenic zebrafish

Evolutionarily conserved Tcf/Lef transcription factors (Lef1, Tcf7, Tcf7l1, and Tcf7l2) mediate gene expression regulated by Wnt/β-catenin signaling, which has multiple roles in early embryogenesis, organogenesis, adult tissue homeostasis, and tissue regeneration. However, the spatiotemporal dynamics of Tcf/Lef activity during these events remain poorly understood. We generated stable transgenic zebrafish lines carrying a new Wnt/β-catenin signaling reporter, Tcf/Lef-miniP:dGFP. The reporter revealed the transcriptional activities of four Tcf/Lef members controlled by Wnt/β-catenin signaling, which were expressed in known Wnt/β-catenin signaling-active sites during embryogenesis, organ development and growth, and tissue regeneration. We used the transgenic lines to demonstrate the contribution of Tcf/Lef-mediated Wnt/β-catenin signaling to the development of the anterior lateral line, dorsal and secondary posterior lateral lines, and gill filaments. Thus, these reporter lines are highly useful tools for studying Tcf/Lef-mediated Wnt/β-catenin signaling-dependent processes.

Aportación de una gammacámara portátil en la localización intraoperatoria del ganglio centinela en el cáncer de mama

AimTo show our experience using the portable gamma camera during surgery in the detection of the sentinel node in breast cancer. Material and methodsA total of 46 women diagnosed with breast cancer were included. This study was carried out by means of the periareolar intradermal injection. Planar images and SPECT/CT were acquired. On the following day, the sentinel nodes were localized intraoperatively with a portable gamma camera and a gamma probe. Images of the anterior axillary line projection and lateral axillary line projection were acquired before and after extraction of all the sentinel nodes. ResultsThe images acquired with the portable gamma-camera during the operation showed no activity in all of the patients after the removal of the sentinel nodes. In four out of the 46 cases, the portable gamma camera provided relevant information during the procedure (it detected a sentinel node in two cases in which it had not been detected prior to the surgery and it oriented the surgeon in the localization of low activity nodes when the probe screening had been negative). ConclusionsThe use of the intraoperative portable gamma camera to detect the sentinel node in breast cancer patients is useful in order to ensure, independently of the surgeon, that all the sentinel nodes have been removed. It can also help guide the surgeon in the event of a single low activity sentinel node.

Heterogeneity and dynamics of lateral line afferent innervation during development in zebrafish (Danio rerio)

The lateral line system of larval zebrafish is emerging as a model to study a range of topics in neurobiology, from hair cell regeneration to sensory processing. However, despite numerous studies detailing the patterning and development of lateral line neuromasts, little is known about the organization of their connections to afferent neurons and targets in the hindbrain. We found that as fish grow and neuromasts proliferate over the body surface, the number of afferent neurons increases linearly. The number of afferents innervating certain neuromasts increases over time, while it decreases for other neuromasts. The ratio of afferent neurons to neuromasts differs between the anterior and posterior lateral line system, suggesting potential differences in sensitivity threshold or spatial resolution. A single afferent neuron routinely contacts a group of neuromasts, suggesting that different afferent neurons can convey information about receptive fields along the body. When afferent projections are traced into the hindbrain, where a distinct somatotopy has been previously described, we find that this general organization is absent at the Mauthner cell. We speculate that directional input from the lateral line is less important at an early age, whereas the speed of the escape response is paramount, and that directional responses arise later in development. By quantifying morphological connections in the lateral line system, this study provides a detailed foundation to understand how hydrodynamic information is processed and ultimately translated into appropriate motor behaviors.

Morphological structure and peripheral innervation of the lateral line system in the Siberian sturgeon (Acipenser baerii)

Light and scanning electron microscopy (SEM) were used to study the epidermal lateral line system of the Siberian sturgeon (Acipenser baerii Brandt, 1869). This system consists of mechanoreceptive neuromasts, ampullae and the electroreceptive organ. The neuromasts are located in 5 pairs of cephalic and 1 pair of trunk canals and superficially in the middle and posterior pit lines that lie dorsomedially along the top of the skull immediately adjacent to the otic ampullae field. Both canal neuromasts and pit organ superficial neuromasts have opposite polarized hair cells that are parallel along the axis of the canal and pit line, respectively. However, they differ in both size and shape and in the density and length of the hair bundles. The ampullae are confined on the head, adjacent to the neuromast lines. The morphological structure of the ampullae in the Siberian sturgeon is similar to the ampullae in elasmobranchs and other primitive fish. Nevertheless, it has a relatively large mucus-filled ampulla, and a shorter and narrower canal leading to a small opening to the outer epidermal surface. We also present new information concerning the peripheral innervation of lateral line receptors in sturgeons. The receptors of the lateral line system are innervated by 2 pairs of cranial nerves: anterior and posterior lateral line nerves. The peripheral processes of the anterior lateral line nerve form superficial ophthalmic, buccal, otic and anteroventral rami. The peripheral processes of the posterior lateral line nerve form middle, supratemporal and lateral rami.

Ambiguities in the identification of batoid lateral line systems clarified by innervation

Innervation of the lateral line canal system in seven batoid species (representing seven families in three orders) provided a reliable basis for the identification of each canal element. Previous topographic definitions of the canal elements have failed to recognize homologies within batoids for the scapular and hyomandibular canals. The former is innervated by the posterior lateral line nerve and the latter is innervated by an external mandibular branch of the anterior lateral line nerve. This indicates that the scapular canal is represented only by the scapular loop in Myliobatoidei.

Characterisation, expression and ontogeny of interleukin-6 and its receptors in zebrafish (Danio rerio)

Interleukin-6 (IL-6) is one of the most pleiotropic cytokines due to its importance in both innate and adaptive immune responses and other physiological processes. In this study, we identified the zebrafish (Danio rerio) IL-6 homologue by investigating the synteny between the human (Homo sapiens), the fugu (Takifugu rubripes) and the zebrafish genome. Although zebrafish IL-6 showed a low sequence homology with other IL-6 sequences in other species, it presented a high structural similarity to human IL-6. We also analysed IL-6 expression in several different tissues, along with analysis of the expression of the genes that form the IL-6 receptor complex, IL-6R and gp130. After treatment with bacterial or viral stimuli, zebrafish IL-6 expression was modulated in a manner similar to that of other proinflammatory molecules, such as IL-1β and TNF-α. The expression of IL-6, IL-6R and gp130 was also studied during the ontogeny of zebrafish larvae using quantitative PCR and in situ hybridisation. Our results indicated that the transcripts were detected very early, increased during the first week of life and were predominantly expressed in the head, epidermis and neuromasts of the anterior and posterior lateral line system, suggesting their involvement in the normal development of these tissues.

Expression patterns of lgr4 and lgr6 during zebrafish development

Leucine-rich repeat (LRR)-containing G protein-coupled receptors (LGRs) belong to the superfamily of G protein-coupled receptors, and are characterized by the presence of seven transmembrane domains and an extracellular domain that contains a series of LRR motifs. Three Lgr proteins – Lgr4, Lgr5, and Lgr6 – were identified as members of the LGR subfamily. Mouse Lgr4 has been implicated in the formation of various organs through regulation of cell proliferation during development, and Lgr5 and Lgr6 are stem cell markers in the intestine or skin. Although the expression of these three genes has already been characterized in adult mice, their expression profiles during the embryonic and larval development of the organism have not yet been defined. We cloned two zebrafish lgr genes using the zebrafish genomic database. Phylogenetic analyses showed that these two genes are orthologs of mammalian Lgr4 and Lgr6. Zebrafish lgr4 is expressed in the neural plate border, Kupffer’s vesicle, neural tube, otic vesicles, midbrain, eyes, forebrain, and brain ventricular zone by 24 h post-fertilization (hpf). From 36 to 96 hpf, lgr4 expression is detected in the midbrain–hindbrain boundary, otic vesicles, pharyngeal arches, cranial cartilages such as Meckel’s cartilages, palatoquadrates, and ceratohyals, cranial cavity, pectoral fin buds, brain ventricular zone, ciliary marginal zone, and digestive organs such as the intestine, liver, and pancreas. In contrast, zebrafish lgr6 is expressed in the notochord, Kupffer’s vesicle, the most anterior region of diencephalon, otic vesicles, and the anterior and posterior lateral line primordia by 24 hpf. From 48 to 72 hpf, lgr6 expression is confined to the anterior and posterior neuromasts, otic vesicles, pharyngeal arches, pectoral fin buds, and cranial cartilages such as Meckel’s cartilages, ceratohyals, and trabeculae. Our results provide a basis for future studies aimed at analyzing the functions of zebrafish Lgr4 and Lgr6 in cell differentiation and proliferation during organ development.

Transgene excision in zebrafish using the phiC31 integrase

AbstractSingle optical section of a 72 hour post‐fertilization T2K‐XpGbR (EF1‐alpha‐attP‐GFP‐attB‐DsRed‐Express) transgenic zebrafish larva obtained by laser scanning confocal microscopy. Injection of phiC31 integrase messenger RNA at the one‐cell stage induces recombination of the transgene in a mosaic fashion, resulting in excision of the green fluorescent protein cassette and expression of DsRed fluorescent protein. Here recombination is evident in the lens and neuromasts of the anterior lateral line. See the article by Lister in this issue.

Ultrastructure and distribution of superficial neuromasts of blind cavefish, Phreatichthys andruzzii, juveniles

Transmission and scanning electron microscopy (TEM, SEM) were used to study the ultrastructure of superficial neuromasts in 15 six-month old blind cavefish juveniles, Phreatichthys andruzzii (Cyprinidae). In five specimens examined with SEM, the number of superficial neuromasts over the fish body (480-538) was recorded. They were localized mainly on the head (362-410), including the dorsal surface, the mentomandibular region, and laterally from the mouth to the posterior edge of the operculum. Neuromasts were also present laterally on the trunk and near the caudal fin (116-140). A significantly higher number of neuromasts were present on the head compared to the trunk (t-test, P < 0.05). Superficial neuromasts of the head and those along the trunk were similar in ultrastructure. Each neuromast comprised sensory hair cells surrounded by nonsensory support cells (mantle cells and supporting basal cells) with the whole covered by a cupula. Each hair cell was pear-shaped, 15-21 microm high and 4-6 microm in diameter, with a single long kinocilium and several short stereocilia. Most support cells were elongated, with nuclei occupying a large portion of the cytoplasm. In the margin of the neuromast, mantle cells were particularly narrow. Both types of support cells had well-developed Golgi apparatus and rough endoplasmic reticulum. The number of hair cells and nonsensory support cells of the anterior lateral line (head) did not differ significantly from those of the posterior lateral line (trunk) (t-test, P > 0.05).

Multisensory integration in the tadpole medulla arises from discrete and overlapping regions of endorgan projection.

Vestibular and auditory nuclei in the medulla are inherently multimodal in developing bullfrogs (Rana catesbeiana), responding to pressure, particle motion, and lateral line input. While brainstem and midbrain connectivity of these nuclei have been well mapped across metamorphic development, developmental changes in projection patterns from individual endorgans in the otic capsule (sacculus, utricle, lagena, and amphibian and basilar papilla) to the medulla are less well understood. Using small (26 nl) focal injections of neurobiotin in preganglionic branchlets for specific endorgans, we were able to map input from the separate peripheral organs to different medullary nuclei (medial, superior and lateral vestibular nuclei, the dorsal medullary nucleus, and the anterior lateral line nucleus). Some secondary trans‐synaptic label of the reticular gray and cerebellum was also noted. Projection patterns are correlated with different patterns of medullary responses to vibratory and to pressure stimulation. Endo...

Development of the central and peripheral nervous systems in the lamprey

Vertebrate brains are highly organized structures that show remarkable diversity throughout the animal groups. Among the vertebrates, the agnathan animals, which diverged from the gnathostomes early in the evolution of the vertebrates, occupy a key phylogenetic position in order to clarify the origin and evolution of the brain. We found that the lamprey brain has the basic molecular mechanisms necessary to form brain compartments. Conversely, the telencephalon and cerebellum display gnathostome-specific developmental mechanisms. We also propose that, in contrast to those of gnathostomes, the maxillary ramus of the trigeminal nerve and buccal ramus of the nerve on the anterior lateral line are not fused in the developing lamprey. Thus, the development of the central nervous system and the framework of the peripheral nerve around the oral region are thought to have improved in the course of the agnathan-gnathostome transition.