The role of jab1, a putative downstream effector of the neurotrophic cytokine macrophage migration inhibitory factor (MIF) in zebrafish inner ear hair cell development.

Macrophage migration inhibitory factor (MIF) is a neurotrophic cytokine essential for inner ear hair cell (HC) development and statoacoustic ganglion (SAG) neurite outgrowth, and SAG survival in mouse, chick and zebrafish. Another neurotrophic cytokine, Monocyte chemoattractant protein 1 (MCP1) is known to synergize with MIF; but MCP1 alone is insufficient to support mouse/chick SAG neurite outgrowth or neuronal survival. Because of the relatively short time over which the zebrafish inner ear develops (~30hpf), the living zebrafish embryo is an ideal system to examine mif and mcp1 cytokine pathways and interactions. We used a novel technique: direct delivery of antisense oligonucleotide morpholinos (MOs) into the embryonic zebrafish otocyst to discover downstream effectors of mif as well as to clarify the relationship between mif and mcp1 in inner ear development. MOs for mif, mcp1 and the presumptive mif and mcp1 effector, c-Jun activation domain-binding protein-1 (jab1), were injected and then electroporated into the zebrafish otocyst 25-48hours post fertilization (hpf). We found that although mif is important at early stages (before 30hpf) for auditory macular HC development, jab1 is more critical for vestibular macular HC development before 30hpf. After 30hpf, mcp1 becomes important for HC development in both maculae.

Ontogenetic development of the inner ear saccule and utricle in the Lusitanian toadfish: Potential implications for auditory sensitivity.

Studies addressing structure-function relationships of the fish auditory system during development are sparse compared to other taxa. The Batrachoididae has become an important group to investigate mechanisms of auditory plasticity and evolution of auditory-vocal systems. A recent study reported ontogenetic improvements in the inner ear saccule sensitivity of the Lusitanian toadfish, Halobatrachus didactylus, but whether this results from changes in the sensory morphology remains unknown. We investigated how the macula and organization of auditory receptors in the saccule and utricle change during growth in this species. Inner ear sensory epithelia were removed from the end organs of previously PFA-fixed specimens, from non-vocal posthatch fry (<1.4 cm, standard length) to adults (>23 cm). Epithelia were phalloidin-stained and analysed for area, shape, number and orientation patterns of hair cells (HC), and number and size of saccular supporting cells (SC). Saccular macula area expanded 41x in total, and significantly more (relative to body length) among vocal juveniles (2.3-2.9 cm). Saccular HC number increased 25x but HC density decreased, suggesting that HC addition is slower relative to epithelial growth. While SC density decreased, SC apical area increased, contributing to the epithelial expansion. The utricule revealed increased HC density (striolar region) and less epithelial expansion (5x) with growth, contrasting with the saccule that may have a different developmental pattern due to its larger size and main auditory functions. Both macula shape and HC orientation patterns were already established in the posthatch fry and retained throughout growth in both end organs. We suggest that previously reported ontogenetic improvements in saccular sensitivity might be associated with changes in HC number (not density), size and/or molecular mechanisms controlling HC sensitivity. This is one of the first studies investigating the ontogenetic development of the saccule and utricle in a vocal fish and how it potentially relates to auditory enhancement for acoustic communication.

Zebrafish pax5 regulates development of the utricular macula and vestibular function.

The zebrafish otic vesicle initially forms with only two sensory epithelia, the utricular and saccular maculae, which primarily mediate vestibular and auditory function, respectively. Here, we test the role of pax5, which is preferentially expressed in the utricular macula. Morpholino knockdown of pax5 disrupts vestibular function but not hearing. Neurons of the statoacoustic ganglion (SAG) develop normally. Utricular hair cells appear to form normally but a variable number subsequently undergo apoptosis and are extruded from the otic vesicle. Dendrites of the SAG persist in the utricle but become disorganized after hair cell loss. Hair cells in the saccule develop and survive normally. Otic expression of pax5 requires pax2a and fgf3, mutations in which cause vestibular defects, albeit by distinct mechanisms. Thus, pax5 works in conjunction with fgf3 and pax2a to establish and/or maintain the utricular macula and is essential for vestibular function.

Stereological study of postnatal development in the mouse utricular macula.

This study describes the morphometric changes taking place in the utricular macula of mice with ages in geometric progression from 1 to 512 days after birth. By using design-based stereological methods, the total volume and surface area of the sensory epithelium as well the total number of the hair cells and supporting cells were estimated. Finally, the numerical density, volume density, and mean volume of the individual cell types were determined. The major changes were found in the number of the individual cell types during the first couple of weeks, and a mature composition of cell types was not attained until 16 days after birth. There was no change in the total number of cells and no decline in the number of hair cells within the time period studied.

The inner ear macular sensory epithelia of the Daubenton's bat.

The inner ear macular sensory epithelia of the Daubenton's bat were examined quantitatively to estimate the area and total number of hair cells. Ultrastructural examination of the sensory epithelium reveals two main types of hair cells: the chalice-innervated hair cell and the bouton-innervated hair cell. The existence of an intermediate type, with a nerve ending covering the lateral side of the hair cell, indicates that the chalice-innervated hair cells are derived from bouton-innervated hair cells. Thus, at least a part of the bouton-innervated hair cells forms a transitional stage. A number of immature as well as apoptotic hair cells were observed. It is suggested that a continuous production of new hair cells takes place in mature individuals, probably based on transdifferentiation of supporting cells.

Vesicular bodies in fish maculae are artifacts not contributing to otolith growth.

The presence, morphology and possible origin of vesicle-like bodies (VBs) within the inner ear macular otolithic membrane of developmental stages of cichlid fish Oreochromis mossambicus and neonate (i.e. functionally fully developed except the reproductive organs) swordtail fish Xiphophorus helleri were analyzed by means of transmission and scanning electron microscopy (TEM and SEM, respectively) employing various fixation procedures. Some authors believe that these VBs are involved in the formation of the organic phase of inner ear otoliths (or statoliths in birds and mammals). Decreasing the osmolarity of the fixation medium from a value rather close to that of native fresh water fish tissue (i.e. 250 mOsm and 290--300 mOsm, respectively) to a value of fixatives mostly employed in TEM studies (ca. 190 mOsm), the amount of VBs increased and the components of sensory inner ear tissue increasingly dilated. Whilst a conventional prefixation with aldehydes followed by osmium tetroxide postfixation yielded numerous VBs, only few of them were observed when the tissue was fixed with aldehydes and osmium tetroxide simultaneously. Therefore, the results demonstrate that inner ear sensory epithelia are extremely sensitive to altering fixation media. On this background it must be concluded that VBs are fixative (i.e. glutaraldehyde) induced artificial structures, so-called membrane blisters. Thus, the protein matrix of otoliths (and possibly that of statoliths in higher vertebrates) is rather provided by secretion processes than by the release of vesicles.

Developmental study of rat vestibular neuronal circuits during a spaceflight of 17 days.

The aim of this study was to investigate the potential plasticity of the vestibular system, in structural and biochemical terms, at the level of the gravity receptors (the sensory hair cells), the primary neurons relaying the sensory signals (the vestibular ganglion neurons) and their projections into the vestibular nuclei. We studied the biochemical differentiation of the sensory cells and of the vestibular ganglion by investigating which calcium-binding proteins were present. We studied the development of peripheral synaptic connections of the efferent system by investigating the distribution of CGRP (calcitonin-gene related-peptide) and we also studied the cerebellar synaptic connections in the vestibular nuclei, as identified by the presence of calbindin. Putative changes were studied after a 17-day episode of microgravity (Neurolab STS-90), in developing rats between postnatal days 8 and 25. The extent to which these changes could be caused by alterations in gravity was determined by examining sensory and nervous structures not involved in gravity detection, the cochlea and the cochlear nuclei.

Hair-cell numbers continue to increase in the utricular macula of the early posthatch chick.

It is generally assumed that hair-cell numbers do not increase in the vestibular epithelia of postembryonic birds after hatching. However, for the domestic chicken, it is not known when or if hair-cell numbers ever reach a steady state level during life. The numbers of hair cells in the utricular maculae of chickens from embryonic day (E) 7 to posthatch day (PH) 112 were therefore counted directly. Hair-cell numbers increase approximately 15 fold between E7 and PH2, from an average of 1,858/macula at E7 to 27,017 at PH2. Between PH2 and PH112 hair-cell numbers increase by a further 36%, to 36,650/macula. A mathematical description of the increase in hair-cell numbers observed with time predicts a half life of 29.88 days for a utricular hair cell and a steady-state turnover value of 850 hair cells/day by approximately PH60. The patterns of hair and supporting cells in the postembryonic utricular macula were also assessed. The ratios of supporting cells and hair cells, the average number of supporting cells around each hair cell, and the average number of hair cells each supporting cell contacts at PH2, PH16 and approximately 2.5 years of age are not significantly different. In contrast to the mitotically quiescent basilar papilla where all supporting cells contact at least one hair cell, 7.6% of supporting cells in the extrastriolar region of the postembryonic utricular macula do not make apical contact with a hair cell. These results indicate that hair-cell numbers in the utricular macula increase significantly after hatching, and support the concept that contact-mediated inhibition influences the proliferative potential of inner-ear supporting cells.

Hair cell orientation and a possible intraepithelial growth of the macula lagenae in the inner ear of the European eel.

The hair cell orientation of the macula lagenae in eels at three different stages of life was examined with the light microscope. Part of the posterior periphery of all three stages was occupied by a few rows of sensory cells having their kinocilium pointing in a direction opposite to the direction of the adjacent sensory cells. The width of this peripheral belt remained the same at all three stages of life, in spite of a considerable growth of the sensory epithelium, and the belt was always confined to the margin. This indicates an intraepithelial growth of at least part of the neuroepithelium of the lagenar macula of the European eel.