Otic Development Research Articles

The preplacodal region: an ectodermal domain with multipotential progenitors that contribute to sense organs and cranial sensory ganglia

The otic primordium belongs to a group of related structures, the sensory placodes that contribute to the paired sense organs - ear, eye and olfactory epithelium - and to the distal parts of the cranial sensory ganglia. Recent evidence suggests that despite their diversity, all placodes share a common developmental origin and a common molecular mechanism which initiates their formation. At the base of placode induction lies the specification of a unique "placode field", termed the preplacodal region and acquisition of this "preplacodal state" is required for ectodermal cells to undergo otic development. Here I review the molecular mechanisms that sequentially subdivide the ectoderm to give rise to the placode territory.

Comparative analysis of Gata3 and Gata2 expression during chicken inner ear development

The inner ear is a complex sensory organ with hearing and balance functions. Gata3 and Gata2 are expressed in the inner ear, and to gain more insight into their roles in otic development, we made a detailed expression analysis in chicken embryos. At early stages, their expression was highly overlapping. At later stages, Gata2 expression became prominent in vestibular and cochlear nonsensory epithelia. In contrast to Gata2, Gata3 was mainly expressed in the developing sensory epithelia, reflecting the importance of this factor in the sensory-neural development of the inner ear. While the later expression patterns of both Gata3 and Gata2 were highly conserved between chicken and mouse, important differences were observed especially with Gata3 during early otic development, providing indications of divergent molecular control during placode invagination in mice and chickens. We also found indications that the regulatory hierarchy observed in mouse, where Gata3 is upstream of Gata2 and Fgf10, could be conserved in chicken.

Expression of marker genes during early ear development in medaka

Induction of the otic placode involves a number of regulatory interactions. Early studies revealed that the induction of this program is initiated by instructive signals from the mesendoderm as well as from the adjacent hindbrain. Further investigations on the molecular level identified in zebrafish Fgf3, Fgf8, Foxi1, Pax8, Dlx3b and Dlx4b genes as key players during the induction phase. Thereafter an increasing number of genes participates in the regulatory interactions finally resulting in a highly structured sensory organ. Based on data from zebrafish we selected medaka genes with presumptive functions during early ear development for an expression analysis. In addition we isolated Foxi1 and Dlx3b gene fragments from embryonic cDNA. Altogether we screened the spatio-temporal distribution of more than 20 representative marker genes for otic development in medaka embryos, with special emphasis on the early phases. Whereas the spatial distribution of these genes is largely conserved between medaka and zebrafish, our comparative analysis revealed several differences, in particular for the timing of expression.

Gata3 is required for early morphogenesis and Fgf10 expression during otic development

Inner ear develops from an induced surface ectoderm placode that invaginates and closes to form the otic vesicle, which then undergoes a complex morphogenetic process to form the membranous labyrinth. Inner ear morphogenesis is severely affected in Gata3 deficient mouse embryos, but the onset and basis of the phenotype has not been known. We show here that Gata3 deficiency leads to severe and unique abnormalities during otic placode invagination. The invagination problems are accompanied often by the formation of a morphological boundary between the dorsal and ventral otic cup and by the precocious appearance of dorsal endolymphatic characteristics. In addition, the endolymphatic domain often detaches from the rest of the otic epithelium during epithelial closure. The expression of several cell adhesion mediating genes is altered in Gata3 deficient ears suggesting that Gata3 controls adhesion and morphogenetic movements in early otic epithelium. Inactivation of Gata3 leads also to a loss of Fgf10 expression in otic epithelium and auditory ganglion demonstrating that Gata3 is an important regulator of Fgf-signalling during otic development.

EYA1 Expression in the Developing Inner Ear

We sought to determine the developmental anatomy and EYA1 protein distribution in the inner ear of Xenopus laevis. Xenopus laevis embryos were stained with monoclonal antibodies and imaged with confocal microscopy. At stage 27, the otocyst fully forms, with strong tubulin staining of early sensory cells at its ventromedial aspect. Neuronal ingrowth follows at stage 33/34. At stage 50, the semicircular canals are complete. EYA1 localizes to the anterior aspect of the otocyst from stages 37 to 44. By stage 50, EYA1 distribution is localized primarily to the sensory maculae and the endolymphatic duct of the developing inner ear. Whole mount confocal imaging of the developing Xenopus inner ear delineates the exact timing of otic development, sensory cell differentiation, and innervation. EYA1 protein expression has a distinct distribution pattern at the anterior aspect of the developing otocyst in stages 41 and 44. Later stages have a more localized pattern, in which EYA1 is detected only in the sensory epithelium and endolymphatic duct. This specific pattern of expression indicates a possible role in the determination of the anterior-posterior orientation of the inner ear, as well as a later role in sensory cell differentiation.

Wnt-dependent regulation of inner ear morphogenesis is balanced by the opposing and supporting roles of Shh

The inner ear is partitioned along its dorsal/ventral axis into vestibular and auditory organs, respectively. Gene expression studies suggest that this subdivision occurs within the otic vesicle, the tissue from which all inner ear structures are derived. While the specification of ventral otic fates is dependent on Shh secreted from the notochord, the nature of the signal responsible for dorsal otic development has not been described. In this study, we demonstrate that Wnt signaling is active in dorsal regions of the otic vesicle, where it functions to regulate the expression of genes (Dlx5/6 and Gbx2) necessary for vestibular morphogenesis. We further show that the source of Wnt impacting on dorsal otic development emanates from the dorsal hindbrain, and identify Wnt1 and Wnt3a as the specific ligands required for this function. The restriction of Wnt target genes to the dorsal otocyst is also influenced by Shh. Thus, a balance between Wnt and Shh signaling activities is key in distinguishing between vestibular and auditory cell types.

Molecular characterization of conditionally immortalized cell lines derived from mouse early embryonic inner ear

Inner ear sensory hair cells (HCs), supporting cells (SCs), and sensory neurons (SNs) are hypothesized to develop from common progenitors in the early embryonic otocyst. Because little is known about the molecular signals that control this lineage specification, we derived a model system of early otic development: conditionally immortalized otocyst (IMO) cell lines from the embryonic day 9.5 Immortomouse. This age is the earliest stage at which the otocyst can easily be separated from surrounding mesenchymal, nervous system, and epithelial cells. At 9.5 days post coitum, there are still pluripotent cells in the otocyst, allowing for the eventual identification of both SN and HC precursors--and possibly an elusive inner ear stem cell. Cell lines derived from primitive precursor cells can also be used as blank canvases for transfections of genes that can affect lineage decisions as the cells differentiate. It is important, therefore, to characterize the "baseline state" of these cell lines in as much detail as possible. We characterized seven representative "precursor-like" IMO cell populations and the uncloned IMO cells, before cell sorting, at the molecular level by polymerase chain reaction (PCR) and immunocytochemistry (IHC), and one line (IMO-2B1) in detail by real-time quantitative PCR and IHC. Many of the phenotypic markers characteristic of differentiated HCs or SCs were detected in IMO-2B1 proliferating cells, as well as during differentiation for up to 30 days in culture. These IMO cell lines represent a unique model system for studying early stages of inner ear development and determining the consequences of affecting key molecular events in their differentiation.

Mouse FGF15 is the ortholog of human and chick FGF19, but is not uniquely required for otic induction

The inner ear develops from an ectodermal placode that is specified by inductive signals from the adjacent neurectoderm and underlying mesoderm. In chick, fibroblast growth factor ( Fgf)- 19 is expressed in mesoderm underlying the presumptive otic placode, and human FGF19 induces expression of otic markers in a tissue explant containing neural plate and surface ectoderm. We show here that mouse Fgf15 is the sequence homolog of chick and human Fgf19/FGF19. In addition, we show that FGF15, like FGF19, is sufficient to induce expression of otic markers in a chick explant assay, suggesting that these FGFs are orthologs. Mouse embryos lacking Fgf15, however, do not have otic abnormalities at E9.5–E10.5, suggesting that Fgf15 is not uniquely required for otic induction or early patterning of the otocyst. To compare FGF15 and FGF19 signaling components and assess where signals potentially redundant with FGF15 might function, we determined the expression patterns of Fgf15 and Fgf19. Unlike Fgf19, Fgf15 is not expressed in mesoderm underlying the presumptive otic placode, but is expressed in the adjacent neurectoderm. Fgfr4, which encodes the likely receptor for both FGF19 and FGF15, is expressed in the neurectoderm of both species, and is also expressed in the mesoderm only in chick. These results suggest the hypotheses that during otic induction, FGF19 signals in either an autocrine fashion to the mesoderm or a paracrine fashion to the neurectoderm, whereas FGF15 signals in an autocrine fashion to the neurectoderm. Thus, the FGFs that signal to the neurectoderm are the best potential candidates for redundancy with FGF15 during mouse otic development.

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.

Expression of mouse fibroblast growth factor and fibroblast growth factor receptor genes during early inner ear development

The inner ear, which mediates hearing and equilibrium, develops from an ectodermal placode located adjacent to the developing hindbrain. Induction of the placode and its subsequent morphogenesis and differentiation into the inner ear epithelium and its sensory neurons, involves signalling interactions within and between otic and non-otic tissues. Several members of the fibroblast growth factor (FGF) family play important roles at various stages of otic development; however, there are additional family members that have not been evaluated. In this study, we surveyed the expression patterns of 18 mouse Fgf and 3 Fgf receptor (Fgfr) genes during early otic development. Two members of the Fgf family, Fgf4 and Fgf16, and all three tested members of the Fgfr family, Fgfr2c, Fgfr3c, and Fgfr4, were expressed in tissues relevant to inner ear development. Fgf4 transcripts were expressed in the preplacodal and placodal ectoderm, suggesting potential roles in placode induction and/or maintenance. Fgf16 was expressed in the posterior otic cup and vesicle, suggesting roles in otic cell fate decisions and/or axis formation.

Molecular Basis of Otic Commitment and Morphogenesis: A Role for Homeodomain-Containing Transcription Factors and Signaling Molecules

Abstract This chapter reviews the patterning processes in the developing inner ear. Pattern formation commences in the forming otic placode and thus takes place in parallel with and is influenced by the ear induction. The commitment steps that were inferred from classical transplantation and explantation experiments are now assumed to be directly related to pattern formation and are addressed first. The next part concentrates on transcription factors, which are regionally expressed at the early stages of otic development (placode, pit, vesicle) and divides these morphologically homogeneous structures into fields or compartments committed to form individual parts of the future inner ear. Furthermore, the role of regulatory loops between signaling molecules and transcription factors during pattern formation are discussed. The last part discusses the importance of patterning genes for the morphogenetic processes that shape the inner ear. Here, data on inactivation of some of the patterning genes ( Nkx5-1 , Dlx5 , Pax2 , and others) in the mouse are reviewed.

A review of inner ear fate maps and cell lineage studies.

A renewed interest in the development of the inner ear has provided more data on the fate and cell lineage relationships of the tissues making up this complex structure. The inner ear develops from a simple ectodermal thickening of the head called the otic placode, which undergoes a great deal of growth and differentiation to form a multichambered nonsensory epithelium that houses the six to nine sensory organs of the inner ear. Despite a large number of studies examining otic development, there have been surprisingly few fate maps generated. The published fate maps encompass four species and range from preotic to otocyst stages. Although some of these studies were consistent with a compartment and boundary model, other studies reveal extensive cell mixing during development. Cell lineage studies have been done in fewer species. At the single cell level the resulting clones in both chicks and frogs appear somewhat restricted in terms of distribution. We conclude that up until late placode stages there are no clear lineage restriction boundaries, meaning that cells seem to mix extensively at these early stages. At late placode stages, when the otic cup has formed, there are at least two boundaries located dorsally in the forming otocyst but none ventrally. These conclusions are consistent with all the fate maps and reconciles the chick and frog data. These results suggest that genes involved in patterning the inner ear may have dynamic and complex expression patterns.

Fgf3 and Fgf8 are required together for formation of the otic placode and vesicle

Fgf3 has long been implicated in otic placode induction and early development of the otocyst; however, the results of experiments in mouse and chick embryos to determine its function have proved to be conflicting. In this study, we determined fgf3 expression in relation to otic development in the zebrafish and used antisense morpholino oligonucleotides to inhibit Fgf3 translation. Successful knockdown of Fgf3 protein was demonstrated and this resulted in a reduction of otocyst size together with reduction in expression of early markers of the otic placode. fgf3 is co-expressed with fgf8 in the hindbrain prior to otic induction and, strikingly, when Fgf3 morpholinos were co-injected together with Fgf8 morpholinos, a significant number of embryos failed to form otocysts. These effects were made manifest at early stages of otic development by an absence of early placode markers (pax2.1 and dlx3) but were not accompanied by effects on cell division or death. The temporal requirement for Fgf signalling was established as being between 60% epiboly and tailbud stages using the Fgf receptor inhibitor SU5402. However, the earliest molecular event in induction of the otic territory, pax8 expression, did not require Fgf signalling, indicating an inductive event upstream of signalling by Fgf3 and Fgf8. We propose that Fgf3 and Fgf8 are required together for formation of the otic placode and act during the earliest stages of its induction.

The nuclear receptor Nor-1 is essential for proliferation of the semicircular canals of the mouse inner ear.

Nor-1 belongs to the nur subfamily of nuclear receptor transcription factors. The precise role of Nor-1 in mammalian development has not been established. However, recent studies indicate a function for this transcription factor in oncogenesis and apoptosis. To examine the spatiotemporal expression pattern of Nor-1 and the developmental and physiological consequences of Nor-1 ablation, Nor-1-null mice were generated by insertion of the lacZ gene into the Nor-1 genomic locus. Disruption of the Nor-1 gene results in inner ear defects and partial bidirectional circling behavior. During early otic development, Nor-1 is expressed exclusively in the semicircular canal forming fusion plates. After formation of the membranous labyrinth, Nor-1 expression in the vestibule is limited to nonsensory epithelial cells localized at the inner edge of the semicircular canals and to the ampullary and utricular walls. In the absence of Nor-1, the vestibular walls fuse together as normal; however, the endolymphatic fluid space in the semicircular canals is diminished and the roof of the ampulla appears flattened due to defective continual proliferative growth of the semicircular canals.

Zebrafish fgf3 and fgf8 Encode Redundant Functions Required for Otic Placode Induction

Members of the fibroblast growth factor (FGF) family of peptide ligands have been implicated in otic placode induction in several vertebrate species. Here, we have functionally analyzed the roles of fgf3 and fgf8 in zebrafish otic development. The role of fgf8 was assessed by analyzing acerebellar (ace) mutants. fgf3 function was disrupted by injecting embryos with antisense morpholino oligomers (MO) specifically designed to block translation of fgf3 transcripts. Disruption of either fgf3 or fgf8 causes moderate reduction in the size of the otic vesicle. Injection of fgf3-MO into ace/ace mutants causes much more severe reduction or complete loss of otic tissue. Moreover, preplacode cells fail to express pax8 and pax2.1, indicating disruption of early stages of otic induction in fgf3-depleted ace/ace mutants. Both fgf3 and fgf8 are normally expressed in the germring by 50% epiboly and are induced in the primordium of rhombomere 4 by 80% epibloy. In addition, fgf3 is expressed during the latter half of gastrulation in the prechordal plate and paraxial cephalic mesendoderm, tissues that either pass beneath or persist near the prospective otic ectoderm. Conditions that alter the pattern of expression of fgf3 and/or fgf8 cause corresponding changes in otic induction. Loss of maternal and zygotic one-eyed pinhead (oep) does not alter expression of fgf3 or fgf8 in the hindbrain, but ablates mesendodermal sources of fgf signaling and delays otic induction by several hours. Conversely, treatment of wild-type embryos with retinoic acid greatly expands the periotic domains of expression of fgf3, fgf8, and pax8 and leads to formation of supernumerary and ectopic otic vesicles. These data support the hypothesis that fgf3 and fgf8 cooperate during the latter half of gastrulation to induce differentiation of otic placodes.

Identification of synergistic signals initiating inner ear development.

Tissue manipulation experiments in amphibians more than 50 years ago showed that induction of the inner ear requires two signals: a mesodermal signal followed by a neural signal. However, the molecules mediating this process have remained elusive. We present evidence for mesodermal initiation of otic development in higher vertebrates and show that the mesoderm can direct terminal differentiation of the inner ear in rostral ectoderm. Furthermore, we demonstrate the synergistic interactions of the extracellular polypeptide ligands FGF-19 and Wnt-8c as mediators of mesodermal and neural signals, respectively, initiating inner ear development.

Genetic Patterning of Embryonic Inner Ear Development

The commitment of the otocyst to form balance (pars superior) and hearing (pars inferior) receptors is under the control of patterning genes. Intersecting patterns of gene expression has been proposed to explain the regionalization of inner ear sensory receptor development. Insights into the roles of patterning genes is being acquired from the analysis of inner ear development of null mutation mice. An example of the consequences of the loss of expression of a single patterning gene is the effect of a null mutation of paired-box gene 2 (Pax2) on formation of the cochlea. Pax2 transcripts are expressed in the pars inferior of the otocyst and null mutant inner ears show agenesis of the cochlea. This is an example of the profound effect that loss of a single patterning gene can have on inner ear development. However, more typically there is redundancy of gene action during inner ear development as exemplified by the overlapping pattern of expression of two closely related homeobox-containing genes (i.e. Hmx2 and Hmx3) in the pars superior of the otocyst. Hmx2 and Hmx3 genes are both expressed early in otic development with Hmx3 transcripts present in the placode. However, null mutation of the Hmx3 patterning gene produced only a limited vestibular defect, and did not result in the agenesis of the vestibule. This result suggests that there is redundancy in genes that have similar patterns of regional expression in the otic anlagen.

Genetic Analysis of Tissue Interactions Required for Otic Placode Induction in the Zebrafish

Development of the vertebrate inner ear begins during gastrulation with induction of the otic placode. Several embryonic tissues, including cephalic mesendoderm, notochord, and hindbrain, have been implicated as potential sources of otic-inducing signals. However, the relative contributions of these tissues have not been determined, nor have any genes affecting placode induction been identified. To address these issues, we analyzed otic placode induction in zebrafish mutants that are deficient in prospective otic-inducing tissues. Otic development was monitored by examining mutant embryos for morphological changes and, in some cases, by visualizing expression patterns ofdlx-3orpax-2.1in preotic cells several hours before otic placode formation. Incyclops(cyc−) mutants, which develop with a partial deficiency of prechordal mesendoderm, otic induction is delayed by up to 1 h. Inone-eyed pinhead(oep−) mutants, which are more completely deficient in prechordal mesendoderm, otic induction is delayed by 1.5 h, and morphology of the otic vesicles is abnormal. Expression of marker genes in other regions of the neural plate is normal, suggesting that ablation of prechordal mesendoderm selectively inhibits otic induction. In contrast, the timing and morphology of otic development is not affected by mutations inno tail(ntl) orfloating head(flh), which prevent notochord differentiation. Similarly, a mutation invalentino(val), which blocks early differentiation of rhombomeres 5 and 6 in the hindbrain, does not delay otic induction, although subsequent patterning of the otic vesicle is impaired. To test whether inductive signals from one tissue can compensate for loss of another, we generated double or triple mutants with various combinations of the above mutations. In none of the multiple mutants do theflhorvalmutations exacerbate delays in placode induction, althoughvaldoes contribute additively to defects in subsequent patterning of the otic vesicle. In contrast, mutants homozygous for bothoepandntl, which interact synergistically to disrupt differentiation of cephalic and axial mesendoderm, show a delay in otic development of about 3 h. These data suggest that cephalic mesendoderm, including prechordal mesendoderm and anterior paraxial mesendoderm, provides the first otic-inducing signals during gastrulation, whereas chordamesoderm plays no discernible role in this process. Becauseval−mutants are deficient for only a portion of the hindbrain, we cannot rule out a role for that tissue in otic placode induction. However, if the hindbrain does provide otic-inducing signals, they apparently differ quantitatively or qualitatively from the signals required for vesicle patterning, asvaldisrupts only the latter.

Induction of cell growth by insulin and insulin-like growth factor-I is associated with Jun expression in the otic vesicle.

The present report investigates the cellular mechanisms involved in the regulation of cell proliferation by insulin and insulin-like growth factor-I (IGF-I) in the developing inner ear. The results show that insulin and IGF-I stimulate cell proliferation in the otic vesicle. This effect is associated with the induction of the expression of the nuclear proto-oncogene c-jun. The temporal profile of Jun expression coincided with the proliferative period of growth of the otic vesicle. IGF-I promoted the hydrolysis of a membrane glycosyl-phosphatidylinositol, which was characterised as the endogenous precursor for inositol phosphoglycan (IPG). Both purified IPG and a synthetic analogue, 6-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-D-myoinositol-1,2-cyclic phosphate (C3), were able to mimic the effects of IGF-I on Jun expression. Anti-IPG antibodies blocked the effects of IGF-I, which were rescued by the addition of IPG or its analogue. These results suggest that the sequence involving the hydrolysis of membrane glycolipids and the expression of c-jun and c-fos proto-oncogenes is part of the mechanism that activates cell division in response to insulin and IGF-I during early organogenesis of the avian inner ear. The implications of these observations for otic development and regeneration are briefly discussed.

Two regulatory genes, cNkx5-1 and cPax2, show different responses to local signals during otic placode and vesicle formation in the chick embryo.

The early stages of otic placode development depend on signals from neighbouring tissues including the hindbrain. The identity of these signals and of the responding placodal genes, however, is not known. We have identified a chick homeobox gene cNkx5-1, which is expressed in the otic placode beginning at stage 10 and exhibits a dynamic expression pattern during formation and further differentiation of the otic vesicle. In a series of heterotopic transplantation experiments, we demonstrate that cNkx5-1 can be activated in ectopic positions. However, significant differences in otic development and cNkx5-1 gene activity were observed when placodes were transplanted into the more rostral positions within the head mesenchyme or into the wing buds of older hosts. These results indicate that only the rostral tissues were able to induce and/or maintain ear development. Ectopically induced cNkx5-1 expression always reproduced the endogenous pattern within the lateral wall of the otocyst that is destined to form vestibular structures. In contrast, cPax2 which is expressed in the medial wall of the early otic vesicle later forming the cochlea never resumed its correct expression pattern after transplantation. Our experiments illustrate that only some aspects of gene expression and presumably pattern formation during inner ear development can be established and maintained ectopically. In particular, the dorsal vestibular structures seem to be programmed earlier and differently from the ventral cochlear part.