Competence, specification and commitment in otic placode induction

The inner ear is induced from cranial ectoderm adjacent to the hindbrain. Despite almost a century of study, the molecular mechanisms of inner ear induction remain obscure. We have identified four genes expressed very early in the anlage of the inner ear, the otic placode. Pax-2, Sox-3, BMP-7 and Notch are all expressed in placodal ectoderm from the 4-5 somite stage (ss) onwards, well before the otic placode becomes morphologically visible at the 12-14ss. We have used these four molecular markers to show that cranial ectoderm becomes specified to form the otic placode at the 4-6ss, and that this ectoderm is committed to a placodal fate by the 10ss. We also demonstrate that much of the embryonic ectoderm is competent to generate an otic placode if taken at a sufficiently early age. We have mapped the location of otic placode-inducing activity along the rostrocaudal axis of the embryo, and have determined that this activity persists at least until the 10ss. Use of the four molecular otic placode markers suggests that induction of the otic placode in birds occurs earlier than previously thought, and proceeds in a series of steps that are independently regulated

Notch signaling augments the canonical Wnt pathway to specify the size of the otic placode

The inner ear derives from a patch of ectoderm defined by expression of the transcription factor Pax2. We recently showed that this Pax2^+ ectoderm gives rise not only to the otic placode but also to the surrounding cranial epidermis, and that Wnt signaling mediates this placode-epidermis fate decision. We now present evidence for reciprocal interactions between the Wnt and Notch signaling pathways during inner ear induction. Activation of Notch1 in Pax2+ ectoderm expands the placodal epithelium at the expense of cranial epidermis, whereas loss of Notch1 leads to a reduction in the size of the otic placode. We show that Wnt signaling positively regulates Notch pathway genes such as Jag1, Notch1 and Hes1, and we have used transgenic Wnt reporter mice to show that Notch signaling can modulate the canonical Wnt pathway. Gain- and loss-of-function mutations in the Notch and Wnt pathways reveal that some aspects of otic placode development - such as Pax8 expression and the morphological thickening of the placode - can be regulated independently by either Notch or Wnt signals. Our results suggest that Wnt signaling specifies the size of the otic placode in two ways, by directly upregulating a subset of otic genes, and by positively regulating components of the Notch signaling pathway, which then act to augment Wnt signaling

Spalt4 mediates invagination and otic placode gene expression in cranial ectoderm

Vertebrate placodes are regions of thickened head ectoderm that contribute to paired sensory organs and cranial ganglia. We demonstrate that the transcription factor Spalt4 (also known as Sall4) is broadly expressed in chick preplacodal epiblast and later resolves to otic, lens and olfactory placodes. Ectopic expression of Spalt4 by electroporation is sufficient to induce invagination of non-placodal head ectoderm and prevent neurogenic placodes from contributing to cranial ganglia. Conversely, loss of Spalt4 function in the otic placode results in abnormal otic vesicle development. Intriguingly, Spalt4 appears to initiate a placode program appropriate for the axial level but is not involved in later development of specific placode fates. Fgfs can regulate Spalt4, since implantation of Fgf2 beads into the area opaca induces its expression. The results suggest that Spalt4 is involved in early stages of placode development, initiating cranial ectodermal invagination and region-specific gene regulatory networks

Identification and characterization of mouse otic sensory lineage genes

Vertebrate embryogenesis gives rise to all cell types of an organism through the development of many unique lineages derived from the three primordial germ layers. The otic sensory lineage arises from the otic vesicle, a structure formed through invagination of placodal non-neural ectoderm. This developmental lineage possesses unique differentiation potential, giving rise to otic sensory cell populations including hair cells, supporting cells, and ganglion neurons of the auditory and vestibular organs. Here we present a systematic approach to identify transcriptional features that distinguish the otic sensory lineage (from early otic progenitors to otic sensory populations) from other major lineages of vertebrate development. We used a microarray approach to analyze otic sensory lineage populations including microdissected otic vesicles (embryonic day 10.5) as well as isolated neonatal cochlear hair cells and supporting cells at postnatal day 3. Non-otic tissue samples including periotic tissues and whole embryos with otic regions removed were used as reference populations to evaluate otic specificity. Otic populations shared transcriptome-wide correlations in expression profiles that distinguish members of this lineage from non-otic populations. We further analyzed the microarray data using comparative and dimension reduction methods to identify individual genes that are specifically expressed in the otic sensory lineage. This analysis identified and ranked top otic sensory lineage-specific transcripts including Fbxo2, Col9a2, and Oc90, and additional novel otic lineage markers. To validate these results we performed expression analysis on select genes using immunohistochemistry and in situ hybridization. Fbxo2 showed the most striking pattern of specificity to the otic sensory lineage, including robust expression in the early otic vesicle and sustained expression in prosensory progenitors and auditory and vestibular hair cells and supporting cells

Rhombomere rotation reveals that multiple mechanisms contribute to the segmental pattern of hindbrain neural crest migration

Hindbrain neural crest cells adjacent to rhombomeres 2 (r2), r4 and r6 migrate in a segmental pattern, toward the first, second and third branchial arches, respectively. Although all rhombomeres generate neural crest cells, those arising from r3 and r5 deviate rostrally and caudally (J. Sechrist, G. Serbedzija, T. Scherson, S. Fraser and M. Bronner-Fraser (1993) Development 118, 691–703). We have altered the rostrocaudal positions of the cranial neural tube, adjacent ectoderm/mesoderm or presumptive otic vesicle to examine tissue influences on this segmental migratory pattern. After neural tube rotation, labeled neural crest cells follow pathways generally appropriate for their new position after grafting. For example, when r3 and r4 were transposed, labeled r3 cells migrated laterally to the second branchial arch whereas labeled r4 cells primarily deviated caudally toward the second arch, with some cells moving rostrally toward the first. In contrast to r4 neural crest cells, transposed r3 cells leave the neural tube surface in a polarized manner, near the r3/4 border. Surprisingly, some labeled neural crest cells moved directionally toward small ectopic otic vesicles that often formed in the ectoderm adjacent to grafted r4. Similarly, they moved toward grafted or displaced otic vesicles. In contrast, surgical manipulation of the mesoderm adjacent to r3 and r4 had no apparent effects. Our results offer evidence that neural crest cells migrate directionally toward the otic vesicle, either by selective attraction or pathway-derived cues

Basal lamina heparan sulphate proteoglycan is involved in otic placode invagination in chick embryos

Producción CientíficaInvagination of other cup-shaped organ primordia. It is known that the cellular cytoskele- ton plays a Abstract Formation of the otocyst from the otic placode appears to differ from limited role in otic placode invagination, whilst the extracellular matrix underlying the otic pri- mordium intervenes in the folding process. In this study we have analysed the role of the basal lamina heparan sulphate proteoglycan in otic primordium invagination. At 10 H.H. stage, heparan sulphate proteoglycan im- the otic epithelium. Our findings support the theory that otic primordium invagination may be regulated, at least in part, by the basal lamina components, which might contribute towards anchoring the otic epithelium to adja- cent structures. Key words Otic development • Heparinase • Microinjection • Epithelial folding • Extracellular matrix munomarking begins to appear on the otic placode basal lamina, increasing noticeably at 13 H.H. stage, coincid- ing with maximum folding of the otic epithelium, and is still present at later stages. Enzyme degradation of hepa- ran sulphate proteoglycan in the otic primordium basal lamina, by means of microinjection with heparinase III prior to folding, significantly disrupts invagination of the otic placode, which remains practically flat, with a sig- nificant reduction in the depth of the otic pit and an in- crease in the diameter of the otic opening. The immuno- cytochemistry analysis revealed a notable depletion of basal lamina heparan sulphate proteoglycan in the otic primordia microinjected with heparinase, with no statis- tically significant differences observed in the volume or rate of cell proliferation in the otic epithelium relative to the control, which suggests that heparan sulphate prot- eoglycan disruption does not interfere with the epithelial growth. In addition, a study of apoptosis distribution by the TUNEL method confirmed that treatment with hepa- rinase does not cause interference with cell survival i

Pattern of expression of the jun family of transcription factors during the early development of the inner ear: implications in apoptosis

8 pages, 6 figures, 1 table.Jun transcription factors have been implicated in the regulation of cell proliferation, differentiation and apoptosis. We have investigated the relationship between Jun expression and cell death in the developing chicken inner ear. c-jun and junD transcripts were expressed in the epithelium of the otic placode and otic vesicle. c-jun expression was restricted to the dorsal area of the otic pit (stages 14-17), dorsal otic vesicle and cochleo-vestibular ganglion (stages 18-20). junD expression was transient and occurred in the dorsal and upper medial aspects of the otic pit and otic cup, but it was down-regulated in the otic vesicle. A parallel TUNEL analysis revealed that expression of c-jun co-located within areas of intense apoptosis. Furthermore, phosphorylation of c-Jun at serine-63 by Jun amino-terminal-kinases was detected in the dorsal otic pit, otic vesicle and cochleo-vestibular ganglion. c-Jun protein exhibited DNA binding activity, as assessed by gel mobility shift assays. The association between c-Jun and apoptosis was further demonstrated by studying nerve growth factor-induced apoptosis in cultured otic vesicles. Nerve growth factor-induced cell death and c-Jun phosphorylation that were suppressed by insulin-like growth factor-I and by viral-mediated overexpression of Raf, which had survival effects. In conclusion, the precise regulation of the expression and activity of Jun proteins in the otic primordium suggests that it may operate as a fundamental mechanism during organogenesis.L.A. and Y.L. held a CSIC research contract, C.S. was supported by Ministerio de Educación y Cultura and S.C. by the Comunidad de Madrid. This work was supported by grants from Dirección General de Investigación, Ciencia y Tecnología (PM96-0075 and PB95-0086), Europharma (Boehringer Ingelheim Inc.) and Junta de Castilla y León to F.G and I. V.-N.Peer reviewe

Spatial and temporal segregation of auditory and vestibular neurons in the otic placode

AbstractThe otic placode generates the auditory and vestibular sense organs and their afferent neurons; however, how auditory and vestibular fates are specified is unknown. We have generated a fate map of the otic placode and show that precursors for vestibular and auditory cells are regionally segregated in the otic epithelium. The anterior-lateral portion of the otic placode generates vestibular neurons, whereas the posterior-medial region gives rise to auditory neurons. Precursors for vestibular and auditory sense organs show the same distribution. Thus, different regions of the otic placode correspond to particular sense organs and their innervating neurons. Neurons from contiguous domains rarely intermingle suggesting that the regional organisation of the otic placode dictates positional cues to otic neurons. But, in addition, vestibular and cochlear neurogenesis also follows a stereotyped temporal pattern. Precursors from the anterior-lateral otic placode delaminate earlier than those from its medial-posterior portion. The expression of the proneural genes NeuroM and NeuroD reflects the sequence of neuroblast formation and differentiation. Both genes are transiently expressed in vestibular and then in cochlear neuroblasts, while differentiated neurons express Islet1, Tuj1 and TrkC, but not NeuroM or NeuroD. Together, our results indicate that the position of precursors within the otic placode confers identity to sensory organs and to the corresponding otic neurons. In addition, positional information is integrated with temporal cues that coordinate neurogenesis and sensory differentiation

Pax2/8 proteins coordinate sequential induction of otic and epibranchial placodes through differential regulation of foxi1, sox3 and fgf24

AbstractVertebrate cranial placodes contribute vitally to development of sensory structures of the head. Amongst posterior placodes, the otic placode forms the inner ear whereas nearby epibranchial placodes produce sensory ganglia within branchial clefts. Though diverse in fate, these placodes show striking similarities in their early regulation. In zebrafish, both are initiated by localized Fgf signaling plus the ubiquitous competence factor Foxi1, and both express pax8 and sox3 in response. It has been suggested that Fgf initially induces a common otic/epibranchial field, which later subdivides in response to other signals. However, we find that otic and epibranchial placodes form at different times and by distinct mechanisms. Initially, Fgf from surrounding tissues induces otic expression of pax8 and sox3, which cooperate synergistically to establish otic fate. Subsequently, pax8 works with related genes pax2a/pax2b to downregulate otic expression of foxi1, a necessary step for further otic development. Additionally, pax2/8 activate otic expression of fgf24, which induces epibranchial expression of sox3. Knockdown of fgf24 or sox3 causes severe epibranchial deficiencies but has little effect on otic development. These findings clarify the roles of pax8 and sox3 and support a model whereby the otic placode forms first and induces epibranchial placodes through an Fgf-relay