Otic Placode Research Articles

Pax2 modulates proliferation during specification of the otic and epibranchial placodes

The inner ear and epibranchial ganglia of vertebrates arise from a shared progenitor domain that is induced by FGF signalling, the posterior placodal area (PPA), before being segregated by Wnt signalling. One of the first genes activated in the PPA is the transcription factor Pax2. Loss-of- and gain-of function studies have defined a role for Pax2 in placodal morphogenesis and later inner ear development, but have not addressed the role Pax2 plays during the formation and maintenance of the PPA. To understand the role of Pax2 during the development of the PPA, we used over-expression and repression of Pax2. Both gave rise to a smaller otocyst and repressed the formation of epibranchial placodes. In addition, cell cycle analysis revealed that Pax2 suppression reduced proliferation of the PPA. Our results suggest that Pax2 functions in the maintenance but not the induction of the PPA. One role of Pax2 is to maintain proper cell cycle proliferation in the PPA.

Restoration of auditory evoked responses by human ES-cell-derived otic progenitors

Deafness is a condition with a high prevalence worldwide, produced primarily by the loss of the sensory hair cells and their associated spiral ganglion neurons (SGNs). Of all the forms of deafness, auditory neuropathy is of a particular concern. This condition, defined primarily by damage to the SGNs with relative preservation of the hair cells 1, is responsible for a substantial proportion of patients with hearing impairment 2. While the loss of hair cells can be circumvented partially by a cochlear implant, no routine treatment is available for sensory neuron loss since poor innervation limits the prospective performance of an implant 3. Using stem cells to recover the damaged sensory circuitry is a potential therapeutic strategy. Here, we present a protocol to induce differentiation from human embryonic stem cells (hESCs) using signals involved in the initial specification of the otic placode. We obtained two types of otic progenitors able to differentiate in vitro into hair cell-like cells and auditory neurons that display expected electrophysiological properties. Moreover, when transplanted into an auditory neuropathy model, otic neuroprogenitors engraft, differentiate and significantly improve auditory evoked response (ABR) thresholds. These results should stimulate further research into the development of a cell-based therapy for deafness.

Neural crest and olfactory system: new prospective.

Sensory neurons in vertebrates are derived from two embryonic transient cell sources: neural crest (NC) and ectodermal placodes. The placodes are thickenings of ectodermal tissue that are responsible for the formation of cranial ganglia as well as complex sensory organs that include the lens, inner ear, and olfactory epithelium. The NC cells have been indicated to arise at the edges of the neural plate/dorsal neural tube, from both the neural plate and the epidermis in response to reciprocal interactions Moury and Jacobson (Dev Biol 141:243-253, 1990). NC cells migrate throughout the organism and give rise to a multitude of cell types that include melanocytes, cartilage and connective tissue of the head, components of the cranial nerves, the dorsal root ganglia, and Schwann cells. The embryonic definition of these two transient populations and their relative contribution to the formation of sensory organs has been investigated and debated for several decades (Basch and Bronner-Fraser, Adv Exp Med Biol 589:24-31, 2006; Basch et al., Nature 441:218-222, 2006) review (Baker and Bronner-Fraser, Dev Biol 232:1-61, 2001). Historically, all placodes have been described as exclusively derived from non-neural ectodermal progenitors. Recent genetic fate-mapping studies suggested a NC contribution to the olfactory placodes (OP) as well as the otic (auditory) placodes in rodents (Murdoch and Roskams, J Neurosci Off J Soc Neurosci 28:4271-4282, 2008; Murdoch et al., J Neurosci 30:9523-9532, 2010; Forni et al., J Neurosci Off J Soc Neurosci 31:6915-6927, 2011b; Freyer et al., Development 138:5403-5414, 2011; Katoh et al., Mol Brain 4:34, 2011). This review analyzes and discusses some recent developmental studies on the OP, placodal derivatives, and olfactory system.

Apoptosis contributes to placode morphogenesis in the posterior placodal area of mice

In the embryonic head of vertebrates, neurogenic and non-neurogenic ectodermal placodes arise from the panplacodal primordium. Whether and how growth processes of the ectodermal layer, changes in the transcriptional precursor cell profile, or positional changes among precursor cells contribute to interplacodal boundary formation is subject to intense investigation. We demonstrate that large scale apoptosis in the multiplacodal posterior placodal area (PPA) of C57BL/6 mice assists in the segregation of otic and epibranchial placodes. Complex patterns of interplacodal apoptosis precede and parallel the structural individualization of high-grade thickened placodes, with the fundamental separation between otic and epibranchial precursor cells being seemingly prevalent. Interplacodal apoptosis between the emerging epibranchial placodes, which express Neurogenin2 prior to their complete structural individualization, comes out most strongly between the epibranchial placodes 1 and 2. Apoptosis then moves from interplacodal to intraplacodal positions in dorsal and, with a delay, ventral parts of the epibranchial placodes. Intraplacodal apoptosis appears to exert corrective actions among premigratory neuroblasts, and helps to eliminate the epibranchial placodes. The present findings confirm and extend earlier observations in Tupaia belangeri (Washausen et al. in Dev Biol 278:86-102, 2005), regarded as an intermediate between primates and other eutherian orders. Having now available maps of apoptosis in the PPA of embryonic mice, further investigations into the functions of inter- and intraplacodal apoptosis can be carried out in an experimentally and genetically more accessible mammalian model organism.

Retinoic acid-driven Hox1 is required in the epidermis for forming the otic/atrial placodes during ascidian metamorphosis

Retinoic acid (RA)-mediated expression of the homeobox gene Hox1 is a hallmark of the chordate central nervous system (CNS). It has been suggested that the RA-Hox1 network also functions in the epidermal ectoderm of chordates. Here, we show that in the urochordate ascidian Ciona intestinalis, RA-Hox1 in the epidermal ectoderm is necessary for formation of the atrial siphon placode (ASP), a structure homologous to the vertebrate otic placode. Loss of Hox1 function resulted in loss of the ASP, which could be rescued by expressing Hox1 in the epidermis. As previous studies showed that RA directly upregulates Hox1 in the epidermis of Ciona larvae, we also examined the role of RA in ASP formation. We showed that abolishment of RA resulted in loss of the ASP, which could be rescued by forced expression of Hox1 in the epidermis. Our results suggest that RA-Hox1 in the epidermal ectoderm played a key role in the acquisition of the otic placode during chordate evolution.

Evolution of electrosensory ampullary organs: conservation of Eya4 expression during lateral line development in jawed vertebrates

The lateral line system of fishes and amphibians comprises two ancient sensory systems: mechanoreception and electroreception. Electroreception is found in all major vertebrate groups (i.e. jawless fishes, cartilaginous fishes, and bony fishes); however, it was lost in several groups including anuran amphibians (frogs) and amniotes (reptiles, birds, and mammals), as well as in the lineage leading to the neopterygian clade of bony fishes (bowfins, gars, and teleosts). Electroreception is mediated by modified "hair cells," which are collected in ampullary organs that flank lines of mechanosensory hair cell containing neuromasts. In the axolotl (a urodele amphibian), grafting and ablation studies have shown a lateral line placode origin for both mechanosensory neuromasts and electrosensory ampullary organs (and the neurons that innervate them). However, little is known at the molecular level about the development of the amphibian lateral line system in general and electrosensory ampullary organs in particular. Previously, we identified Eya4 as a marker for lateral line (and otic) placodes, neuromasts, and ampullary organs in a shark (a cartilaginous fish) and a paddlefish (a basal ray-finned fish). Here, we show that Eya4 is similarly expressed during otic and lateral line placode development in the axolotl (a representative of the lobe-finned fish clade). Furthermore, Eya4 expression is specifically restricted to hair cells in both neuromasts and ampullary organs, as identified by coexpression with the calcium-buffering protein Parvalbumin3. As well as identifying new molecular markers for amphibian mechanosensory and electrosensory hair cells, these data demonstrate that Eya4 is a conserved marker for lateral line placodes and their derivatives in all jawed vertebrates.

Mutual repression between Gbx2 and Otx2 in sensory placodes reveals a general mechanism for ectodermal patterning

In the vertebrate head, central and peripheral components of the sensory nervous system have different embryonic origins, the neural plate and sensory placodes. This raises the question of how they develop in register to form functional sense organs and sensory circuits. Here we show that mutual repression between the homeobox transcription factors Gbx2 and Otx2 patterns the placode territory by influencing regional identity and by segregating inner ear and trigeminal progenitors. Activation of Otx2 targets is necessary for anterior olfactory, lens and trigeminal character, while Gbx2 function is required for the formation of the posterior otic placode. Thus, like in the neural plate antagonistic interaction between Otx2 and Gbx2 establishes positional information thus providing a general mechanism for rostro-caudal patterning of the ectoderm. Our findings support the idea that the Otx/Gbx boundary has an ancient evolutionary origin to which different modules were recruited to specify cells of different fates.

Sox10‐iCreERT2: A mouse line to inducibly trace the neural crest and oligodendrocyte lineage

SOX10 is a well-conserved and widely expressed transcription factor involved in the regulation of embryonic development and in the determination of cell fate. As it is expressed in neural crest cells, their derivatives and the oligodendrocyte lineage, mutations of the protein contribute to a variety of diseases like neurocristopathies, peripheral demyelinating neuropathies, and melanoma. Here, we report the generation of an inducible Sox10-iCreER(T2) BAC transgenic mouse line that labels, depending on the timepoint of induction, distinct derivatives of the otic placode and the neural crest as well as cells of the oligodendrocyte lineage. Surprisingly, we could show a neural crest origin of pericytes in the brain. Besides its use for fate-mapping, the Sox10-iCreER(T2) mouse line is a powerful tool to conditionally inactivate genes in the neural crest cells, their progeny and/or the oligodendrocyte lineage in a time-dependent fashion to gain further insights into their function and contribution to diseases.

Conditions that influence the response to Fgf during otic placode induction

Despite the vital importance of Fgf for otic induction, previous attempts to study otic induction through Fgf misexpression have yielded widely varying and contradictory results. There are also discrepancies regarding the ability of Fgf to induce otic tissue in ectopic locations, raising questions about the sufficiency of Fgf and the degree to which other local factors enhance or restrict otic potential. Using heat shock-inducible transgenes to misexpress Fgf3 or Fgf8 in zebrafish, we found that the stage, distribution and level of misexpression strongly influence the response to Fgf. Fgf misexpression during gastrulation can inhibit or promote otic development, depending on context, whereas misexpression after gastrulation leads to expansion of otic markers throughout preplacodal ectoderm surrounding the head. Elevated Fgf also expands expression of the putative competence factor Foxi1, which is required for Fgf to expand other otic markers. Misexpression of downstream factors Pax2a or Pax8 also expands otic markers but cannot bypass the requirement for Fgf or Foxi1. Co-misexpression of Pax2/8 with Fgf8 potentiates formation of ectopic otic vesicles expressing a full range of otic markers. These findings document the variables critically affecting the response to Fgf and clarify the roles of foxi1 and pax2/8 in the otic response.

Neural crest stem cells and their potential application in a therapy for deafness

Neurosensory hearing loss is a common condition that has major social and economic implications. Recent advances in stem cell research and in cochlear implantation are offering renewed hopes to people suffering from damage to the auditory hair cells and their associated neurons. Several putative donor cell types are currently being explored, including embryonic stem cells, different types of adult stem cell and the recently described induced-pluripotent stem cells. In this review, we draw attention to the potential application of neural crest stem cells for the treatment of deafness. This population shares a similar developmental origin with the cells of the otic placode, the molecular machinery controlling their maturation and differentiation is comparable and they can produce related sensory neurons. More importantly, pockets of neural crest stem cells remain in the adult body in regions of relatively easy access, facilitating their use for autologous transplantation and therefore avoiding the need for immunosuppression and the problems of tissue rejection. Their exploration and application to hearing conditions could facilitate the development of a clinically-viable, cell-based.

Neural crest stem cells and their potential application in a therapy for deafness

Neurosensory hearing loss is a common condition that has major social and economic implications. Recent advances in stem cell research and in cochlear implantation are offering renewed hopes to people suffering from damage to the auditory hair cells and their associated neurons. Several putative donor cell types are currently being explored, including embryonic stem cells, different types of adult stem cell and the recently described induced-pluripotent stem cells. In this review, we draw attention to the potential application of neural crest stem cells for the treatment of deafness. This population shares a similar developmental origin with the cells of the otic placode, the molecular machinery controlling their maturation and differentiation is comparable and they can produce related sensory neurons. More importantly, pockets of neural crest stem cells remain in the adult body in regions of relatively easy access, facilitating their use for autologous transplantation and therefore avoiding the need for immunosuppression and the problems of tissue rejection. Their exploration and application to hearing conditions could facilitate the development of a clinically-viable, cell-based.

Dual embryonic origin of the mammalian otic vesicle forming the inner ear

The inner ear and cochleovestibular ganglion (CVG) derive from a specialized region of head ectoderm termed the otic placode. During embryogenesis, the otic placode invaginates into the head to form the otic vesicle (OV), the primordium of the inner ear and CVG. Non-autonomous cell signaling from the hindbrain to the OV is required for inner ear morphogenesis and neurogenesis. In this study, we show that neuroepithelial cells (NECs), including neural crest cells (NCCs), can contribute directly to the OV from the neural tube. Using Wnt1-Cre, Pax3(Cre/+) and Hoxb1(Cre/+) mice to label and fate map cranial NEC lineages, we have demonstrated that cells from the neural tube incorporate into the otic epithelium after otic placode induction has occurred. Pax3(Cre/+) labeled a more extensive population of NEC derivatives in the OV than did Wnt1-Cre. NEC derivatives constitute a significant population of the OV and, moreover, are regionalized specifically to proneurosensory domains. Descendents of Pax3(Cre/+) and Wnt1-Cre labeled cells are localized within sensory epithelia of the saccule, utricle and cochlea throughout development and into adulthood, where they differentiate into hair cells and supporting cells. Some NEC derivatives give rise to neuroblasts in the OV and CVG, in addition to their known contribution to glial cells. This study defines a dual cellular origin of the inner ear from sensory placode ectoderm and NECs, and changes the current paradigm of inner ear neurosensory development.

Fgf and Hh signalling act on a symmetrical pre-pattern to specify anterior and posterior identity in the zebrafish otic placode and vesicle.

Specification of the otic anteroposterior axis is one of the earliest patterning events during inner ear development. In zebrafish, Hedgehog signalling is necessary and sufficient to specify posterior otic identity between the 10 somite (otic placode) and 20 somite (early otic vesicle) stages. We now show that Fgf signalling is both necessary and sufficient for anterior otic specification during a similar period, a function that is completely separable from its earlier role in otic placode induction. In lia(-/-) (fgf3(-/-)) mutants, anterior otic character is reduced, but not lost altogether. Blocking all Fgf signalling at 10-20 somites, however, using the pan-Fgf inhibitor SU5402, results in the loss of anterior otic structures and a mirror image duplication of posterior regions. Conversely, overexpression of fgf3 during a similar period, using a heat-shock inducible transgenic line, results in the loss of posterior otic structures and a duplication of anterior domains. These phenotypes are opposite to those observed when Hedgehog signalling is altered. Loss of both Fgf and Hedgehog function between 10 and 20 somites results in symmetrical otic vesicles with neither anterior nor posterior identity, which, nevertheless, retain defined poles at the anterior and posterior ends of the ear. These data suggest that Fgf and Hedgehog act on a symmetrical otic pre-pattern to specify anterior and posterior otic identity, respectively. Each signalling pathway has instructive activity: neither acts simply to repress activity of the other, and, together, they appear to be key players in the specification of anteroposterior asymmetries in the zebrafish ear.

Zona Pellucida Domain-Containing Protein β-Tectorin is Crucial for Zebrafish Proper Inner Ear Development

BackgroundThe zona pellucida (ZP) domain is part of many extracellular proteins with diverse functions from structural components to receptors. The mammalian β-tectorin is a protein of 336 amino acid residues containing a single ZP domain and a putative signal peptide at the N-terminus of the protein. It is 1 component of a gel-like structure called the tectorial membrane which is involved in transforming sound waves into neuronal signals and is important for normal auditory function. β-Tectorin is specifically expressed in the mammalian and avian inner ear.Methodology/Principal FindingsWe identified and cloned the gene encoding zebrafish β-tectorin. Through whole-mount in situ hybridization, we demonstrated that β-tectorin messenger RNA was expressed in the otic placode and specialized sensory patch of the inner ear during zebrafish embryonic stages. Morpholino knockdown of zebrafish β-tectorin affected the position and number of otoliths in the ears of morphants. Finally, swimming behaviors of β-tectorin morphants were abnormal since the development of the inner ear was compromised.Conclusions/SignificanceOur results reveal that zebrafish β-tectorin is specifically expressed in the zebrafish inner ear, and is important for regulating the development of the zebrafish inner ear. Lack of zebrafish β-tectorin caused severe defects in inner ear formation of otoliths and function.

A Sox10 enhancer element common to the otic placode and neural crest is activated by tissue-specific paralogs

The otic placode, a specialized region of ectoderm, gives rise to components of the inner ear and shares many characteristics with the neural crest, including expression of the key transcription factor Sox10. Here, we show that in avian embryos, a highly conserved cranial neural crest enhancer, Sox10E2, also controls the onset of Sox10 expression in the otic placode. Interestingly, we show that different combinations of paralogous transcription factors (Sox8, Pea3 and cMyb versus Sox9, Ets1 and cMyb) are required to mediate Sox10E2 activity in the ear and neural crest, respectively. Mutating their binding motifs within Sox10E2 greatly reduces enhancer activity in the ear. Moreover, simultaneous knockdown of Sox8, Pea3 and cMyb eliminates not only the enhancer-driven reporter expression, but also the onset of endogenous Sox10 expression in the ear. Rescue experiments confirm that the specific combination of Myb together with Sox8 and Pea3 is responsible for the onset of Sox10 expression in the otic placode, as opposed to Myb plus Sox9 and Ets1 for neural crest Sox10 expression. Whereas SUMOylation of Sox8 is not required for the initial onset of Sox10 expression, it is necessary for later otic vesicle formation. This new role of Sox8, Pea3 and cMyb in controlling Sox10 expression via a common otic/neural crest enhancer suggests an evolutionarily conserved function for the combination of paralogous transcription factors in these tissues of distinct embryological origin.

Developmental expression of the fermitin/kindlin gene family in Xenopus laevis embryos

Fermitin genes are highly conserved and encode cytocortex proteins that mediate integrin signalling. Fermitin 1 (Kindlin1) is implicated in Kindler syndrome, a human skin blistering disorder. We report the isolation of the three Fermitin orthologs from Xenopus laevis embryos and describe their developmental expression patterns. Fermitin 1 is expressed in the skin, otic and olfactory placodes, pharyngeal arches, pronephric duct, and heart. Fermitin 2 is restricted to the somites and neural crest. Fermitin 3 is expressed in the notochord, central nervous system, cement gland, ventral blood islands, vitelline veins, and myeloid cells. Our findings are consistent with the view that Fermitin 1 is generally expressed in the skin, Fermitin 2 in muscle, and Fermitin 3 in hematopoietic lineages. Moreover, we describe novel sites of Fermitin gene expression that extend our knowledge of this family. Our data provide a basis for further functional analysis of the Fermitin family in Xenopus laevis.

The role of Foxi3 in otic placode induction

The role of Foxi3 in otic placode induction

PAPC and the Wnt5a/Ror2 pathway control the invagination of the otic placode in Xenopus

BackgroundParaxial protocadherin (PAPC) plays a crucial role in morphogenetic movements during gastrulation and somitogenesis in mouse, zebrafish and Xenopus. PAPC influences cell-cell adhesion mediated by C-Cadherin. A putative direct adhesion activity of PAPC is discussed. PAPC also promotes cell elongation, tissue separation and coordinates cell mass movements. In these processes the signaling function of PAPC in activating RhoA/JNK and supporting Wnt-11/PCP by binding to frizzled 7 (fz7) is important.ResultsHere we demonstrate by loss of function experiments in Xenopus embryos that PAPC regulates another type of morphogenetic movement, the invagination of the ear placode. Knockdown of PAPC by antisense morpholinos results in deformation of the otic vesicle without altering otocyst marker expression. Depletion of PAPC could be rescued by full-length PAPC, constitutive active RhoA and by the closely related PCNS but not by classical cadherins. Also the cytoplasmic deletion mutant M-PAPC, which influences cell adhesion, does not rescue the PAPC knockdown. Interestingly, depletion of Wnt5a or Ror2 which are also expressed in the otocyst phenocopies the PAPC morphant phenotype.ConclusionsPAPC signaling via RhoA and Wnt5a/Ror2 activity are required to keep cells aligned in apical-basal orientation during invagination of the ear placode. Since neither the cytoplasmic deletion mutant M-PAPC nor a classical cadherin is able to rescue loss of PAPC we suggest that the signaling function of the protocadherin rather than its role as modulator of cell-cell adhesion is required during invagination of the ear placode.

Notch Signaling Alters Sensory or Neuronal Cell Fate Specification of Inner Ear Stem Cells

Multipotent progenitor cells in the otic placode give rise to the specialized cell types of the inner ear, including neurons, supporting cells, and hair cells. The mechanisms governing acquisition of specific fates by the cells that form the cochleovestibular organs remain poorly characterized. Here we show that whereas blocking Notch signaling with a γ-secretase inhibitor increased the conversion of inner ear stem cells to hair cells by a mechanism that involved the upregulation of bHLH transcription factor, Math1 (mouse Atoh1), differentiation to a neuronal lineage was increased by expression of the Notch intracellular domain. The shift to a neuronal lineage could be attributed in part to continued cell proliferation in cells that did not undergo sensory cell differentiation due to the high Notch signaling, but also involved upregulation of Ngn1. The Notch intracellular domain influenced Ngn1 indirectly by upregulation of Sox2, a transcription factor expressed in many neural progenitor cells, and directly by an interaction with an RBP-J binding site in the Ngn1 promoter/enhancer. The induction of Ngn1 was blocked partially by mutation of the RBP-J site and nearly completely when the mutation was combined with inhibition of Sox2 expression. Thus, Notch signaling had a significant role in the fate specification of neurons and hair cells from inner ear stem cells, and decisions about cell fate were mediated in part by a differential effect of combinatorial signaling by Notch and Sox2 on the expression of bHLH transcription factors.

Ephrin/ephrin receptor expression during early stages of mouse inner ear development

Ephrins and their tyrosine kinase receptors (Ephs) are a highly conserved family of signaling proteins with various functions during embryonic development. Among others, Eph/ephrin signaling is involved in regulating axon guidance, cell migration, and tissue border formation through inducing modifications of the actin cytoskeleton and cell adhesion. During development ephrins and Ephs are expressed in spatially and temporarily regulated patterns in a wide range of tissues. Here, we analyzed the expression of seven members of the Eph and four member of the ephrin family during early stages of mouse inner ear development by whole-mount in situ hybridization. We detected expressions of EphA2, EphA4, EphA7, EphB1, ephrinA4, and ephrinA5 in and around the forming otic placode between embryonic day (E) 8.5 and E10, and report their detailed expression patterns. Our results reveal dynamic expression of several members of the ephrin/Eph family consistent with functions in otic placode development, invagination and neuroblast delamination.