David H. Nichols

The development of vestibulocochlear efferents and cochlear afferents in mice.

We have reinvestigated the embryonic development of the vestibulocochlear system in mice using anterograde and retrograde tracing techniques. Our studies reveal that rhombomeres 4 and 5 include five motor neuron populations. One of these, the abducens nucleus, will not be dealt with here. Rhombomere 4 gives rise to three of the remaining populations: the facial branchial motor neurons; the vestibular efferents; and the cochlear efferents. The migration of the facial branchial motor neurons away from the otic efferents is completed by 13.5 days post coitum (dpc). Subsequently the otic efferents separate into the vestibular and cochlear efferents, and complete their migration by 14.5 dpc. In addition to their common origin, all three populations have perikarya that migrate via translocation through secondary processes, form a continuous column upon completion of their migrations, and form axonal tracts that run in the internal facial germ. Some otic efferent axons travel with the facial branchial motor nerve from the internal facial genu and exit the brain with that nerve. These data suggest that facial branchial motor neurons and otic efferents are derived from a common precursor population and use similar cues for pathway recognition within the brain. In contrast, rhombomere 5 gives rise to the fourth population to be considered here, the superior salivatory nucleus, a visceral motor neuron group. Other differences between this group and those derived from rhombomere 4 include perikaryal migration as a result of translocation first through primary processes and only then through secondary processes, a final location lateral to the branchial motor/otic efferent column, and axonal tracts that are completely segregated from those of the facial branchial and otic efferents throughout their course inside the brain. Analysis of the peripheral distribution of the cochlear efferents and afferents show that efferents reach the spiral ganglion at 12.5 dpc when postmitotic ganglion cells are migrating away from the cochlear anlage. The efferents begin to form the intraganglionic spiral bundle by 14.5 dpc and the inner spiral bundle by 16.5 dpc in the basal turn. They have extensive collaterals among supporting cells of the greater epithelial ridge from 16.5 dpc onwards. Afferents and efferents in the basal turn of the cochlea extend through all three rows of outer hair cells by 18.5 dpc. Selective labeling of afferent fibers at 20.5 dpc (postnatal day 1) shows that although some afferents are still in early developmental stages, some type II spiral ganglion cells already extend for long distances along the outer hair cells, and some type I spiral ganglion cells end on a single inner hair cell. These data support previous evidence that in mice the early outgrowth of afferent and efferent fibers is essentially achieved by birth.

Atoh1 Null Mice Show Directed Afferent Fiber Growth to Undifferentiated Ear Sensory Epithelia Followed by Incomplete Fiber Retention

Inner ear hair cells have been suggested as attractors for growing afferent fibers, possibly through the release of the neurotrophin brain‐derived neurotrophic factor (BDNF). Atoh1 null mice never fully differentiate hair cells and supporting cells and, therefore, may show aberrations in the growth and/or retention of their innervation. We investigated the distribution of cells positive for Atoh1‐ or Bdnf‐mediated β‐galactosidase expression in Atoh1 null and Atoh1 heterozygotic mice and correlated the distribution of these cells with their innervation. Embryonic day (E) 18.5 Atoh1 null and heterozygotic littermates show Atoh1‐ and BDNF‐β‐galactosidase–positive cells in comparable distributions in the canal cristae and the cochlea apex. Atoh1‐β‐galactosidase–positive but only occasional Bdnf‐β‐galactosidase–positive cells are found in the utricle, saccule, and cochlea base of Atoh1 null mutant mice. Absence of Bdnf‐β‐galactosidase expression in the utricle and saccule of Atoh1 null mice is first noted at E12.5, a time when Atoh1‐β‐galactosidase expression is also first detected in these epithelia. These data suggest that expression of Bdnf is dependent on ATOH1 protein in some but does not require ATOH1 protein in other inner ear cells. Overall, the undifferentiated Atoh1‐ and Bdnf‐β‐galactosidase–positive cells show a distribution reminiscent of that in the six sensory epithelia in control mice, suggesting that ear patterning processes can form discrete patches of Atoh1 and Bdnf expression in the absence of ATOH1 protein. The almost normal growth of afferent and efferent fibers in younger embryos suggests that neither fully differentiated hair cells nor BDNF are necessary for the initial targeted growth of fibers. E18.5 Atoh1 null mice have many afferent fibers to the apex of the cochlea, the anterior and the posterior crista, all areas with numerous Bdnf‐β‐galactosidase–positive cells. Few fibers remain to the saccule, utricle, and the base of the cochlea, all areas with few or no Bdnf‐β‐galactosidase–positive cells. Thus, retention of fibers is possible with BDNF, even in the absence of differentiated hair cells. Developmental Dynamics 233:570–583, 2005.

DiI reveals a prenatal arrival of efferents at the differentiating otocyst of mice

We have reinvestigated the time of arrival of efferent fibers at the developing otocyst of mice employing diffusion of the lipophilic dye DiI in fixed tissue. In contrast to almost all previous reports, our data indicate a prenatal arrival of efferent fibers. A few efferent fibers were found to enter the eighth nerve root at embryonic day (ED) 10 1/2. Retrogradely labelled efferent cell bodies were at this stage coextensive with those of the facial motor nucleus, but started to segregate by ED 12. In contrast to retrogradely labelled facial motor neurons, labelled efferent neurons were bilaterally distributed in the hindbrain with a few projecting to both otocysts as early as ED 12. Anterograde labelling from the brain showed efferent fibers in the vestibular ganglion by ED 11. Invasion of the future vestibular sensory epithelia started by ED 12. Growth cones of efferent fibers had also reached the future cochlear sensory epithelium but invasion was only achieved by a few filopodia at this stage. The early arrival of efferents at the future sensory epithelia demonstrated here may allow an as yet unexplored interaction of efferent fibers with the proliferating and/or differentiating hair cells.

Lmx1a is required for segregation of sensory epithelia and normal ear histogenesis and morphogenesis

At embryonic day 8.5, the LIM-homeodomain factor Lmx1a is expressed throughout the otic placode but becomes developmentally restricted to non-sensory epithelia of the ear (endolymphatic duct, ductus reuniens, cochlea lateral wall). We confirm here that the ears of newborn dreher (Lmx1adr) mutants are dysmorphic. Hair cell markers such as Atoh1 and Myo7 reveal, for the first time, that newborn Lmx1a mutants have only three sensory epithelia: two enlarged canal cristae and one fused epithelium comprising an amalgamation of the cochlea, saccule, and utricle (a “cochlear-gravistatic” endorgan). The enlarged anterior canal crista develops by fusion of horizontal and anterior crista, whereas the posterior crista fuses with an enlarged papilla neglecta that may extend into the cochlear lateral wall. In the fused endorgan, the cochlear region is distinguished from the vestibular region by markers such as Gata3, the presence of a tectorial membrane, and cochlea-specific innervation. The cochlea-like apex displays minor disorganization of the hair and supporting cells. This contrasts with the basal half of the cochlear region, which shows a vestibular epithelium-like organization of hair cells and supporting cells. The dismorphic features of the cochlea are also reflected in altered gene expression patterns. Fgf8 expression expands from inner hair cells in the apex to most hair cells in the base. Two supporting cell marker proteins, Sox2 and Prox1, also differ in their cellular distribution between the base and the apex. Sox2 expression expands in mutant canal cristae prior to their enlargement and fusion and displays a more diffuse and widespread expression in the base of the cochlear region, whereas Prox1 is not detected in the base. These changes in Sox2 and Prox1 expression suggest that Lmx1a expression restricts and sharpens Sox2 expression, thereby defining non-sensory and sensory epithelium. The adult Lmx1a mutant organ of Corti shows a loss of cochlear hair cells, suggesting that the long-term maintenance of hair cells is also disrupted in these mutants.

Migratory routes and fates of cells transcribing the Wnt‐1 gene in the murine hindbrain

To investigate the origins, migrations, and fates of Wnt‐1–expressing cells in the murine hindbrain, mice carrying a Wnt‐1 enhancer/lacZ transgene were observed from embryonic day (E) 8 through postnatal day 18. The transgene‐stained ventricular layer waxed and waned prior to and following migrations from it. Stained cells migrated first external to the hindbrain as neural crest and then within it to form typical populations of the rhombic lip, as well as others not recognized as lip derivatives. Migrations originated in a temporally defined sequence, many from discrete rhombomeres. All moved first radially, then rostrally and/or ventrally, ipsi‐, or contralaterally, in the mantle or marginal layers. These movements ultimately formed elements of several nuclei, aligned in four longitudinal bands: dorsal (including the gracile, cuneate, cochlear, and vestibular nuclei, plus cerebellar granular cells), dorsal intermediate (including trigeminal sensory, parvicellular reticular, and deep cerebellar nuclei), ventral intermediate (including lateral and intermediate reticular nuclei), and ventral (including the raphe obscurus and pontine nuclei). Transgene staining often persisted long enough to identify stained cells in their definitive, adult nuclei. However, staining was transient. The strength of the staining, however, was in its ability to reveal origins and migrations in both whole‐mounts and sections, in single cell detail. The present results will permit analyses of the effects of genetic manipulations on Wnt‐1 lineage cells. Developmental Dynamics 235:285–300, 2006.

Developmental Expression of Kcnq4 in Vestibular Neurons and Neurosensory Epithelia

Sensory signal transduction of the inner ear afferent neurons and hair cells (HCs) requires numerous ionic conductances. The KCNQ4 voltage-gated M-type potassium channel is thought to set the resting membrane potential in cochlear HCs. Here we describe the spatiotemporal expression patterns of Kcnq4 and the associated alternative splice forms in the HCs of vestibular labyrinth. Whole mount immunodetection, qualitative and quantitative RT-PCR were performed to characterize the expression patterns of Kcnq4 transcripts and proteins. A topographical expression and upregulation of Kcnq4 during development was observed and indicated that Kcnq4 is not restricted to either a specific vestibular structure or cell type, but is present in afferent calyxes, vestibular ganglion neurons, and both type I and type II HCs. Of the four alternative splice variants, Kcnq4_v1 transcripts were the predominant form in the HCs, while Kcnq4_v3 was the major variant in the vestibular neurons. Differential quantitative expression of Kcnq4_v1 and Kcnq4_v3 were respectively detected in the striolar and extra-striolar regions of the utricle and saccule. Analysis of gerbils and rats yielded results similar to those obtained in mice, suggesting that the spatiotemporal expression pattern of Kcnq4 in the vestibular system is conserved among rodents. Analyses of vestibular HCs of Bdnf conditional mutant mice, which are devoid of any innervation, demonstrate that regulation of Kcnq4 expression in vestibular HCs is independent of innervation.

Partial behavioral compensation is revealed in balance tasked mutant mice lacking otoconia.

We describe for the first time behavioral tests which show that mammals with congenital absence of otoconia can learn a motor task that normally relies on gravity perception. The mouse mutation tilted (tlt) occurs in the otopetrin 1 gene (Otop1(tlt/tlt)) and eliminates an essential component necessary for the formation of otoconia. Our data show that even in the absence of otoconia, tlt mutant mice, like normal mice, learn to cross a bar suspended between two boxes and, with practice, improve their speed of crossing. Despite this learned compensatory skills, tlt mutant mice show balance impairments, such as falling from the bar, not observed in wild type (WT) or heterozygous (het) Otop1(+/)(tlt) littermates. The tlt mutant mice also use their tail as additional support, a behavior that is rarely exhibited in the control littermates. Interestingly, the Otop1(+/)(tlt) heterozygous littermates show in many aspects an intermediate phenotype between wild type and tlt mutant mice, suggestive of a gene dosage effect. Overall, these data support the notion that mammals can use other otic and extraotic receptors such as semicircular canals and limb proprioreceptors, respectively, to compensate for the absence of otoconia-mediated gravity perception in a balance task.

Organ of corti and stria vascularis: Is there an interdependence for survival?

Cochlear hair cells and the stria vascularis are critical for normal hearing. Hair cells transduce mechanical stimuli into electrical signals, whereas the stria is responsible for generating the endocochlear potential (EP), which is the driving force for hair cell mechanotransduction. We questioned whether hair cells and the stria interdepend for survival by using two mouse models. Atoh1 conditional knockout mice, which lose all hair cells within four weeks after birth, were used to determine whether the absence of hair cells would affect function and survival of stria. We showed that stria morphology and EP remained normal for long time despite a complete loss of all hair cells. We then used a mouse model that has an abnormal stria morphology and function due to mutation of the Mitf gene to determine whether hair cells are able to survive and transduce sound signals without a normal electrochemical environment in the endolymph. A strial defect, reflected by missing intermediate cells in the stria and by reduction of EP, led to systematic outer hair cell death from the base to the apex after postnatal day 18. However, an 18-mV EP was sufficient for outer hair cell survival. Surprisingly, inner hair cell survival was less vulnerable to reduction of the EP. Our studies show that normal function of the stria is essential for adult outer hair cell survival, while the survival and normal function of the stria vascularis do not depend on functional hair cells.

Peptides bind to eosinophils in the rat stomach

An immunological role for eosinophils has been well established. However, roles for eosinophils in the physiological functions of the organs they populate are little explored.

Carboxyfluorescein and Biotin Neuromedin C Analogues: Synthesis and Applications

Two neuromedin C (NC) analogues were constructed by Fmoc synthesis and in situ coupling of 4(5)-carboxyfluorescein or biotin to the N-terminus. Both displayed full agonism in an amylase release assay and cross-reacted fully with a NC-specific antiserum. Biotin NC functioned in a streptavidin-capture ELISA. Carboxyfluorescein NC was used to probe receptor localization in rat stomach. Specific NC binding sites, which did not interact with substance P, angiotensin I, or neurokinin A, were labeled in the antrum. Identity of NC binding sites was confirmed by microautoradiography. The specifically labeled cells were all found in the lamina propria and at least some of cells were identified as eosinophils.