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.

Morphological characteristics of the human macula sacculi.

The surface morphology of 20 human maculae sacculi is presented. Individual data on the total area, shape and the relative area of the pars superior are given. The mean area of 14 adult maculae was (mean +/- SD) 2.35 +/- 0.31 mm2. The ratio between the length and width of the macula was 2.54 +/- 0.28. The pars superior was significantly larger than the pars inferior. The percentage of pars superior was 56.4 +/- 4.7. As regards the shape of the macula, there was a large interindividual variability. The findings are discussed, taking into consideration comparative anatomy as well as spatial orientation.

Morphometry of otoconia in the utricle and saccule of developing Japanese quail.

The development of otoconia in the utricular and saccular maculae from initial embryonic formation to adult stages was examined in Japanese quails. Both the morphology and size of the otoconia were quantified at different developmental stages. It was observed that the otoconia were initially formed on embryologic stage E5 in the saccule and E6 in the utricle. Otolith mass areas increased in a sigmoidal growth pattern, with saccular otolith areas being smaller than the utricular mass areas. Saccular otolith masses reached adult values at embryonic stage E12 and utricular areas reached adult values at post-hatch day 7. Mature individual otoconia were characterized by a barrel shape with two trihedral faceted ends. However, initial formation of otoconia at E5 (saccular) and E6 (utricular) maculae was characterized by a double fluted morphology that consisted of an hourglass shape with extended fins forming trihedral angles of 120 degrees. Double fluted otoconia rapidly filled, so that by embryonic day 8 mature otoconia dominated the maculae for the remainder of development through adulthood. Thus, a progression from double fluted to mature forms was noted. Mature utricular otoconia in adult quails averaged 11 microm in length and 5 microm in width, with length/width ratios of approximately 2.5:1, for all size ranges. Saccular otoconia were smaller, having about 70% the size of utricular otoconia in both length and width. During development, the average size and range of individual otoconia increased nearly linearly for both otolith organs. In the utricular macula, large otoconia were concentrated in the lateral regions of the epithelium. In contrast, otoconia of various sizes were distributed uniformly across the surface of the saccular macula.

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.

Efferent neurons to the macular lagena in the embryonic chick.

The lipophilic dye, DiI, was placed into the macula lagena of paraformaldehyde-fixed embryonic chicks. Retrogradely labeled cells were found bilaterally in the pontine reticular formation (RF) between the dorsal facial nucleus and the abducens nerve root. This location is similar to that of the dorsomedial group of efferent cells that project to the basilar papilla. No lagenar efferent neurons, however, were found near the superior olivary nucleus where the ventrolateral group of cochlear efferents is located. Whether efferent neurons in the pontine RF send collaterals to both the basilar papilla and to the macula lagena has yet to be determined.

Development of the utricular macula in the mouse.

There have been many studies reporting on the development of the utricular macula using transmission electron microscopy. This study demonstrates the morphological development of the utricular macula in the mouse using scanning electron microscopy. Utricular maculae from CBA/CBA mice were studied from the 11.5th gestational day to 2 weeks after birth. Between the 11.5th and 12.5th gestational day there are numerous microvilli and a primary cilium on the surface of the otocyst. Sensory hairs appear on the 13.5th gestational day. The kinocilium and stereocilia are short and the latter of almost equal in length. The polarity of the kinocilium is first noticed after the 16.5th gestational day. The formation of otoconia starts on the 14.5th gestational day. They are shaped like a dumb-bell and are oval and columnar. Otoconia with a hexagonal form are recognized after the 16.5th gestational day.

The pattern of ciliary development in fetal mouse vestibular receptors. A qualitative and quantitative SEM study.

The development of vestibular receptors in the mouse was studied by scanning electron microscopy between the 13th gestation day to birth. On the 13th gestation day, the utricle was entirely covered with microvilli, which were often grouped around small kinocilia at the center of the macula. The vertical cristae were not clearly differentiated at this stage. On the 15th gestation day, the opposite orientation of ciliary tufts in the utricle indicated the beginnings of the striola. During the whole period studied, gradients in differentiation of ciliary tufts were observed between the center and the periphery of the utricle, and the top and base of the cristae. The auxiliary structures (otolithic membrane and cupula) began to appear at the same time as the first ciliary tufts differentiated. Otoliths, still immature, were only observed as from the 16th gestation day. Differentiation of ciliary tufts on the utricle appeared to be progressive during the fetal period. However, between the 16th and 17th gestation days, a pause in the differentiation of ciliary tufts was registered. A day later, there was a pause in the increase of the utricular sensory surface, which coincided with a temporary stabilization of the decrease in the thickness of the sensory epithelium.

A study of the development of utricular and saccular maculae in man and in rat.

The ultrastructural development of utricular and saccular maculae in man and rat is described. In the human fetus with a crown-rump length (CRL) of 50 mm, the utricular and saccular epithelia consist of a pseudostratified layer of dark and light cells without nerve endings. In human fetuses of 110 and 220 mm CRL, as well as in 17- and 18-day-old rat embryos, it is already possible to identify sensory and supporting cells in various stages of differentiation. Nerve endings can also be found. Ciliogenesis is present in both sensory and supporting cells of the 17- and 18-day-old rat embryos. During epithelial differentiation a heavy calcium deposition occurs on vestibular cells of both man and rat. Otoconial mineralization increases gradually as epithelial maturation advances. Phagocytosis of otoconia by free macrophages occurs in 50-mm CRL human fetuses. Vestibular nerve myelination is asynchronous and is present in 110-mm CRL human fetuses and in 30-minute postpartum rats.

SEM observations on development of human otoconia during the first trimester of gestation.

The early development of human otoconial membranes was studied by scanning electron microscopy and X-ray microanalysis; 87 specimens, ranging from 7 to 12 weeks gestational age were examined. A few otoconia, which contained calcium, were present in the utricle by the end of the seventh week. By 8 weeks, both the saccule and utricle contained otoconia and differentiation of the macular neuroepithelia was well underway. At all stages, the utricular otoconia appeared more mature and were more varied in size and shape than saccular otoconia. In 12-week specimens, the maculae were covered by otoconial membranes with clearly defined crystalline and gelatinous layers. Near the end of the first trimester, newly-formed otoconia were present on the inferior utricular wall. X-ray analysis showed that a marked increase in otoconial calcium content occurs during the period from 7 to 12 weeks gestational age.

Otoconial formation in the fetal rat.

The development of otoconia in the fetal rat was investigated by scanning and transmission electron microscopy and by x-ray elemental analysis. The transmission electron microscopical results indicate that primitive otoconia are highly organic appearing but are trigonal in cross section, indicating that they already possess a three-fold axis of symmetry and a complement of calcite. These otoconia develop into spindle-shaped units which accrue fibrous, organic material at an angle to their surfaces. Dumbbell-shaped otoconia, with distinct central cores and peripheral zones, result. These otoconia then mature to the adult crystal configuration having a more cylindrical body and pointed ends. The existence of trigonal, spindle- and dumbbell-shaped otoconia was verified by scanning electron microscopy of fresh-frozen material. Tissues prepared for transmission electron microscopy proved (by elemental analysis) to have been decalcified inadvertently, fortuitously revealing the arrangement of the organic material. Subsequent transmission electron microscopy of dumbbell-shaped otoconia not exposed to fluids during embedment showed that calcite deposits mimicked the arrangement of the organic material. X-ray elemental analysis demonstrated that calcium was present in lower quantities in the central core than peripherally. Findings are interpreted to indicate that organic material is essential to otoconial seeding and directs otoconial growth.

Observations of human fetal otoconial membranes.

Intact otoconial membranes from 31 human fetuses ranging in gestational age from 14 to 36 weeks were studied. At all stages of development specimens from different individuals showed marked variations in overall shape. During the course of the second and third trimesters, both saccular and utricular otoconial membranes were found to increase three to fourfold in surface area and more than twofold in weight. Near the end of gestation the fetal specimens were about equal in area and weight to otoconial membranes from children up to 13 years of age. However, the crystal layers of the fetal membranes has less prominently developed surface contours than usually observed in children and adults, indicating that maturational changes continue after the time of birth.

Observations of otoconial membranes from human infants.

The microdissection technique was used to study otoconial membrane from 30 human infants ranging from newborn to 2 years of age. Both saccular and utricular membranes were quite variable in overall shape. During the neonatal period, the gelatinous layer of the otoconial membrane appears to thicken and become less adherent to the macular surface than in the fetal period. In many infants older than 6 weeks, otoconial membranes were found at autopsy to be completely dislodged from the maculae, with abnormally large saccular otoconia present in four specimens. Fourteen of the infants studied died of sudden infant death syndrome and 16 died of other causes. The incidence of detached otoconial membranes was approximately the same in both groups. Although the majority of these membranes were apparently dislodged post mortem, the present findings suggest that otoconial membranes are susceptible to pathological alteration due to disease or head trauma.