The ultrastructure and innervation of the ear of the gar, Lepisosteus osseus

The endorgans of the inner ear of the gar were examined using transmission and scanning electron microscopy as well as nerve staining. The ultrastructure of the sensory hair cells and supporting cells of the gar ear are similar to cells in other bony fishes, whereas there are significant differences between the gar and other bony fishes in the orientations patterns of the sensory hair cells on the saccular and lagenar sensory epithelia. The saccular sensory epithelium has two regions, a main region and a secondary region ventral to the main region. The ciliary bundles on the main region are divided into two groups, one oriented dorsally and the other ventrally. Furthermore, as a result of curvature of the saccular sensory epithelium, the dorsal and ventral ciliary bundles on the rostral portion of the epithelium are rotated ninety degrees and are thus oriented on the animal's rostro-caudal axis. Hair cells on the secondary region are generally oriented ventrally. The lagenar epithelium has three groups of sensory hair cells. The groups on the rostral and caudal ends of the macula are oriented dorsally, whereas the middle group is oriented ventrally. Hair cell orientations on the utricular epithelium and macula neglecta are similar to those in other bony fishes. Nerve fiber diameters can be divided into three size classes, 1-8 microns, 9-13 microns, and 14 microns or more, with the smallest size class containing the majority of fibers. The distribution of the various classes of fiber diameters is not the same in nerve branches to each of the end organs. Similarly, the ratio of hair cells to axons differs in each end organ. The highest hair cell to axon ratio is in the utricle (23:1) and the smallest is in the macula neglecta (7:1). The number of sensory hair cells far exceed the number of eighth nerve axons in all sensory epithelia.     

The polarization of inner ear ciliary bundles from a scorpaeniform fish

The polarization of ultrastructural ciliary bundles from hair cells in the inner ear of the sea scorpion Taurulus bubalis was studied using a scanning electron microscope, revealing arrays of ciliary bundles with diverse orientations on each of the sensory epithelia. Members of this order are known to produce sound, though results of this study show no significant variation from the standard receptor patterns found in the hearing system of many silent marine teleosts. This is the first time that the ultrastructure of T. bubalis has been studied, and this work presents a new set of polarization patterns, which provide anatomical information important in understanding electrophysiological aspects of fish hearing from an ecological perspective.     

Sensory surface of the saccule and lagena in the ears of ostariophysan fishes

The inner ears of a few fishes in the teleost superorder Ostariophysi are structurally unlike those of most other teleosts. Scanning electron microscopy was used to determine if other ostariophysans share these unusual features. Examined were the families Cyprinidae, Characidae, and Gymnotidae (all of the series Otophysi), and Chanidae (of the sister series Anotophysi), representing the four major ostariophysan lineages, the auditory organs of which have not yet been well described. Among the Otophysi, the saccular and lagenar otolith organs are similar to those reported for other ostariophysans. The lagena is generally the larger of the two organs. The saccular sensory epithelium (macula) contains long ciliary bundles on the sensory hair cells in the caudal region, and short bundles in the rostral region. The saccule and the lagena each have hair cells organized into two groups having opposing directional orientations. In contrast, Chanos, the anotophysan, has a saccular otolith larger than the lagenar otolith, and ciliary bundles that are more uniform in size over most of its saccular macula. Most strikingly, its saccular macula has hair cells organized into groups oriented in four directions instead of two, in a pattern very similar to that in many nonostariophysan teleosts. We suggest that the bi-directional pattern seen consistently in the Otophysi is a derived development related to particular auditory capabilities of these species.     

The polarisation of hair cells from the ear of the European bass (Dicentrarchus labrax)

The polarisation of ciliary bundles on the macula of the saccule in the European bass (Dicentrarchus labrax L) has been studied using a scanning electron microscope (SEM). These data show that D. labrax possesses ciliary bundles arranged in four dichotomous quadrants with a standard orientation, comparable to hearing generalists from the order Perciformes. The spacing between ciliary bundles was investigated in three size classes of fish, with the results indicating that the addition of receptor cells in the ear of D. labrax continues for at least the first 2 years of development. The lengths of the kinocilia from ciliary bundles in each quadrant of the macula were also studied, and found to be of uniform length. In addition, we look at the internal structure of the afferent using transmission electron microscopy (TEM), revealing the nucleated cell body and peripheral nerve fibres of the saccule consistent with other TEM examinations of saccular ultrastructure. This information is required to gain an insight into the inner ear of D. labrax, as part of a larger study of the morphology and physiology of the hearing systems of both vertebrate and invertebrate marine animals.     

Mechanism of hyperthermia effects on CNS development: rostral gene expression domains remain, despite severe head truncation; and the hindbrain/otocyst relationship is altered

To study the mechanism of hyperthermia on the development of the rostral neural tube, we used a model in which closely-staged presomite 9.5-day rat embryos were exposed in culture to 43 degrees C for 13 min, and then cultured further for 12-48 hr. This treatment had little effect on the development of the rest of the embryo, but resulted in a spectrum of brain defects, the most severe being a lack of all forebrain and midbrain structures. Whole-mount in situ hybridisation was used to monitor the expression domains of Otx2, Emx2, Krox20, and hoxb1. These showed that there were no ectopic expression patterns, for any gene at any stage examined. Even in those embryos which apparently lacked all forebrain and midbrain structures, there were expression domains of Otx2 and Emx2 in the most rostral neural tissue, and these retained their nested dorso-ventral boundaries, showing that cells fated to form rostral brain were not wholly eliminated. Thus, heat-induced rostral neural tube truncation is of a quite different mechanism from the respecification proposed for retinoic acid, despite their very similar phenotypes. In the hindbrain region of treated embryos, we observed decreased intensity of Krox20, staining and an abnormal relationship developed between the position of hoxb1 expression and the otocyst and pharyngeal arches. In the most extreme cases, this domain was shifted to be more caudal than the rostral edge of the otocyst, while the otocyst retained its normal position relative to the pharyngeal arches. We interpret this as a growth imbalance between neuroepithelium and overlying tissues, perhaps due to a disruption of signals from the midbrain/hindbrain boundary.     

Morphological study on the inferior olivary nuclear complex of the donkey (Equus asinus)

This study provides basic data on the normal structure of the inferior olivary complex (IOC) of the donkey, Equus asinus, at the light microscopic level. In common with that of other mammals, the donkey IOC consisted of three major nuclei and four minor groups of cells. The former was comprised of the medial and dorsal accessory olives (MAO and DAO, respectively) and the principal olive (PO), and the latter was comprised of the dorsal cap, nucleus beta, ventrolateral outgrowth and dorsomedial cell column. The MAO had the longest rostral to caudal representation and formed the caudal pole of IOC. The DAO was located dorsally to the MAO in the caudal half of the IOC. In the rostral half, the DAO bended ventrally and merged with the dorsal lamella of PO. More rostrally, the DAO lost its connection with the dorsal lamella and then conversely connected with the ventral lamella of PO. The DAO formed the rostral pole of the IOC. The PO extended through the rostral half of the IOC. The dorsal cap was a small group of cells. Overall, the donkey IOC is similar to that of other mammals.     

A review of inner ear fate maps and cell lineage studies

A renewed interest in the development of the inner ear has provided more data on the fate and cell lineage relationships of the tissues making up this complex structure. The inner ear develops from a simple ectodermal thickening of the head called the otic placode, which undergoes a great deal of growth and differentiation to form a multichambered nonsensory epithelium that houses the six to nine sensory organs of the inner ear. Despite a large number of studies examining otic development, there have been surprisingly few fate maps generated. The published fate maps encompass four species and range from preotic to otocyst stages. Although some of these studies were consistent with a compartment and boundary model, other studies reveal extensive cell mixing during development. Cell lineage studies have been done in fewer species. At the single cell level the resulting clones in both chicks and frogs appear somewhat restricted in terms of distribution. We conclude that up until late placode stages there are no clear lineage restriction boundaries, meaning that cells seem to mix extensively at these early stages. At late placode stages, when the otic cup has formed, there are at least two boundaries located dorsally in the forming otocyst but none ventrally. These conclusions are consistent with all the fate maps and reconciles the chick and frog data. These results suggest that genes involved in patterning the inner ear may have dynamic and complex expression patterns.     

Ventromedial focus of cell death is absent during development of Xenopus and zebrafish inner ears

We present the normal patterns of programmed cell death in the developing inner ears of a primitive anuran, Xenopus laevis, and an ostariophysan fish, Danio rerio. A prominent ventromedial focus of cell death was described previously in the developing chicken and mouse otocysts. We hypothesize that this focus of cell death might be associated with a signaling center that directs morphogenesis of the surrounding tissue. Amphibian and fish ear anatomies differ considerably from those of birds and mammals, particularly in the structures derived from the ventral part (pars inferior) of the otic vesicle. We reasoned that these anatomical differences between species might result from a difference in the size, location, or presence of a putative morphogenetic signaling center. Using in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) to detect apoptotic cells, we show that developing Xenopus and zebrafish ears have apoptotic cells in the eighth cranial ganglia, the developing sensory patches, and in various positions in the otocyst wall. However, both species lack the persistent ventromedial hot spot of cell death that is prominently situated between the pars superior and pars inferior in the chicken and mouse otocysts.     

The development of the hypophysial portal system in the White-crowned Sparrow,Zonotrichia leucophrys gambelii

Summary The development of the hypophysial portal system has been studied in 35 embryos and 45 nestlings of the White-crowned Sparrow. The primordium of the hypophysis is vascularized by the infundibular (primary) capillary plexus, supplied by the right and left infundibular arteries, which, in the embryo, are constant branches of the right and left internal carotid arteries.The cellular proliferation and differentiation of the pars distalis into rostral and caudal lobes is accompanied by a penetration of portal vessels from the infundibular (primary) capillary plexus into these lobes beginning on the fifth day of incubation. The cellular proliferation of the rostral lobe of the pars distalis and development of the rostral group of the portal vessels precedes that of the caudal lobe of the pars distalis and the development of the caudal group of the portal vessels.The periglandular vessels, which originate in younger embryos from the infundibular (primary) capillary plexus, apparently become a part of the portal vessels.The portal vessels are the sole blood supply to the developing pars distalis of the White-crowned Sparrow; there is no evidence of a direct arterial supply at anytime during embryonic development. The neural-lobe artery appears at the end of incubation as a secondary branch of the right and left infundibular arteries. The rostral and caudal groups of the portal vessels are well-developed at the end of incubation (17–29 mm CRL) when aldehyde-fuchsin positive neurosecretory material first appears in the supraoptic and paraventricular nuclei, in the median eminence and in the neural lobe.The differentiation of the median eminence into rostral and caudal divisions begins at the end of the nestling period although its adult form is not achieved until later. The formation of the portal zone begins at the end of incubation (17–29 mm CRL) and is completed by the time of fledging.Dedicated to Professor Dr. W. Bargmann in honor of his 60th birthday.The investigations reported herein were supported by a research grant (HE 07240 NEUA) from the National Institutes of Health to Professor Vitums, by funds for biological and medical research made available by State of Washington Initiative Measure No 171 to Professor Vitums, by a research grant from the Deutsche Forschungsgemeinschaft to Professor Oksche, by aresearch grant (NB 01353) from the National Institutes of Health to Professor Farner, and by a Research Career Development Award from the National Institute of Arthritis and Metabolic Diseases (5 K 3 AM-18,370) to Professor King. We are grateful to Professor Bargmann for his generosity in making available the facilities of the Anatomisches Institut Kiel for this investigation. We wish to thank Frau Karin Graap and Mrs. Dianne Reno for technical assistance and Miss Janice Austin for the preparation of the drawings.     

Dorsoventral differences in cAMP levels and correlated changes in the subcellular distribution of the PKA catalytic domain,provide further evidence that PKA signaling coordinates dorsoventral patterning of the otocyst

Dorsoventral (DV) patterning of the otocyst gives rise to formation of the morphologically and functionally complex membranous labyrinth composed of unique dorsal and ventral sensory organs. DV patterning results from extracellular signaling by secreted growth factors, which presumably form reciprocal concentration gradients across the DV axis of the otocyst. Previous work suggested a model in which two important growth factors, bone morphogenetic protein (BMP) and SHH, undergo crosstalk through an intersecting pathway to coordinate DV patterning. cAMP‐dependent protein kinase A (PKA) lies at the heart of this pathway. Here, we provide further evidence that PKA signaling coordinates DV patterning, showing that both BMPs and SHH regulate cAMP levels, with BMPs increasing levels in the dorsal otocyst and SHH decreasing levels in the ventral otocyst. This, in turn, results in regional changes in the subcellular distribution of the catalytic domain of PKA, as well as DV regulation of PKA activity, increasing it dorsally and decreasing it ventrally. These new results fill an important gap in our previous understanding of how ligand signaling acts intracellularly during otocyst DV patterning and early morphogenesis, thereby initiating the series of events leading to formation of the inner ear sensory organs that function in balance and hearing.     

The pituitary gland of the coelacanth fish Latimeria chalumnae Smith: General structure and adenohypophysial cell types

The pituitary gland of Latimeria chalumnae is situated rostroventral to the telencephalon. The hollow pituitary stalk is bent forward and is ventrally connected to a saccus-vasculosus-like organ, rostrally to a neurointermediate lobe. The infundibular lumen protrudes far into the neurohypophysial lobules. The elongated principal part (pars cerebralis) of the pars distalis is partly embedded in a dorsal depression of the pars intermedia and caudally invaded by the neurohypophysis. It may be divided into rostral and proximal pars distalis and includes a ramified hypophysial cleft, which continues rostrally as a duct with adjacent islets of pars distalis tissue (parts of a pars buccalis). The adenohypophysis consists of cell cords and follicles. Eight tinctorial cell types can be distinguished: in the rostral islets: large basophils with acidophil globules, in the rostral pars distalis: small basophils, large basophils with amphiphil characters and erythrosin-, orange G-positive acidophils; in the proximal pars distalis: orange G-positive acidophils and small and large basophils, having similar staining properties; in the pars intermedia: one amphiphil cell type.     

Normal and abnormal pathfinding of facial nerve fibers in the chick embryo.

Development of the facial nerve was studied in normal chicken embryos and after surgical disruption of ingrowing sensory facial nerve fibers at 38-72 h of incubation. Disruption of facial nerve fibers by otocyst removal often induced a rostral deviation of the facial nerve and ganglion to the level of the trigeminal ganglion. Cell bodies of the geniculate ganglion trailed their deviating neurites and occupied an abnormal rostral position adjacent to the trigeminal ganglion. Deviating facial nerve fibers were labeled with the carbocyanine fluorescent tracer DiI in fixed tissue. Labeled fibers penetrated the cranium adjacent to the trigeminal ganglion, but they did not follow the trigeminal nerve fibers into the brain stem. Rather, after entering the cranium, they projected caudally to their usual site of entrance and proceeded towards their normal targets. This rostral deviation of the facial nerve was observed only after surgery at 48-72 h of incubation, but not in cases with early otocyst removal (38-48 h). A rostral deviation of the facial nerve was seen in cases with partial otocyst removal when the vestibular nerve was absent. The facial nerve followed its normal course when the vestibular nerve persisted. We conclude that disruption of the developing facial pathway altered the routes of navigating axons, but did not prevent pathfinding and innervation of the normal targets. Pathfinding abilities may not be restricted to pioneering axons of the facial nerve; later-developing facial nerve fibers also appeared to have positional information. Our findings are consistent with the hypothesis that navigating axons may respond to multiple guidance cues during development. These cues appear to differ as a function of position of the navigating axon.     

Ectopic noggin blocks sensory and nonsensory organ morphogenesis in the chicken inner ear

Bone morphogenetic protein 4 (Bmp4) is expressed during multiple stages of development of the chicken inner ear. At the otocyst stage, Bmp4 is expressed in each presumptive sensory organ, as well as in the mesenchymal cells surrounding the region of the otocyst that is destined to form the semicircular canals. After the formation of the gross anatomy of the inner ear, Bmp4 expression persists in some sensory organs and restricted domains of the semicircular canals. To address the role of this gene in inner ear development, we blocked BMP4 function(s) by delivering one of its antagonists, Noggin, to the developing inner ear in ovo. Exogenous Noggin was delivered to the developing otocyst by using a replication-competent avian retrovirus encoding the Noggin cDNA (RCAS-N) or implanting beads coated with Noggin protein. Noggin treatment resulted in a variety of phenotypes involving both sensory and nonsensory components of the inner ear. Among the nonsensory structures, the semicircular canals were the most sensitive and the endolymphatic duct and sac most resistant to exogenous Noggin. Noggin affected the proliferation of the primordial canal outpouch, as well as the continual outgrowth of the canal after its formation. In addition, Noggin affected the structural patterning of the cristae, possibly via a decrease of Msx1 and p75NGFR expression. These results suggest that BMP4 and possibly other BMPs are required for multiple phases of inner ear development.     

Vascularization of the pituitary of the Australian lungfish and Neoceratodus forsteri

The vascularization of the brain and the pituitary region of the Australian lungfish, Neoceratodus forsteri is described from serial section reconstruction. The distal lobe has no direct arterial blood supply and receives blood solely from a pituitary portal system basically similar to that of other sarcopterygians. The primary capillary plexus of the median eminence receives its arterial blood from the infundibular arteries, which on their way distribute some small branches to the prechiasmatic region. The primary plexus also receives capillaries from the adjacent pial hypothalamic plexus. The primary capillary plexus of the median eminence comprises a rostral 'uncovered' and caudal 'covered' part which are not sharply delineated. Distinct portal vessels connect the 'uncovered' rostral part of the primary plexus with the secondary capillary plexus supplying the rostral subdivision of the pars distalis. The 'covered' caudal part of the primary plexus merges into the proximal subdivision of the pars distalis, apparently without formation of distinct portal vessels. The primary plexus has some connections with the plexus intermedius via a hypophysial stem capillary plexus. The plexus intermedius has a substantial arterial supply and gives off capillaries to the parenchyma of the pars intermedia. The adenohypophysis is drained into an unpaired hypophysial vein. The significance of the vascular pathways is discussed from comparative, functional, and evolutionary viewpoints.