Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear.

Murine models are ideal for studying cochlear gene transfer, as many hearing loss-related mutations have been discovered and mapped within the mouse genome. However, because of the small size and delicate nature, the membranous labyrinth of the mouse is a challenging target for the delivery of viral vectors. To minimize injection trauma, we developed a procedure for the controlled release of adeno-associated viruses (AAVs) into the scala media of adult mice. This procedure poses minimal risk of injury to structures of the cochlea and middle ear, and allows for near-complete preservation of low and middle frequency hearing. In this study, transduction efficiency and cellular specificity of AAV vectors (serotypes 1, 2, 5, 6 and 8) were investigated in normal and drug-deafened ears. Using the cytomegalovirus promoter to drive gene expression, a variety of cell types were transduced successfully, including sensory hair cells and supporting cells, as well as cells in the auditory nerve and spiral ligament. Among all five serotypes, inner hair cells were the most effectively transduced cochlear cell type. All five serotypes of AAV vectors transduced cells of the auditory nerve, though serotype 8 was the most efficient vector for transduction. Our findings indicate that efficient AAV inoculation (via the scala media) can be performed in adult mouse ears, with hearing preservation a realistic goal. The procedure we describe may also have applications for intra-endolymphatic drug delivery in many mouse models of human deafness.

Expression of transgenes in primary neurons from chick peripheral and central nervous systems by retroviral infection of early embryos.

Many cell biological studies require the expression of transgenes in cells in culture, but it is difficult to obtain uniform, stable, and efficient expression of transgenes in primary neurons. We have approached this problem by adapting from developmental biologists the avian retroviral vector, RCAS. This vector allows the introduction of a transgene by infection early in chick embryonic development. Transgenes that are less than 2.6 kb in size can be cloned through an adapter vector, SLAX 12 NCO, and into the RCAS retroviral vector with relative ease. The vector is then used to produce active virus, and the virus is injected into the neural tube or ventricles of stage 10 embryos. By infecting the neuronal precursor cells while they are still mitotic, the retrovirus and accompanying transgene are introduced into the genome and subsequently spread by replication, shedding of new virus, and infection of other cells. Embryos are incubated from the time of injection until E9-E12 and peripheral and central nervous system neurons are dissected out and grown in culture using standard techniques. In this manner, the majority of the sympathetic and dorsal root ganglion neurons can be induced to express the trangene. A similar result, at lower efficiencies, is obtained for central nervous system neurons.

Lineage analysis in the chicken inner ear shows differences in clonal dispersion for epithelial, neuronal, and mesenchymal cells.

The epithelial components of the vertebrate inner ear and its associated ganglion arise from the otic placode. The cell types formed include neurons, hair-cell mechanoreceptors, supporting cells, secretory cells that make endolymphatic fluid or otolithic membranes, and simple epithelial cells lining the fluid-filled cavities. The epithelial sheet is surrounded by an inner layer of connective and vascular tissues and an outer capsule of bone. To explore the mechanisms of cell fate specification in the ear, retrovirus-mediated lineage analysis was performed after injecting virus into the chicken otocyst on embryonic days 2.5-5.5. Because lineage analysis might reveal developmental compartments, an effort was made to study clonal dispersion by sampling infected cells from different parts of the same ear, including the auditory ganglion, cochlea, saccule, utricle, and semicircular canals. Lineage relationships were confirmed for 75 clones by amplification and sequencing of a variable DNA tag carried by each virus. While mesenchymal clones could span different structural parts of the ear, epithelial clones did not. The circumscribed epithelial clones indicated that their progenitors were not highly migratory. Ganglion cell clones, in contrast, were more dispersed. There was no evidence for a common lineage between sensory cells and their associated neurons, a prediction based on a proposal that the ear sensory organs and fly mechanosensory organs are evolutionarily homologous. As expected, placodal derivatives were unrelated to adjacent mesenchymal cells or to nonneuronal cells of the ganglion. Within the otic capsule, fibroblasts and cartilage cells could be related by lineage.

A fate map of chick otic cup closure reveals lineage boundaries in the dorsal otocyst.

The vertebrate inner ear is structurally complex, consisting of fluid-filled tubules and sensory organs that subserve the functions of hearing and balance. The epithelial parts of the inner ear are derived from the otic placode, which deepens to form a cup before closing to form the otic vesicle. We fate-mapped the rim of the otic cup to monitor the cellular movements associated with otocyst formation and to aid in interpreting the changing gene expression patterns of the early otic field. Twelve sites around the rim, defined as positions of a clock face, were targeted by iontophoretic injection of fluorescent, lipophilic dye. Labeled cells were imaged 24 and 48 h after injection. The data show that the entire dorsal rim of the otic cup becomes the endolymphatic duct (ED), while the posteroventral rim becomes the lateral otocyst wall. Two intersecting boundaries of lineage restriction were identified near the dorsal pole: one bisecting the ED into anterior and posterior halves and the other defining its lateral edge. We hypothesize that signaling across compartment boundaries may play a critical role in duct specification. This model is discussed in the context of mouse mutants that are defective in both hindbrain development and ED outgrowth.

Molecular genetics of pattern formation in the inner ear: do compartment boundaries play a role?

The membranous labyrinth of the inner ear establishes a precise geometrical topology so that it may subserve the functions of hearing and balance. How this geometry arises from a simple ectodermal placode is under active investigation. The placode invaginates to form the otic cup, which deepens before pinching off to form the otic vesicle. By the vesicle stage many genes expressed in the developing ear have assumed broad, asymmetrical expression domains. We have been exploring the possibility that these domains may reflect developmental compartments that are instrumental in specifying the location and identity of different parts of the ear. The boundaries between compartments are proposed to be the site of inductive interactions required for this specification. Our work has shown that sensory organs and the endolymphatic duct each arise near the boundaries of broader gene expression domains, lending support to this idea. A further prediction of the model, that the compartment boundaries will also represent lineage-restriction compartments, is supported in part by fate mapping the otic cup. Our data suggest that two lineage-restriction boundaries intersect at the dorsal pole of the otocyst, a convergence that may be critical for the specification of endolymphatic duct outgrowth. We speculate that the patterning information necessary to establish these two orthogonal boundaries may emanate, in part, from the hindbrain. The compartment boundary model of ear development now needs to be tested through a variety of experimental perturbations, such as the removal of boundaries, the generation of ectopic boundaries, and/or changes in compartment identity.

Cell proliferation and cell death in the developing chick inner ear: spatial and temporal patterns.

Morphogenesis of the inner ear is a complex process in which the balance of cell division and death is presumed to play an important role. Surprisingly, there are no reports of a systematic comparison of these two processes in individual ears at different stages of development. This study presents such an analysis for the chicken otocyst at stages 13-29 (embryonic days 2.5-6). To detect proliferating cells, we used exposure to bromodeoxyuridine. To detect apoptotic cells, we used nuclear condensation and fragmentation or terminal dUTP nick-end labeling (TUNEL). The spatial and temporal locations of proliferating and dying cells were mapped across serial sections, revealing regional differences in proliferation within the otocyst epithelium that are more complex than previously reported. In addition, almost all of the previously identified "hot spots" of cell death correspond spatially to regions of reduced cell proliferation. An exception is the ventromedial hot spot of cell death, which is intermingled with proliferating cells when it first appears at stages 19-23 before becoming a cold spot of proliferation. The results further show that the inferior part of the otocyst has a high level of proliferation, whereas the superior part does not. Since the superior part of the otocyst demonstrates outward expansion that is comparable to the inferior part, it appears that regional outgrowth of the otic vesicle is not necessarily coupled to cell proliferation. This study provides a basis for exploring the regulation and function of cell proliferation and cell death during inner ear morphogenesis.

Development of the vertebrate ear: insights from knockouts and mutants.

The three divisions of the ear (outer, middle and inner) each have an important role in hearing, while the inner ear is also crucial for the sense of balance. How these three major components arise and coalesce to form the peripheral elements of the senses of hearing and balance is now being studied using molecular-genetic approaches. This article summarizes data from studies of knockout and mutant animals in which one or more divisions of the ear are abnormal. The data confirm that development of all three divisions of the ear depends on the genes involved in hindbrain segmentation and segment identity. Genes that are regionally expressed in the inner ear can, when absent or mutated, yield selective ablation of specific inner-ear structures or cell types.

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.

Hair cells and supporting cells share a common progenitor in the avian inner ear.

Sensory organs of the vertebrate inner ear contain two major cell types: hair cells (HCs) and supporting cells (SCs). To study the lineage relationships between these two populations, replication-defective retroviral vectors encoding marker genes were delivered to the otic vesicle of the chicken embryo. The resulting labeled clones were analyzed in the hearing organ of the chicken, called the basilar papilla (BP), after cellular differentiation. BPs were allowed to develop for 2 weeks after delivery of the retrovirus, were removed, and were processed histochemically as whole mounts to identify clones of cells. Clusters of labeled cells were evident in the sensory epithelium, the nonsensory epithelium, and in adjacent tissues. Labeled cell types included HCs, two morphologically distinct types of SCs, homogene cells, border cells, hyaline cells, ganglion cells, and connective tissue cells. Each clone was sectioned and cell-type identification was performed on sensory clones expressing retrovirally transduced beta-galactosidase. Cell composition was determined for 41 sensory clones, most of which contained both HCs and SCs. Clones containing one HC and one SC were observed, suggesting that a common progenitor exists that can remain bipotential up to its final mitotic division. The possibility that these two cell types may also arise from a mitotic precursor during HC regeneration in the mature basilar papilla is consistent with their developmental history.

Transferring clients from intensive case management: impact on client functioning.

The effects of transferring clients from assertive community treatment to a less intensive (step-down) case management program were examined. Service use decreased significantly after transfer to the step-down program, and no negative effects of transfer on hospital use or client functioning were evident. Critical elements for successful step-down are suggested and discussed.

The expression domain of two related homeobox genes defines a compartment in the chicken inner ear that may be involved in semicircular canal formation.

Homeobox-containing genes encode a class of proteins that control patterning in developing systems, in some cases by acting as selector genes that define compartment identity. In an effort to demonstrate a similar role for such genes during ear development in the chicken, we present a detailed expression study of two related homeobox-containing genes, SOHo-1 and GH6, using in situ hybridization. At otocyst stages the two genes define a broad lateral domain of expression, which may represent a developmental compartment. Three-dimensional computer reconstructions of SOHo-1 expression at these and later stages revealed that the lateral domain becomes progressively restricted to the three semicircular canals. Thus, SOHo-1 and GH6 are among a small group of markers for a specific structural component of the inner ear. The gene expression domain initially includes the sensory regions of the semicircular canals, known as the cristae ampullaris, but none of the other four sensory organs which were recognizable by BMP4 expression during early morphogenesis (stages 19-24). Significantly, two of the sensory organs (the superior and posterior cristae) were found at the limits, or boundaries, of the SOHo-1/GH6 expression domain, suggesting that compartment boundaries may be involved in specifying sensory organ location as well as identity. Maintained expression at the boundaries may aid in specifying the location of canal outgrowth. These concepts are presented as a formal model which emphasizes that patterning information could be provided at the boundaries of gene expression domains in the inner ear.

Involvement of programmed cell death in morphogenesis of the vertebrate inner ear.

An outstanding challenge in developmental biology is to reveal the mechanisms underlying the morphogenesis of complex organs. A striking example is the developing inner ear of the vertebrate, which acquires a precise three-dimensional arrangement of its constituent epithelial cells to form three semicircular canals, a central vestibule and a coiled cochlea (in mammals). In generating a semicircular canal, epithelial cells seem to 'disappear' from the center of each canal. This phenomenon has been variously explained as (i) transdifferentiation of epithelium into mesenchyme, (ii) absorption of cells into the expanding canal or (iii) programmed cell death. In this study, an in situ DNA-end labeling technique (the TUNEL protocol) was used to map regions of cell death during inner ear morphogenesis in the chicken embryo from embryonic days 3.5-10. Regions of cell death previously identified in vertebrate ears have been confirmed, including the ventromedial otic vesicle, the base of the endolymphatic duct and the fusion plates of the semicircular canals. New regions of cell death are also described in and around the sensory organs. Reducing normal death using retrovirus-mediated overexpression of human bcl-2 causes abnormalities in ear morphogenesis: hollowing of the center of each canal is either delayed or fails entirely. These data provide new evidence to explain the role of cell death in morphogenesis of the semicircular canals.

In vivo gene transfer into the embryonic inner ear using retroviral vectors.

Retrovirus-mediated gene transfer holds great promise for elucidating key genes in the development and function of the inner ear. Retroviral vectors offer a number of advantages over other gene transfer methods including stable and efficient integration into the host genome, high levels of transcription and restriction of expression to a target area. Because of the wide variety of recombinant retroviral vectors currently available, this review outlines which vectors are appropriate for particular applications. Successful strategies for infecting the ear are reviewed and current drawbacks and future directions are discussed.

Cell fate specification in the inner ear.

From its origin as a single ectodermal patch, the inner ear becomes a labyrinth of chambers housing six to eight sensory organs. Along the way, specific cell fates are realized. The secrets underlying these cell fate specifications are beginning to be revealed through the application of several molecular-genetic approaches. Recent papers describing such approaches have included gene expression studies in the early otic epithelium and inner ear sensory epithelia. large-scale screens of zebrafish mutants to identify ear defects, and targeted gene perturbations of neurotrophins. of their receptors or of the Brn-3.1 transcription factor in mice.

Standard atlas of the gross anatomy of the developing inner ear of the chicken.

During development, the chicken inner ear undergoes a series of morphological changes which give rise to the various structures found in the adult, including the mature semicircular canals, utricle, saccule, cochlear duct, endolymphatic duct and sac, and neurons of the eighth cranial nerve ganglion. Beginning as a hollow epithelial sphere, the inner ear is sculpted into this complex labyrinth of fluid-filled ducts punctuated by their associated sensory end organs. In this report, the three-dimensional complexity of the developing inner ear of the chicken embryo is documented in the form of a standard atlas. The protocol involved fixation, dehydration, and clearing of embryonic heads harvested at daily intervals, followed by injection of an opaque dye (enamel paint suspension) into the fluid ducts of the inner ear. The position of the ear is shown relative to surface landmarks at seven different stages of development, ranging from embryonic day 5 (E5) to E18. Also shown are higher-power photomicrographs of the inner ear in isolation taken at daily intervals at E3-E17 and viewed from two orthogonal positions. Three orthogonal views are shown at 6-hour intervals during the critical stages of semicircular canal formation (E6-E7). Quantitative measurements of the linear dimensions of the inner ear (dorsoventral, anteroposterior, and mediolateral axes) as a function of time indicate a linear increase in the growth of the ear from E3 through E18. This atlas should prove valuable for evaluating mutant phenotypes in inner ear morphogenesis following gene perturbation experiments in the chicken.

High efficiency gene transfer into the embryonic chicken CNS using B-subgroup retroviruses.

Attempts to use replication-competent retroviruses to target genes to the chick CNS have met with limited success for injections performed prior to stage 14 using A- or E-subgroup viruses. This study was aimed at improving CNS infection by varying the stage of injection, viral envelope subgroup, viral titer, and the presence or absence of a transgene and/or the polycation polybrene in the inoculum. RCASBP vectors were injected into the neural tube of stages 3-13 embryos and protein expression was determined 9-48 hr later for forebrain, hindbrain, retina, and inner ear. Optimal injection parameters were defined which balanced good survival rates with high levels of transgene expression at early stages. The results demonstrate nearly complete expression of virus-mediated transgenes in neural tissues at stages 15-21 following injection of B-envelope RCASBP with polybrene at stages 7.5-12. This technique can now be applied to study the roles of genes in cell-autonomous events such as cell connectivity, physiology, and differentiation, as well as neural patterning and regional identity.