Distinct roles for hindbrain and paraxial mesoderm in the induction and patterning of the inner ear revealed by a study of vitamin-A-deficient quail

The hindbrain and cranial paraxial mesoderm have been implicated in the induction and patterning of the inner ear, but the precise role of the two tissues in these processes is still not clear. We have addressed these questions using the vitamin-A-deficient (VAD) quail model, in which VAD embryos lack the posterior half of the hindbrain that normally lies next to the inner ear. Using a battery of molecular markers, we show that the anlagen of the inner ear, the otic placode, is induced in VAD embryos in the absence of the posterior hindbrain. By performing grafting and ablation experiments in chick embryos, we also show that cranial paraxial mesoderm which normally lies beneath the presumptive otic placode is necessary for otic placode induction and that paraxial mesoderm from other locations cannot induce the otic placode. Two members of the fibroblast growth factor family, FGF3 and FGF19, continue to be expressed in this mesodermal population in VAD embryos, and these may be responsible for otic placode induction in the absence of the posterior hindbrain. Although the posterior hindbrain is not required for otic placode induction in VAD embryos, the subsequent patterning of the inner ear is severely disrupted. Several regional markers of the inner ear, such as Pax2, EphA4, SOHo1 and Wnt3a, are incorrectly expressed in VAD otocysts, and the sensory patches and vestibulo-acoustic ganglia are either greatly reduced or absent. Exogenous application of retinoic acid prior to 30 h of development is able rescue the VAD phenotype. By performing such rescue experiments before and after 30 h of development, we show that the inner ear defects of VAD embryos correlate with the absence of the posterior hindbrain. These results show that induction and patterning of the inner ear are governed by separate developmental processes that can be experimentally uncoupled from each other.     

Fgf3 and Fgf10 are required for mouse otic placode induction

The inner ear, which contains the sensory organs specialised for audition and balance, develops from an ectodermal placode adjacent to the developing hindbrain. Tissue grafting and recombination experiments suggest that placodal development is directed by signals arising from the underlying mesoderm and adjacent neurectoderm. In mice, Fgf3 is expressed in the neurectoderm prior to and concomitant with placode induction and otic vesicle formation, but its absence affects only the later stages of otic vesicle morphogenesis. We show here that mouse Fgf10 is expressed in the mesenchyme underlying the prospective otic placode. Embryos lacking both Fgf3 and Fgf10 fail to form otic vesicles and have aberrant patterns of otic marker gene expression, suggesting that FGF signals are required for otic placode induction and that these signals emanate from both the hindbrain and mesenchyme. These signals are likely to act directly on the ectoderm, as double mutant embryos showed normal patterns of gene expression in the hindbrain. Cell proliferation and survival were not markedly affected in double mutant embryos, suggesting that the major role of FGF signals in otic induction is to establish normal patterns of gene expression in the prospective placode. Finally, examination of embryos carrying three out of the four mutant Fgf alleles revealed intermediate phenotypes, suggesting a quantitative requirement for FGF signalling in otic vesicle formation.     

The first steps towards hearing: mechanisms of otic placode induction

The entire inner ear, together with the neurons that innervate it, derive from a simple piece of ectoderm on the side of the embryonic head the otic placode. In this review, we describe the current state of the field of otic placode induction. Several lines of evidence suggest that all craniofacial sensory organs, including the inner ear, derive from a common "pre-placodal region" early in development. We review data showing that assumption of a pre-placodal cell state correlates with the competence of embryonic ectoderm to respond to otic placode inducing signals, such as members of the fibroblast growth factor (FGF) family. We also review evidence for FGF-independent signals that contribute to the induction of the otic placode. Finally, we review recent evidence suggesting that Wnt signals may act after FGF signaling to mediate a cell fate decision between otic placode and epidermis.     

Analysis of FGF-Dependent and FGF-Independent Pathways in Otic Placode Induction

The inner ear develops from a patch of thickened cranial ectoderm adjacent to the hindbrain called the otic placode. Studies in a number of vertebrate species suggest that the initial steps in induction of the otic placode are regulated by members of the Fibroblast Growth Factor (FGF) family, and that inhibition of FGF signaling can prevent otic placode formation. To better understand the genetic pathways activated by FGF signaling during otic placode induction, we performed microarray experiments to estimate the proportion of chicken otic placode genes that can be up-regulated by the FGF pathway in a simple culture model of otic placode induction. Surprisingly, we find that FGF is only sufficient to induce about 15% of chick otic placode-specific genes in our experimental system. However, pharmacological blockade of the FGF pathway in cultured chick embryos showed that although FGF signaling was not sufficient to induce the majority of otic placode-specific genes, it was still necessary for their expression in vivo. These inhibitor experiments further suggest that the early steps in otic placode induction regulated by FGF signaling occur through the MAP kinase pathway. Although our work suggests that FGF signaling is necessary for otic placode induction, it demonstrates that other unidentified signaling pathways are required to co-operate with FGF signaling to induce the full otic placode program.     

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.     

Fgf8 and Fgf3 are required for zebrafish ear placode induction,maintenance and inner ear patterning

The vertebrate inner ear develops from initially 'simple' ectodermal placode and vesicle stages into the complex three-dimensional structure which is necessary for the senses of hearing and equilibrium. Although the main morphological events in vertebrate inner ear development are known, the genetic mechanisms controlling them are scarcely understood. Previous studies have suggested that the otic placode is induced by signals from the chordamesoderm and the hindbrain, notably by fibroblast growth factors (Fgfs) and Wnt proteins. Here we study the role of Fgf8 as a bona-fide hindbrain-derived signal that acts in conjunction with Fgf3 during placode induction, maintenance and otic vesicle patterning. Acerebellar (ace) is a mutant in the fgf8 gene that results in a non-functional Fgf8 product. Homozygous mutants for acerebellar (ace) have smaller ears that typically have only one otolith, abnormal semi-circular canals, and behavioral defects. Using gene expression markers for the otic placode, we find that ace/fgf8 and Fgf-signaling are required for normal otic placode formation and maintenance. Conversely, misexpression of fgf8 or Fgf8-coated beads implanted into the vicinity of the otic placode can increase ear size and marker gene expression, although competence to respond to the induction appears restricted. Cell transplantation experiments and expression analysis suggest that Fgf8 is required in the hindbrain in the rhombomere 4-6 area to restore normal placode development in ace mutants, in close neighbourhood to the forming placode, but not in mesodermal tissues. Fgf3 and Fgf8 are expressed in hindbrain rhombomere 4 during the stages that are critical for placode induction. Joint inactivation of Fgf3 and Fgf8 by mutation or antisense-morpholino injection causes failure of placode formation and results in ear-less embryos, mimicking the phenotype we observe after pharmacological inhibition of Fgf-signaling. Fgf8 and Fgf3 together therefore act during induction and differentiation of the ear placode. In addition to the early requirement for Fgf signaling, the abnormal differentiation of inner ear structures and mechanosensory hair cells in ace mutants, pharmacological inhibition of Fgf signaling, and the expression of fgf8 and fgf3 in the otic vesicle demonstrate independent Fgf function(s) during later development of the otic vesicle and lateral line organ. We furthermore addressed a potential role of endomesomerm by studying mzoep mutant embryos that are depleted of head endomesodermal tissue, including chordamesoderm, due to a lack of Nodal-pathway signaling. In these embryos, early placode induction proceeds largely normally, but the ear placode extends abnormally to midline levels at later stages, suggesting a role for the midline in restricting placode development to dorsolateral levels. We suggest a model of zebrafish inner ear development with several discrete steps that utilize sequential Fgf signals during otic placode induction and vesicle patterning.     

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.     

Tissues and signals involved in the induction of placodal Six1 expression in Xenopus laevis

Ectodermal placodes, from which many cranial sense organs and ganglia develop, arise from a common placodal primordium defined by Six1 expression. Here, we analyse placodal Six1 induction in Xenopus using microinjections and tissue grafts. We show that placodal Six1 induction occurs during neural plate and neural fold stages. Grafts of anterior neural plate but not grafts of cranial dorsolateral endomesoderm induce Six1 ectopically in belly ectoderm, suggesting that only the neural plate is sufficient for inducing Six1 in ectoderm. However, extirpation of either anterior neural plate or of cranial dorsolateral endomesoderm abolishes placodal Six1 expression indicating that both tissues are required for its induction. Elevating BMP-levels blocks placodal Six1 induction, whereas ectopic sources of BMP inhibitors expand placodal Six1 expression without inducing Six1 ectopically. This suggests that BMP inhibition is necessary but needs to cooperate with additional factors for Six1 induction. We show that FGF8, which is expressed in the anterior neural plate, can strongly induce ectopic Six1 in ventral ectoderm when combined with BMP inhibitors. In contrast, FGF8 knockdown abolishes placodal Six1 expression. This suggests that FGF8 is necessary and together with BMP inhibitors sufficient to induce placodal Six1 expression in cranial ectoderm, implicating FGF8 as a central component in generic placode induction.     

The role of Paraxial Protocadherin in Xenopus otic placode development

Vertebrate inner ear develops from its rudiment, otic placode, which later forms otic vesicle and gives rise to tissues comprising the entire inner ear. Although several signaling molecules have been identified as candidates responsible for inner ear specification and patterning, many details remain elusive. Here, we report that Paraxial Protocadherin (PAPC) is required for otic vesicle formation in Xenopus embryos. PAPC is expressed strictly in presumptive otic placode and later in otic vesicle during inner ear morphogenesis. Knockdown of PAPC by dominant-negative PAPC results in the failure of otic vesicle formation and the loss of early inner ear markers Sox9 and Tbx2, suggesting the requirement of PAPC in the early stage of otic vesicle development. However, PAPC alone is not sufficient to induce otic placode formation.     

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.     

Differential expression of LIM domain-only (LMO) genes in the developing mouse inner ear

The vertebrate inner ear, a complex sensory organ with vestibular and auditory functions, is derived from a single ectoderm structure called the otic placode. Currently, the molecular mechanisms governing the differentiation and specification of the otic epithelium are poorly understood. We present here a detailed expression study of LMO1-4 in the developing mouse inner ear using a combination of in situ hybridization and immunohistochemistry. LMO1 is specifically expressed in the vestibular and cochlear hair cells as well as the vestibular ganglia of the developing inner ear. LMO2 expression is detected in the periotic mesenchyme of the developing mouse cochlea from E12.5 to E14.5. The expression of LMO3 expression is first observed in the cochlea at E13.5 and becomes confined to the lesser epithelial ridge (LER) from E14.5 to E17.5. LMO3 is also expressed in some of the vestibular ganglion cells. LMO4 is initially expressed in the dorsolateral portion of the otic vesicle and its expression persists in the semicircular canals, macula, crista, and the spiral ganglia throughout embryogenesis. Thus, the regionalized expression patterns of LMO1-4 are closely associated with the morphogenesis of the inner ear.     

Essential role for PDGF signaling in ophthalmic trigeminal placode induction

Much of the peripheral nervous system of the head is derived from ectodermal thickenings, called placodes, that delaminate or invaginate to form cranial ganglia and sense organs. The trigeminal ganglion, which arises lateral to the midbrain, forms via interactions between the neural tube and adjacent ectoderm. This induction triggers expression of Pax3, ingression of placode cells and their differentiation into neurons. However, the molecular nature of the underlying signals remains unknown. Here, we investigate the role of PDGF signaling in ophthalmic trigeminal placode induction. By in situ hybridization, PDGF receptor beta is expressed in the cranial ectoderm at the time of trigeminal placode formation, with the ligand PDGFD expressed in the midbrain neural folds. Blocking PDGF signaling in vitro results in a dose-dependent abrogation of Pax3 expression in recombinants of quail ectoderm with chick neural tube that recapitulate placode induction. In ovo microinjection of PDGF inhibitor causes a similar loss of Pax3 as well as the later placodal marker, CD151, and failure of neuronal differentiation. Conversely, microinjection of exogenous PDGFD increases the number of Pax3+ cells in the trigeminal placode and neurons in the condensing ganglia. Our results provide the first evidence for a signaling pathway involved in ophthalmic (opV) trigeminal placode induction.