Kate F. Barald

From placode to polarization: new tunes in inner ear development

The highly orchestrated processes that generate the vertebrate inner ear from the otic placode provide an excellent and circumscribed testing ground for fundamental cellular and molecular mechanisms of development. The recent pace of discovery in developmental auditory biology has been unusually rapid, with hundreds of papers published in the past 4 years. This review summarizes studies addressing several key issues that shape our current thinking about inner ear development, with particular emphasis on early patterning events, sensory hair cell specification and planar cell polarity.

Early Embryology of the Vertebrate Ear

Organogenesis of the vertebrate inner ear has been described as “one of the most remarkable displays of precision microengineering in the vertebrate body” (Swanson, Howard, and Lewis 1990). The initial morphological event in ear development in all vertebrates is the formation of the embryonic otic placode, a thickening of the head ectoderm in the region of the developing hindbrain. Through interaction with and incorporation of tissue from several other embryonic sources, the placode develops into the otocyst or otic vesicle, a differentiated structure with sharply defined borders (Noden and Van De Water 1986; Couly, Coltey, and Le Douarin 1993). The epithelium of the otic placode/vesicle also gives rise to the primary neurons of the statoacoustic ganglion, later in development called the cochleovestibular ganglion, the octaval, or the otic ganglion (probably the most appropriate terminology), which contributes to cranial nerve VIII and to the specialized sensory structures known as hair cells (Fig. 3.1).

Retinoic Acid Repression of Bone Morphogenetic Protein 4 in Inner Ear Development

ABSTRACT Bone morphogenetic protein 4 (BMP4) and retinoic acid are important for normal development of the inner ear, but whether they are linked mechanistically is not known. BMP4 antagonists disrupt semicircular canal formation, as does exposure to retinoic acid. We demonstrate that retinoic acid directly down-regulates BMP4 transcription in a mouse inner ear-derived cell line, and we identify a novel promoter in the second intron of the BMP4 gene that is a target of this regulation both in the cell line and in the mouse embryonic inner ear in vivo. The importance of this down-regulation is demonstrated in chicken embryos by showing that the retinoic acid effect on semicircular canal development can be overcome by exogenous BMP4.

Expression of Pax2 and patterning of the chick inner ear.

Early regionalized gene expression patterns within the otocyst appear to correlate with and contribute to development of mature otic structures. In the chick, the transcription factor Pax2 becomes restricted to the dorsal and entire medial side of the otocyst by stage 16/17. The dorsal region of the otocyst forms the endolymphatic duct and sac (ED/ES), and the cochlear duct is derived from the ventromedial region. In the mouse, however, Pax2 expression is reported only in the ventromedial and not the dorsal otocyst. In Pax2 null mice, the cochlea is missing or truncated, but vestibular structures differentiate normally. Here we demonstrate that in the chick, the emerging ED/ES express high levels of Pax2 even when the position of the emerging ED is altered with respect to its environment, either by 180° otocyst rotations about the anterior/posterior axis or transplantation of the otocyst into the hindbrain cavity. However, the Pax2 expression pattern is plastic in the rest of the otic epithelium after 180° rotation of the otocyst. Pax2 is upregulated on the medial side (formerly lateral), and downregulated on the lateral side (formerly medial and expressing Pax2) indicating that Pax2 expression is influenced by the environment. Although Pax2 is upregulated in the epithelium after 180° rotations in the region that should form the cochlear duct, cochlear ducts are truncated or absent, and the ED/ES emerge in a new ventrolateral position. Ablation of the hindbrain at the placode or early otic pit stage alters the timing of regionalized Pax2 expression in the otocyst. The resulting otocysts and ears are generally smaller, vestibular structures are abnormal, ED/ES are missing but cochlear ducts are of normal length. The hindbrain and dorsal periotic mesenchyme provide unique trophic and patterning information to the dorsal otocyst. Our results demonstrate that the ED is the earliest structure patterned in the inner ear and that the hindbrain is important for its specification. We also show that, although normal Pax2 expression is required for cochlear duct development, it is downstream of ventral otocyst patterning events.

Immortalized cell lines from embryonic avian and murine otocysts: Tools for molecular studies of the developing inner ear

Recently, our studies have focused on genes expressed at the earliest stages of inner ear development. Our aim is to identify and characterize genes that are involved in determining the axes of the semicircular canals, in otic crest delamination and in early innervation of the inner ear. Many elegant studies of auditory development have been done in animal models. However, the need for large amounts of well‐characterized embryonic material for molecular studies makes the development of otocyst cell lines with different genetic repertoires attractive. We have therefore derived immortalized otocyst cells from two of the most widely used animal models of ear development: avians and mice. Avian cell isolates were produced from quail otocysts (embryonic stage 19) that were transformed with temperature‐sensitive variants of the Rous sarcoma virus (RSV). Among the individual transformed cells are those that produce neuron‐like derivatives in response to treatment with 10−9 M retinoic acid. Mammalian cell isolates were derived from otocysts, of 9 day (post coitus) embryos of the H2kbtsA58 transgennc mouse (Immortomouse), which carries a temperature‐sensitive variant of the Simian Virus 40 Tumor antigen. The vast majority of cells of the Immortomouse are capable of being immortalized at 33°C, the permissive temperature for transgene expression, in the presence of γterferon. Several putative clones of these cells differentiated into neuron‐like cells after temperature shift and withdrawal of γ‐interferon; another isolate of cells assumed a neuron‐like morphology on exposure to brain‐derived neurotrophic factor even at the permissive temperature. We describe also a cell isolate that expresses the Pax‐2 protein product and two putative cell lines that express the protein product of the chicken equivalent of the Drosophila segmentation gene engrailed. These genes and their protein products are expressed in specific subpopulations of otocyst cells at early stages. Both mouse and quail immortalized cell lines will be used to study inner ear development at the molecular level.

The Role of Steroid Hormones in the NF1 Phenotype : Focus on Pregnancy

The Neurofibromatosis Type 1 (NF1) gene functions as a tumor suppressor gene. Loss of its protein, neurofibromin, in the autosomal dominant disorder NF1 is associated with peripheral nervous system tumors, particularly neurofibromas, benign lesions in which the major cell type is the Schwann Cell (SC). Benign and malignant human tumors found in NF1 patients are heterogeneous with respect to their cellular composition. The number and size of neurofibromas in NF1 patients has been shown to increase during pregnancy, with, in some cases, post‐partum regression, which suggests hormonal involvement in this increase. However, in this review, we consider evidence from the literature that both direct hormonal influence on tumor growth and on angiogenesis may contribute to these effects.

Neurotoxic amyloid beta oligomeric assemblies recreated in microfluidic platform with interstitial level of slow flow

Alzheimers disease is accompanied by progressive, time-dependent changes of three moieties of amyloid beta. In vitro models therefore should provide same conditions for more physiologic studies. Here we observed changes in the number of fibrils over time and studied the correlation between amyloid beta moieties and neurotoxicity. Although the number of fibrils increased dramatically, the change in neurotoxicity with time was small, suggesting that fibrils make little contribution to neurotoxicity. To study the neurotoxicity of diffusible moieties by regulating microenvironments, we created a bio-mimetic microfluidic system generating spatial gradients of diffusible oligomeric assemblies and assessed their effects on cultured neurons. We found amyloid beta exposure produced an atrophy effect and observed neurite extension during the differentiation of neural progenitor cells increased when cells were cultured with continuous flow. The results demonstrate the potential neurotoxicity of oligomeric assemblies and establish a prospective microfluidic platform for studying the neurotoxicity of amyloid beta.

Expression of ZIC genes in the development of the chick inner ear and nervous system

ZIC genes, vertebrate homologues of the Drosophila pair‐rule gene odd‐paired (opa), function in embryonic pattern formation, in the early stages of central nervous system neurogenesis and in cerebellar maturation. Mouse Zic genes are expressed in restricted, and in some cases overlapping, patterns during development, particularly in the central and peripheral nervous systems. We identified chick ZIC2 in a differential display analysis of the auditory system designed to find genes up‐regulated after noise trauma. In this study, we examined the expression of chick ZIC1, ZIC2, and ZIC3 by in situ hybridization in normal inner ear development and in the tissues that influence its development, including the hindbrain, the neural crest, and the periotic mesenchyme. Between Hamburger and Hamilton stages 13 and 24, all three ZIC genes were found in the dorsal periotic mesenchyme adjacent to the developing inner ear. ZIC1 mRNA was expressed in the otocyst epithelium between stages 12 and 24, in some sensory tissue, as well as in a striped pattern in the floorplate of the hindbrain that appears to be complementary to that of Chordin, a gene known to regulate ZIC expression in frogs. Chick ZIC genes are also expressed in the neuroectoderm, paraxial mesenchyme, brain, spinal cord, neural crest, and/or the overlying ectoderm as well as the limb buds. In general, ZIC1 and ZIC2 expression patterns overlapped, although ZIC2 expression was less robust; ZIC3 expression was minimal. These observations suggest that ZIC genes, in addition to their known roles in brain development, may play an important role in the development of the chick inner ear. Developmental Dynamics 702–712, 2003.

Uniform cell seeding and generation of overlapping gradient profiles in a multiplexed microchamber device with normally-closed valves

Generation of stable soluble-factor gradients in microfluidic devices enables studies of various cellular events such as chemotaxis and differentiation. However, many gradient devices directly expose cells to constant fluid flow and that can induce undesired responses from cells due to shear stress and/or wash out of cell-secreted molecules. Although there have been devices with flow-free gradients, they typically generate only a single condition and/or have a decaying gradient profile that does not accommodate long-term experiments. Here we describe a microdevice that generates several chemical gradient conditions on a single platform in flow-free microchambers which facilitates steady-state gradient profiles. The device contains embedded normally-closed valves that enable fast and uniform seeding of cells to all microchambers simultaneously. A network of microchannels distributes desired solutions from easy-access open reservoirs to a single output port, enabling a simple setup for inducing flow in the device. Embedded porous filters, sandwiched between the microchannel networks and cell microchambers, enable diffusion of biomolecules but inhibit any bulk flow over the cells.

DAN directs endolymphatic sac and duct outgrowth in the avian inner ear

Bone morphogenetic proteins (BMPs) are expressed in the developing vertebrate inner ear and participate in inner ear axial patterning and the development of its sensory epithelium. BMP antagonists, such as noggin, chordin, gremlin, cerberus, and DAN (differential screening‐selected gene aberrative in neuroblastoma) inhibit BMP activity and establish morphogenetic gradients during the patterning of many developing tissues and organs. In this study, the role of the BMP antagonist DAN in inner ear development was investigated. DAN‐expressing cell pellets were implanted into the otocyst and the periotic mesenchyme to determine the effects of exogenous DAN on otic development. Similar to the effects on the inner ear seen after exposure of otocysts to the BMP4 antagonist noggin, semicircular canals were truncated or eliminated based upon the site of pellet implantation. Unique to the DAN implantations, however, were effects on the developing endolymphatic duct and sac. In DAN‐treated inner ears, endolymphatic ducts and sacs were merged with the crus or grew into the superior semicircular canal. Both the canal and endolymphatic duct and sac effects were rescued by joint implantation of BMP4‐expressing cells. Electroporation of DAN antisense morpholinos into the epithelium of stage 15–17 otocysts, blocking DAN protein synthesis, resulted in enlarged endolymphatic ducts and sacs as well as smaller semicircular canals in some cases. Taken together, these data suggest a role for DAN both in helping to regulate BMP activity spatially and temporally and in patterning and partitioning of the medial otic tissue between the endolymphatic duct/sac and medially derived inner ear structures. Developmental Dynamics 229:219–230, 2004.