Proneural gene requirement for hair cell differentiation in the zebrafish lateral line

The lateral line system comprises an array of mechanosensory organs, the neuromasts, distributed over the body surface. Each neuromast consists of a patch of mechanosensory hair cells surrounded by support cells. We show that, in the zebrafish, two proneural genes are essential for differentiation of the hair cells, neuroD (nrd) and atonal homolog 1 (ath1). Gene knockdown experiments demonstrate that loss of function of either gene, but not of the related proneural gene neurogenin1 (ngn1), abrogate the appearance of hair cell markers. This is in contrast to other sensory systems, such as the neurons of the lateral line ganglion, where nrd is regulated by ngn1 and not by ath1. Overexpression of ath1 can induce nrd, and the phenotype produced by loss of ath1 function can be partially rescued by injection of nrd mRNA. This supports the conclusion that the activation of nrd probably requires ath1 in the hair cell lineage, whereas in sensory neurons nrd activation requires ngn1. We propose that the emergence of two atonal homologs, ath1 and ngn1, allowed the cellular segregation of mechanoreception and signal transmission that were originally performed by a single cell type as found in insects.     

Immunohistochemical studies on peptide- and amine-containing endocrine cells and nerves in the gut and the rectal gland of the ratfish Chimaera monstrosa

Summary The occurrence and distribution of endocrine cells and nerves were immunohistochemically demonstrated in the gut and rectal gland of the ratfish Chimaera monstrosa (Holocephala). The epithelium of the gut mucosa revealed open-type endocrine cells exhibiting immunoreactivity for serotonin (5HT), gastrin/cholecystokinin (CCK), pancreatic polypeptide (PP)/FMRFamide, somatostatin, glucagon, substance P or gastrin-releasing peptide (GRP). The rectum contained a large number of closed-type endocrine cells in the basal layer of its stratified epithelium; the majority contained 5HT- and GRP-like immunoreactivity in the same cytoplasm, whereas others were immunoreactive for substance P. The rectal gland revealed closed-type endocrine cells located in the collecting duct epithelium. Most of these contained substance P-like immunoreactivity, although some reacted either to antibody against somatostatin or against 5HT. Four types of nerves were identified in the gut and the rectal gland. The nerve cells and fibers that were immunoreactive for vasoactive intestinal peptide (VIP) and GRP formed dense plexuses in the lamina propria, submucosa and muscular layer of the gut and rectal gland. A sparse network of gastrin- and 5HT-immunoreactive nerve fibers was found in the mucosa and the muscular layer of the gut. The present study demonstrated for the first time the occurrence of the closed-type endocrine cells in the mucosa of the rectum and rectal gland of the ratfish. These abundant cells presumably secrete 5HT and/or peptides in response to mechanical stimuli in the gut and the rectal gland. The peptide-containing nerves may be involved in the regulation of secretion by the rectal gland.     

Histology and Ultrastructure of the Cranial Lymphohaemopoietic Tissue in Chimaera monstrosa (Pisces,Holocephali)

In the present ultrastructural study we have extended previous reports on the histological organization and cell components of the lymphohaemopoietic masses occurring in the cranium, mainly in the orbit, the preorbital canal, and the suprapalatal and coiacoid areas of the holocephalan Chimaera monstrosa. Mature and developing granulocytes, monocytes, macrophages, lymphocytes and plasma cells occur in a reticular and/or fibroblastic supporting stroma inside the cartilaginous skeleton. Heterophils, which are the most abundant granulocytes in the cranial tissue, contain two distinct cytoplasmic granular populations, whereas eosinophils show one uniformly electron dense granule type. Heterophils and eosinophils may differentiate from a common precursor producing granules of each cell type in relation to the activity of rough endoplasmic reticulum and Golgi complex. The presence of macrophages, lymphocytes and developing and mature plasma cells suggests an important role of the cranial lymphohaemopoietic tissue in eliciting the immune responses. A phylogenetical relationship between this tissue and the higher vertebrate bone marrow is proposed on the basis of histological similarities between the cell microenvironments governing haemopoietic differentiation in these organs.     

The ultrastructure of cerebral blood capillaries in the raffish,Chimaera monstrosa

Summary Sharks and skates (Chondrichthyes: Elasmobranchii) have a glial blood-brain barrier, while all other vertebrates examined so far have an endothelial barrier. For comparative reasons it is desirable to examine the blood-brain barrier in species from the other subclass of cartilaginous fish, the holocephalans. The ultrastructure of cerebral capillaries in the chimaera (Chondrichthyes: Holocephali) is described in the present study. The endothelial cells are remarkably thick. Fenestrae and transendothelial channels were not observed. The endothelial cells are joined by elaborate tight junctions. The perivascular glial processes are separated by wide spaces (15–60nm) without obliterating junctional complexes. These findings indicate that the chimaera has an endothelial blood-brain barrier.     

The myoseptal system in Chimaera monstrosa: collagenous fiber architecture and its evolution in the gnathostome stem lineage

Recent studies have revealed the 3D morphology and collagen fiber architecture of myosepta in teleostome fishes. Here we present the first data set on the myoseptal structure of a representative of the chondrichthyan clade. We investigate the series of myosepta in the ratfish Chimaera monstrosa (Holocephali) from the anterior to the posterior body using microdissections of cleared and stained specimens, polarized light microscopy of excised myosepta, and histology. The features of the myoseptal system of Chimaera are compared to data from closely related vertebrate groups and are mapped onto a phylogenetic tree to further clarify the characteristics of the myoseptal series in the gnathostome ancestor. The 3D morphology and collagen fiber architecture of the myoseptal series in C. monstrosa resembles that of Teleostomi (Actinopterygii+Sarcopterygii) with regard to several features. Our comparative analysis reveals that some of them have evolved in the gnathostome stem lineage. (1) A series of epineural and epaxial lateral tendons (LTs) along the whole body, and a series of epipleural and hypaxial LTs in the postanal region evolved in the gnathostome stem lineage. (2) The LTs increase in length towards the posterior body (three-fold in Chimaera). Data on Chimaera and some comparative data on actinopterygian fishes indicate that LTs also increase in thickness towards the posterior body, but further data are necessary to test whether this holds true generally. (3) Another conspicuous apomorphic gnathostome feature is represented by multi-layer structures of myosepta. These are formed along the vertebral column by converging medial regions of successive sloping parts of myosepta. (4) The dorsalmost and ventralmost flanking parts of myosepta bear a set of mediolaterally oriented collagen fibers that are present in all gnathostomes but are lacking in outgroups. Preanal hypaxial myosepta are clearly different from epaxial myosepta and postanal hypaxial myosepta in terms of their collagen fiber architecture. In Chimaera, preanal hypaxial myosepta consist of an array of mediolaterally oriented collagen fibers closely resembling the condition in other gnathostome groups and in petromyzontids. Only one series of tendons, the myorhabdoid tendons of the flanking parts of myosepta, have evolved in the stem lineage of Myopterygii (Gnathostomata+Petromyzontida). Similar to LTs, the tendons of this series also increase in length towards the posterior body. In combination with other studies, the present study provides a framework for the design of morphologically based experiments and modeling to further address the function of myosepta and myoseptal tendons in gnathostomes.     

The lateral line system and its innervation in the boxfish Ostracion immaculatus (Tetraodontiformes: Ostraciidae): description and comparisons with other tetraodontiform and perciform conditions

The lateral line system and its innervation were examined in the ostraciid Ostracion immaculatus (Tetraodontiformes), and compared with those in the triacanthodid Triacanthodes anomalus (Tetraodontiformes) and the acropomatid Malakichthys wakiyae (Perciformes). The carapace of O. immaculatus was composed of 6 cephalic and 2 trunk lateral lines, all neuromasts being categorized as “superficial.” Triacanthodes anomalus was identical with O. immaculatus in the absence of the mandibular line and its innervating ramus, whereas in M. wakiyae the line and ramus were present. All neuromasts were “superficial” in the former two, but “canal” in the latter. Judging from the essentially identical lateral line topography and innervation patterns in all three species, the superficial neuromasts in the two tetraodontiforms were considered to have resulted from replacement of canal neuromasts. The number of neuromasts in the cephalic lateral lines of O. immaculatus (106) and T. anomalus (91) were similar, being significantly higher than in M. wakiyae (30). However, the reverse was true for the trunk lateral lines, the two tetraodontiforms having fewer neuromasts (39 in O. immaculatus, 47 in T. anomalus) compared with M. wakiyae (59).     

Object localization through the lateral line system of fish: theory and experiment

Fish acquire information about their aquatic environment by means of their mechanosensory lateral-line system. This system consists of superficial and canal neuromasts that sense perturbations in the water surrounding them. Based on a hydrodynamic model presented here, we propose a mechanism through which fish can localize the source of these perturbations. In doing so we include the curvature of the fish body, a realistic lateral line canal inter-pore distance for the lateral-line canals, and the surface boundary layer. Using our model to explore receptor behavior based on experimental data of responses to dipole stimuli we suggest that superficial and canal neuromasts employ the same mechanism, hence provide the same type of input to the central nervous system. The analytical predictions agree well with spiking responses recorded experimentally from primary lateral-line nerve fibers. From this, and taking into account the central organization of the lateral-line system, we present a simple biophysical model for determining the distance to a source.     

Rapid responses of the cupula in the lateral line of ruffe (<Emphasis Type="Italic">Gymnocephalus cernuus</Emphasis>)

Displacements of cupulae in the supraorbital lateral line canal in ruffe (Gymnocephalus cernuus) have been measured using laser interferometry and by applying transient as well as sinusoidal fluid stimuli in the lateral line canal. The cupular displacement in response to impulses of fluid velocity shows damped oscillations at approximately 120 Hz and a relaxation time-constant of 4.4 ms, commensurate with a quality factor of approximately 1.8. These values are in close agreement with the frequency characteristics determined via sinusoidal fluid stimuli, implying that the nonlinearity of cupular dynamics imposed by the gating apparatus of the sensory hair cells is limited in the range of cupular displacements and velocities measured (100–300 nm; 100–300 m/s). The measurements also show that cupular displacement instantaneously follows the initial waveform of transient stimuli. The functional significance of the observed cupular dynamics is discussed.     

Ultrastructure of lateral line organs in aneurogenic amphibian larvae (Ambystoma)

Summary An ultrastructural study was performed to determine the ability of presumptive lateral line tissue in A. maculatum embryos to migrate and differentiate in the absence of all sensory innervation. In experimental animals, all presumptive neural tissue was surgically removed at Harrison's stage 24, leaving only the posterior portion of the post-auditory placode. At later stages, completely differentiated aneural lateral line organs were found in the midbody region of the larvae. These were morphologically similar to those of control larvae. No signs of atrophy or degeneration of the neurqmasts were seen even after several weeks of nerve-free conditions.The use of the electron microscope of the College of Osteopathic Medicine is gratefully acknowledged. Thanks are due I.M. Korr and G.S.L. Appeltauer for comments and R. Meyers and R. Herbold for technical assistance     

Observations on the lateral-line sense organs of the Salamander Neurergus crocatus crocatus Cope (Amphibia: Urodela)

The lateral-line system of the subadult and adult Neurergus crocatus crocatus Cope is retained throughout the life. It is constructed of pear-shaped sense organs or neuromasts which in the subadults are confined entirely to the epidermis with their apices opening distally to the exterior at the general level of the epidermal surface whereas in the adults they are embedded proximally more than halfway in the dermis with their distal apices opening into shallow grooves slightly below the regular epidermal surface. Dimensional differences are also observed between these organs in both stages. The neuromasts are constructed of 4 distinct cell types: sense cells, basal cells, sustentacular cells and mantle cells. These cells differ morphologically, structurally, topographically and functionally. The sense cells are clup-shaped, sensory, central in position and couched among the extremely elongated supporting basal and sustentacular cells, all of which are covered laterally by the extremely slender protective mantle cells. In both the mature and larvae, differences are observed among these cells in their size, number, location and arrangement. The sense organs are richly-supplied and well-nourished with vascular and nervous supplies.     

First record of the opal chimaera,Chimaera opalescens (Holocephali: Chimaeridae) and revision of the occurrence of the rabbitfish Chimaera monstrosa in the Azores waters

The presence of the opal chimaera, Chimaera opalescens, is reported for the first time in the deep waters of the Azores, with the capture of four specimens by fishermen and the video recording of an additional five individuals. Species identification was supported by the 646 bp sequenced fragment of the mitochondrial gene cytochrome oxidase subunit I. Because C. opalescens is a recently recognised species that had been recurrently misidentified as rabbitfish, Chimaera monstrosa, the historical data of C. monstrosa in the Azores were reviewed to assess the possible presence of both Chimaera species in the region. Although several authors have reported the occurrence of C. monstrosa in the Azorean waters since the 1800s, the majority of these are based on only three specimens caught during the late 1800s. The investigation performed using literature and examination of the museum specimens still available concluded that the most likely scenario is that C. monstrosa is absent from the Azores and past records of that species in the region are most likely misidentifications of C. opalescens.     

Chemical composition of the body fluids andmuscle of the hagfish Myxine glutinosa and the rabbit-fish Chimaera monstrosa

Comprehensive analyses have been made of the plasma and parietal muscle of the marine cyclostome Myxine glutinosa and of the chimaeraoid Chimaera monstrosa , and the sum of some 17 constituents compared with the directly determined osmotic concentration of plasma. Both Myxine and Chimaera plasmas are isosmotic with sea water within 2%. Ions account for at least 96% of the total concentration of the plasma in Myxine and about 60% in Chimaera, but the blood of the latter contains 332 mM urea, compared with 3 mM in Myxine . In muscle a similar divergence exists, the urea concentrations per kilogram muscle water being practically the same as in the respective plasmas of the two species. The mean values in millimoles of the other nitrogenous constituents of muscle are for Myxine and Chimaera (figures for latter in brackets), trimethylamine oxide 87 (189), betaine 65 (38), creatine and creatinine 30 (37), amino acids 291 (43). Using osmotic coefficients the calculated osmotic concentration of muscle exceeds that of plasma in Myxine and possible reasons for this are discussed.
Estimates of inulin space in Myxine muscle have enabled intracellular concentrations of ions to be calculated. Analysis of Myxine bile shows it to have much higher concentrations of sodium, calcium and magnesium, and much lower chloride, than plasma. The resulting large anion deficit is presumably made up chiefly by the bile salt myxinol disulphate.     

Feeding ecology and trophic level of Chimaera monstrosa Linnaeus, 1758 (Holocephali: Chimaeridae) in the Eastern Mediterranean

The feeding ecology of Chimaera monstrosa at a depth of 550?m was studied off S??ac?k Bay, Eastern Mediterranean, in 2008. Forty males and 57 females with a length between 7.80 and 45.5?cm were caught. 33 stomachs contained food items. The diet was composed of 11 prey items belonging to 6 groups: Bryozoa, Cnidaria, Crustacea, Mollusca, Tunicata, and Teleostei, of which Crustacea (IRI% [Index of Relative Importance]=80) and Mollusca (IR%=16) were the most abundant prey groups. The trophic level was estimated at 3.50, which is similar to the results from other studies in the western and northern part of the Mediterranean and the Atlantic Ocean but is higher than in the North Sea.     

On the neurons and synapses of the lateral geniculate nucleus of the monkey,and the effects of eye enucleation

Summary Two neuron types are distinguished by electron microscopy in the lateral geniculate nucleus (LGN) of the monkey-a large cell (P cell) interpreted as a geniculostriate relay cell, and a small cell (I cell) interpreted as an inhibitory interneuron. The I cell, distinguished by its small size, infolded nucleus, small mitochondria, cilium and small granular bodies, forms about 10% of the total neuron population. It could not be determined whether this cell has an axon, but its dendrites, which contain aggregates of flattened vesicles, are thought to form a proportion of the F processes, profiles which are post-synaptic to the retinal (RLP) axons and presynaptic to the dendrites of the P cells. The small dark (RSD) axon terminals of unknown origin contact the dendrites of both cell types.After eye enucleation the P cells of the affected laminae of the LGN shrink and partially withdraw their dendrites from the neuropil. By 29 months' survival, they have only a narrow cytoplasmic rim around the nucleus. A necrotic process also occurs, affecting fine dendrites by 22 days and large profiles by 45 days, but it is not clear whether whole cells are destroyed by this process. At 45 days the I cells are commonly seen to form somatodendritic synapses. The appearance of these synapses is interpreted as the result of a withdrawal to the soma of the presynaptic dendrites.It is concluded that the I cells are probably inhibitory interneurons subject to excitation and presynaptic inhibition by the RLP and RSD axons, and a diagram is presented to demonstrate the possible significance of these connections for the transmission of information through the LGN.The author wishes to thank Dr. J. Campos-Ortega for much practical advice.     

Ultrastructure of the ampullary electroreceptors in lungfish and brachiopterygii

Summary Electron microscopy shows that the electroreceptors of Protopterus dolloi (Dipnoi) and Calamoichthys calabaricus (Brachiopterygii) are very similar to the known ampullary electroreceptors of other primitive fish groups. With Protopterus, however, there is one difference: at the apical surface of the receptor cells, instead of a kinocilium, a club-shaped protrusion is found. The club-shaped protrusion has no fixed number nor a fixed arrangement of microtubules (Fig. 4).The presynaptic ribbons are in direct contact with the presynaptic membrane, and may be invaginations of the presynaptic membrane (Fig. 3).The functional significance of the kinocilium and of the club-shaped protrusion is discussed. Furthermore, a possible biological role of the ampullary electroreceptors in fish coming into frequent contact with the water surface (Dipnoi, Brachiopterygii) is considered.Supported by the Deutsche Forschungsgemeinschaft     

Ultrastructure of synapses and cellular relationships in the oculomotor nucleus of the rhesus monkey

Summary The fine structure of synapses and of cellular relations was examined in the somatic efferent portion of the oculomotor nucleus of the adult rhesus monkey. Axosomatic and axodendritic synapses are characterized by distinct synaptic clefts which usually measure 20–30 nm between pre- and postsynaptic membranes. Cytoplasmic thickenings of pre- and postsynaptic membranes are often observed. Subjunctional bodies are present at both axosomatic and axodendritic synapses. Somatic and dendritic spine synapses are present. Serial synapses are also found, suggesting the operation of presynaptic inhibition in this nucleus. At some synapses the extracellular gap between pre- and postsynaptic membranes is reduced to 5–9 nm. However, junctions similar to the latter are also present between neurons and glia, and at the junctions between adjacent glial elements. The present results provide no evidence for a clear morphological substrate for electrotonic transmission in the somatic efferent portion of the primate oculomotor nucleus.Supported in part by grants from the National Institutes of Health (NS-15320), the Kroc Foundation, and by the Medical Research Service of the Veterans Administration (to S.G.W.), and from the National Science Foundation (BNS 77-28493) and the Muscular Dystrophy Association of America (to G.D.P.)     

Fine structure of the ordinary lateral line organ

Summary The lateral line organ of the spotted shark is characterized by its semi-cylindrical shape. Each organ (neuromast) is so closely apposed to the next that the individual neuromasts are almost continuous. The neuromast is composed of receptor cells, supporting cells and mantle cells. The receptor cells bear one kinocilium and up to 40 stereocilia. Bi-directional arrangement of the receptor cells as occurs in teleosts was demonstrated. Afferent and efferent nerve endings were found at the base of the receptor cells. The supporting cells extend from the basal lamina to the free surface. Long microvilli and a cilium-like ciliary rod project from the top of each supporting cell. The cell contains relatively few elements of the Golgi apparatus and little rough endoplasmic reticulum, but mitochondria and filaments are abundant. The mantle cell limits the lateral margin of the neuromast. It is distinguished from the supporting cell because of its long crescent-shaped nucleus and scarce, short microvilli. Myelinated nerve fibres are found in the subepithelial connective tissue but not in the epithelium.The fine structure of the shark lateral line organ suggests that this organ is in an intermediated step of evolution between that of lamprey and teleost.