Otic Anlage Research Articles

Chicken embryos share mammalian patterns of apoptosis in the posterior placodal area.

In the posterior placodal area (PPA) of C57BL/6N mice and primate-related Tupaia belangeri (Scandentia), apoptosis helps to establish morphologically separated otic and epibranchial placodes. Here, we demonstrate that basically identical patterns of apoptosis pass rostrocaudally through the Pax2+ PPA of chicken embryos. Interplacodal apoptosis eliminates unneeded cells either between the otic anlage and the epibranchial placodes 1, 2 and/or 3, respectively (type A), or between neighbouring epibranchial placodes (type B). These observations support the idea that in chicken embryos, as in mammals, interplacodal apoptosis serves to remove vestigial lateral line placodes (Washausen & Knabe, 2018, Biol Open 7, bio031815). A special case represents the recently discovered Pax2- /Sox2+ paratympanic organ (PTO) placode that has been postulated to be molecularly distinct from and developmentally independent of the ventrally adjacent first epibranchial (or 'geniculate') placode (O'Neill etal. 2012, Nat Commun 3, 1041). We show that Sox2+ (PTO placodal) cells seem to segregate from the Pax2+ geniculate placode, and that absence of Pax2 in the mature PTO placode is due to secondary loss. We further report that, between Hamburger-Hamilton (HH) stages HH14 and HH26, apoptosis in the combined anlage of the first epibranchial and PTO placodes is almost exclusively found within and/or immediately adjacent to the dorsally located PTO placode. Hence, apoptosis appears to support decision-making processes among precursor cells of the early developing PTO placode and, later, regression of the epibranchial placodes 2 and 3.

Otx2, Gbx2, and Fgf8 expression patterns in the chick developing inner ear and their possible roles in otic specification and early innervation

The chick inner ear is a complex structure containing auditory and vestibular sensory organs innervated by neurons of the acoustic-vestibular ganglion. The molecular signals involved in the specification and initial innervation of the otic epithelium are poorly understood. Here, we present a detailed description of the Otx2, Gbx2, and Fgf8 gene expression patterns in the chick developing inner ear, comparing them with the Bmp4 expression, a putative sensory-organ marker. The Otx2 expression was detected in the ventro-lateral wall of the otic anlage and could play a role in the segregation of the saccule and utricle maculae. The relationship between Gbx2 and Fgf8 expression changed during inner ear development but was always related to the macula sacculi innervation and endolymphatic duct formation. Our results also suggest that the maculae of the saccule and lagena, and the medial portion of the macula utriculi could arise within a broad Fgf8-positive domain previously observed at the otocyst stage. The spatial and temporal relationships between these gene expression domains and the initial innervation of the epithelium by some subpopulations of otic axons suggest that expression domain boundaries could be involved in the specification and early innervation of presumptive sensory patches.

Zebrafish foxi1 mediates otic placode formation and jaw development.

The otic placode is a transient embryonic structure that gives rise to the inner ear. Although inductive signals for otic placode formation have been characterized, less is known about the molecules that respond to these signals within otic primordia. Here, we identify a mutation in zebrafish, hearsay, which disrupts the initiation of placode formation. We show that hearsay disrupts foxi1, a forkhead domain-containing gene, which is expressed in otic precursor cells before placodes become visible; foxi1 appears to be the earliest marker known for the otic anlage. We provide evidence that foxi1 regulates expression of pax8, indicating a very early role for this gene in placode formation. In addition, foxi1 is expressed in the developing branchial arches, and jaw formation is disrupted in hearsay mutant embryos.

13-cis-Retinoic acid alters neural crest cells expressing Krox-20 and Pax-2 in macaque embryos.

This study investigates hindbrain and associated neural crest (NCC), otocyst, and pharyngeal arch development in monkey embryos following teratogenic exposure to 13-cis-retinoic acid (cRA). cRA was orally administered (5 mg/kg) to pregnant long-tailed macaques (Macaca fascicularis) between gestational days (GD) 12 and 27. Embryos were surgically collected at desired stages during treatment, analyzed for external morphological changes, and processed for immunohistochemistry. Two transiently expressed nuclear proteins, Krox-20 and Pax-2, were used as markers for the target cellular and anatomical structures. Rhombomere (r) expression patterns of Pax-2 (r4/r6) and Krox-20 (r3/r5) were maintained after cRA treatment, but r4 and r5 were substantially reduced in size. In untreated embryos, Krox-20 immunoreactive NCC derived from r5 migrated caudally around the developing otocyst to contribute to the third pharyngeal arch mesenchyme. In cRA-treated embryos, a subpopulation of NCC rostral to the otocyst also showed Krox-20 immunoreactivity, but there was a substantial reduction in Krox-20 post-otic NCC. Pax-2 immunoreactive NCC migrating from r4 to the second pharyngeal arch were substantially reduced in numbers in treated embryos. Alteration in the otic anlage included delayed invagination, abnormal relationship with the adjacent hindbrain epithelium, and altered expression boundaries for Pax-2. cRA-associated changes in the pharyngeal arch region due to cRA included truncation of the distal portion of the first arch and reduction in the size of the second arch. These alterations in hindbrain, neural crest, otic anlage, and pharyngeal arch morphogenesis could contribute to some of the craniofacial malformations in the macaque fetus associated with exposure to cRA.

Monoclonal antibody GL1 and its possible involvement in the morphogenesis of the otic vesicle.

In a previous study, a monoclonal antibody (MAB) named GL1 was identified that is expressed in a precise pattern during gastrulation and early neurulation stages in chick embryos. In this article we have further investigated the expression pattern of this MAB in the chick embryo. GL1 antigen is present in several organs that seem not to be related developmentally. Among them, GL1 is present during the early steps of the otic placode formation, in the pharyngeal endoderm, in some neural crest cells, in the somites, and in the ventricular surface of the nervous system. The distribution in the nervous system is well patterned with two broad lines of expression in the ventricular side of the metencephalic region, a unique and centered expression in the border between the metencephalon and the myelencephalon and again in two lines running along the myelencephalon and the rostral spinal cord. Additionally, GL1 can be induced by members of the FGF family, and we have used this system to elucidate its role in otic placode formation. The results obtained reveal that GL1 can be a useful marker for the study of developmental processes in the endoderm, the otic anlage, and the apical surface of the nervous system. Biochemical analysis of the antigen recognized by this MAB must be carried out to elucidate the molecular nature of the antigen.

Shaping, invagination, and closure of the chick embryo otic vesicle: Scanning electron microscopic and quantitative study

Scanning electron microscopy, light microscopy, and morphometric analysis were used to study the morphological changes of the otic placode and vesicle before and during invagination and closure processes. Our results reveal that the otic placode undergoes shaping between stages HH9 and HH12; during this period the rostrocaudal axis is shortened, while the lateromedial axis of the placode lengthens. The presence of long cytokinesis bridges during this period suggests that cellular displacements after mitosis may participate in the shaping of the otic placode. The shaping process appears to facilitate the approach of the otic placode to the neural tube. From stage HH12 on, the otic anlage gradually becomes a U-shaped structure with its medial portion in intimate apposition to the rhombencephalic neural tube. The coincidence in time between the beginning of intimate otic anlage-rhombencephalon contact and active invagination suggests that these two processes are related. Changes occurring at the edges of the otic vesicle until their disappearance in stage HH17 suggest that, in addition to a process of invagination, the edges of the otic anlage become bent. During closure, cells at the edges of the otic vesicle differ in apical morphology according to their topographical location: The cells between the rostral and lateral edges have elongated apices, in contrast with the polygonal shape of the cell apices in other places of the edges. In the opposite side (between the caudal and medial edges) cell death is observed. Closure of the otic vesicle conceptualized as a zipper-like model is discussed. We propose that early development of the otic anlage takes place in four stages: 1) shaping (stages HH9-11); 2) triggering of the invagination (stage HH12); 3) early invagination and lateral bending (stages HH13-15); and 4) late invagination and closure (stages HH16-17).

Patterns of Epithelial Cell Death during Early Development of the Human Inner Ear

A light microscopic study of cell death in a developmental series of otic primordia from 23 human embryos (Carnegie stages 9 to 14) was completed. Degenerated cells were noted predominantly in the placode (stages 9 and 10), cup (stages 11 and 12), and otocyst (stages 13 and 14). A systematic camera lucida study of the appearance and topography of degenerating epithelial cells showed four different areas of cell death in the otic primordia that related to 1) invagination and detachment of the otic anlage, 2) early histogenesis of the statoacoustic ganglion, and 3) development of the endolymphatic duct. The possible role of cell death in the morphogenesis of the inner ear related to morphogenetic movements is discussed.

Cell proliferation during early development of the chick embryo otic anlage: quantitative comparison of migratory and nonmigratory regions of the otic epithelium.

During development of the otic anlage, a certain proportion of epithelial cells migrate toward the mesenchymal compartment to form part of the acoustic-vestibular ganglion. The migrating cells are observed only in the zone of the otic anlage that will make contact with the acoustic-vestibular ganglion (so-called ganglion zone). In Hamburger and Hamilton's stages 13 to 16, the number of epithelial cells that migrate is relatively low, but it becomes steadily higher from stage 17 on. In the otic anlage of chick embryos, between developmental stages 9 and 21 (48 to 94 hours of incubation), mitotic index, apical or basal localization within the epithelium of dividing cells, and orientation of the mitotic spindles were analyzed. These features in the ganglion zone were compared with observations in the rest of the otic epithelium, where migratory processes do not take place. In stages 13 to 15, when few epithelial cells are migrating, the mitotic index (MI) in the ganglion zone of the otic anlage is similar to that in nonmigratory regions. In more advanced stages, however, when cell migration becomes accelerated, the MI in the migratory zone of the otic wall is significantly higher than that in the rest of the otic epithelium. This suggests an intimate relationship between the migration of otic epithelial cells and a high rate of cell proliferation, the possible nature of which is discussed. Although the majority of mitoses in the otic anlage are located at the apical surface of the epithelium, from stage 13 onward, a few dividing cells are seen in the basal third of the epithelium. Furthermore, these basal mitoses appear exclusively in the migratory zone of the otic anlage, thus suggesting a possible relationship between epithelial cell migration and basal mitosis. During the developmental period prior to stage 18, no significant differences in mitotic spindle orientation are noted between migratory and nonmigratory zones of the otic anlage. In contrast, in stages of maximal otic epithelial cell migration (stages 19 to 21), the frequency of mitoses with the spindle axis oriented radially is significantly higher in the migratory zone. These findings point toward a close correlation between increased frequency of radial mitotic spindle orientation and intense cell migration, although the exact nature of this relationship is as yet unknown.

An ultrastructural analysis of abnormal otic development in exencephalic mutant mice.

Homozygous loop-tail (Lp/Lp) mice exhibit defects in the otocyst as well as extensive neural dysraphism. At 19 days of gestation, cells in the otic pit of abnormal embryos are flattened and lack the rounded luminal contours characteristic of otic cells in their normal littermates. Apical filaments also are not as prominent as in normal embryos, and there is an increase in densely stained globular material in cells at the ventral lip of the otic pit. With glutaraldehyde-tannic acid fixation, the basal lamina of the otic pit cells shows differences from that of the normal otic pit. In abnormal specimens, the lamina densa is irregular and clumped, and the adjacent less dense area is spotty and lacks the more uniformly arranged and delicate fibrils characteristic of the normal basal lamina. These defects may reflect faulty developmental interactions between the dysraphic neural tube, mesenchymal cells, and otic anlage.

Pattern formation of the otic placode and morphogenesis of the otocyst

The early embryonic development of the inner ear anlage, from the otic placode stage to formation of the otocyst, was documented in CBA/CBA mice by scanning electron microscopy. The otic placode is identified at the eight- to ten-somite stage, as revealed by the surface morphology of the developing ectoderm. The cells in the otic depression are considerably smaller than those of the adjacent ectodermal surface. Rapid invagination of the otic anlage occurs during the ninth gestational day (approximately eight to 20 somites). In most specimens from the tenth gestational day, the otic vesicle has closed toward the ectodermal surface. The surface of the otocyst on the 13th gestational day shows considerable specialization, with regional differences of the surface structure.

Early development of the otocyst in an exencephalic mutant of the mouse.

Development of the otic anlage was studied by means of scanning electron microscopy in exencephalic loop-tail (Lp/Lp) embryos and in their normal (+/+; Lp/+) littermates at 8.5-9.5 days of gestation. In the abnormal embryos, the cells of the otic pit lack the rounded prominent surfaces and dense arrangement of microvilli which characterize the normal otic pit, and the overall configuration of the otic anlage is also distorted. The relatively early manifestation of abnormalities in the otic anlage of this mutant suggests that the defective neural tube may exert an influence as early as the otic placode stage, or that a common factor may simultaneously affect both otic and neural tube development in this mutant.