Utility of HoxB2 enhancer‐mediated Cre activity for functional studies in the developing inner ear

The rhombomere 4(r4)‐restricted expression of the mouse Hoxb2 gene is regulated by a 1.4‐kb enhancer‐containing fragment. Here, we showthat transgenic mouse lines expressing cre driven by this fragment (B2‐r4‐Cre), activated the R26R Cre reporter in rhombomere 4 and the second branchial arch, the epithelium of the first branchial arch, apical ectodermal ridge of the limb buds and the tail region. Of particular interest is Cre activity in the developing inner ear. Cre activity was found in the preotic field and otic placode at E8.5 and otocyst at E9.5–E12.5, in the cochleovestibular and facio‐acoustic ganglia at E10.5 and the vestibular and spiral ganglia and all the otic epithelia derived from the otocyst at E15.5 and P0. Our data suggest that the B2‐r4‐Cre transgenic mice provide an important tool for conditional gene manipulation and lineage tracing in the inner ear. In combination with other transgenic lines expressing cre exclusively in the otic vesicle, the relative contributions of the hindbrain, periotic mesenchyme and otic epithelium in otic development can be dissected. genesis 47:361–365, 2009. © 2009 Wiley‐Liss, Inc.     

Inner ear malformations induced by isotretinoin in hamster fetuses.

Inner ear malformations induced in anotic hamster fetuses following maternal treatment with 50 mg/kg isotretinoin (13-cis-retinoic acid) on gestational day 8 are described. Computer-assisted three dimensional reconstruction was used. Two general types of defective vestibulocochlear development were seen. Defects were bilateral and correlated with extent of middle ear deficiency and severity of mandibular defects. In the more severely affected fetuses the inner ear was limited to an epithelial sac with occasional small projections, no apparent innervation and a correspondingly reduced otic capsule. In most of the fetuses examined the inner ear was less severely affected and was characterized by a reduction in the number of semicircular ducts and alterations in the size and shape of the cochlear duct. These defects are similar to those seen in a child with the isotretinoin embryopathy. Pathogenesis may result from a direct effect on otic epithelium or from faulty inductive interactions with the rhombencephalon or with periotic neural crest cells.     

Lineage analysis in the chicken inner ear shows differences in clonal dispersion for epithelial, neuronal, and mesenchymal cells

The epithelial components of the vertebrate inner ear and its associated ganglion arise from the otic placode. The cell types formed include neurons, hair-cell mechanoreceptors, supporting cells, secretory cells that make endolymphatic fluid or otolithic membranes, and simple epithelial cells lining the fluid-filled cavities. The epithelial sheet is surrounded by an inner layer of connective and vascular tissues and an outer capsule of bone. To explore the mechanisms of cell fate specification in the ear, retrovirus-mediated lineage analysis was performed after injecting virus into the chicken otocyst on embryonic days 2.5-5.5. Because lineage analysis might reveal developmental compartments, an effort was made to study clonal dispersion by sampling infected cells from different parts of the same ear, including the auditory ganglion, cochlea, saccule, utricle, and semicircular canals. Lineage relationships were confirmed for 75 clones by amplification and sequencing of a variable DNA tag carried by each virus. While mesenchymal clones could span different structural parts of the ear, epithelial clones did not. The circumscribed epithelial clones indicated that their progenitors were not highly migratory. Ganglion cell clones, in contrast, were more dispersed. There was no evidence for a common lineage between sensory cells and their associated neurons, a prediction based on a proposal that the ear sensory organs and fly mechanosensory organs are evolutionarily homologous. As expected, placodal derivatives were unrelated to adjacent mesenchymal cells or to nonneuronal cells of the ganglion. Within the otic capsule, fibroblasts and cartilage cells could be related by lineage.     

Origins of inner ear sensory organs revealed by fate map and time-lapse analyses

The inner ear develops from a simple ectodermal thickening called the otic placode into a labyrinth of chambers which house sensory organs that sense sound and are used to maintain balance. Although the morphology and function of the sensory organs are well characterized, their origins and lineage relationships are virtually unknown. In this study, we generated a fate map of Xenopus laevis inner ear at otic placode and otocyst stages to determine the developmental origins of the sensory organs. Our lineage analysis shows that all regions of the otic placode and otocyst can give rise to the sensory organs of the inner ear, though there were differences between labeled quadrants in the range of derivatives formed. A given region often gives rise to cells in multiple sensory organs, including cells that apparently dispersed from anterior to posterior poles and vice versa. These results suggest that a single sensory organ arises from cells in different parts of the placode or otocyst and that cell mixing plays a large role in ear development. Time-lapse videomicroscopy provides further evidence that cells from opposite regions of the inner ear mix during the development of the inner ear, and this mixing begins at placode stages. Lastly, bone morphogenetic protein 4 (BMP-4), a member of the transforming growth factor beta (TGF-beta) family, is expressed in all sensory organs of the frog inner ear, as it is in the developing chicken ear. Inner ear fate maps provide a context for interpreting gene expression patterns and embryological manipulations.     

Increase of ornithine decarboxylase activity elicited by reserpine in the peripheral and central monoaminergic systems of the rat

Ornithine decarboxylase activity was increased about tenfold in adrenal glands and in brain regions preponderantly containing aminergic neurons, by a single dose of 16 mol/kg of reserpine. Maximal enzyme activity in the adrenal glands was observed at about 8 hr after reserpine administration. The ornithine decarboxylase activity-time curves in the brain regions showed a concomitant polyphasic course, with the highest maximum at 12 hr postinjection. Ornithine decarboxylase induction is discussed as an early event in the cascade of molecular events preceding the induction of cell typic enzymes.     

Sonic hedgehog regulates otic capsule chondrogenesis and inner ear development in the mouse embryo

Development of the cartilaginous capsule of the inner ear is dependent on interactions between otic epithelium and its surrounding periotic mesenchyme. During these tissue interactions, factors endogenous to the otic epithelium influence the differentiation of the underlying periotic mesenchyme to form a chondrified otic capsule. We report the localization of Sonic hedgehog (Shh) protein and expression of the Shh gene in the tissues of the developing mouse inner ear. We demonstrate in cultures of periotic mesenchyme that Shh alone cannot initiate otic capsule chondrogenesis. However, when Shh is added to cultured periotic mesenchyme either in combination with otic epithelium or otic epithelial-derived fibroblast growth factor (FGF2), a significant enhancement of chondrogenesis occurs. Addition of Shh antisense oligonucleotide (AS) to cultured periotic mesenchyme with added otic epithelium decreases levels of endogenous Shh and suppresses the chondrogenic response of the mesenchyme cells, while supplementation of Shh AS-treated cultures with Shh rescues cultures from chondrogenic inhibition. We demonstrate that inactivation of Shh by targeted mutation produces anomalies in the developing inner ear and its surrounding capsule. Our results support a role for Shh as a regulator of otic capsule formation and inner ear development during mammalian embryogenesis.     

In vitro development of the embryonic mouse inner ear following exposure to trypsin

Trypsin has been shown to disrupt normal in vitro morphogenesis of embryonic organ rudiments. Otic tissues derived from 11-, 12-, and 13-day-old mouse embryos were exposed to either Ca++- and Mg++-free PBS or 0.25% trypsin dissolved in Ca++- and Mg++-free PBS prior to explanation into organ culture. Trypsin treatment of otic explants disrupted the expression of the normal pattern of inner-ear development in vitro. There was a direct correlation between the embryonic age at time of exposure to trypsin and the severity of dysmorphogenesis of the inner ear. The younger explants showed abnormalities of both vestibular and auditory structures, whereas with increasing embryonic age, abnormalities were confined more to the auditory portion of the inner ear. The results suggest that integrity of the otocyst basal lamina and epitheliomesenchymal tissue interactions are important factors in early otic development. It is postulated that the major effect of trypsin on inner-ear morphogenesis is through disruption of these factors, which may act to regulate the progressive expression of early otic development.     

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.     

Redundant functions of Rac GTPases in inner ear morphogenesis

Development of the mammalian inner ear requires coordination of cell proliferation, cell fate determination and morphogenetic movements. While significant progress has been made in identifying developmental signals required for inner ear formation, less is known about how distinct signals are coordinated by their downstream mediators. Members of the Rac family of small GTPases are known regulators of cytoskeletal remodeling and numerous other cellular processes. However, the function of Rac GTPases in otic development is largely unexplored. Here, we show that Rac1 and Rac3 redundantly regulate many aspects of inner ear morphogenesis. While no morphological defects were observed in Rac3(-/-) mice, Rac1(CKO); Rac3(-/-) double mutants displayed enhanced vestibular and cochlear malformations compared to Rac1(CKO) single mutants. Moreover, in Rac1(CKO); Rac3(-/-) mutants, we observed compromised E-cadherin-mediated cell adhesion, reduced cell proliferation and increased cell death in the early developing otocyst, leading to a decreased size and malformation of the membranous labyrinth. Finally, cochlear extension was severely disrupted in Rac1(CKO); Rac3(-/-) mutants, accompanied by a loss of epithelial cohesion and formation of ectopic sensory patches underneath the cochlear duct. The compartmentalized expression of otic patterning genes within the Rac1(CKO); Rac3(-/-) mutant otocyst was largely normal, however, indicating that Rac proteins regulate inner ear morphogenesis without affecting cell fate specification. Taken together, our results reveal an essential role for Rac GTPases in coordinating cell adhesion, cell proliferation, cell death and cell movements during otic development.     

Changes in the types of collagen synthesized during chondrogenesis of the mouse otic capsule

We have investigated the temporal relationship between the morphological differentiation of the mouse otic capsule and the pattern of collagen synthesis by mouse otocyst-mesenchyme complexes labeled in vitro. In 10.5- to 12-day embryos the mesenchyme surrounding the otocyst was loosely organized except for a few lateroventral condensations; explants from these embryos synthesized only small amounts of collagen. Collagen synthesis by whole explants increased by more than 50% between 12 and 13 days concomitant with metachromatic staining of the lateral periotic mesenchyme. Cartilage specific type II collagen was the predominant collagen synthesized by these explants as confirmed by SDS-PAGE, densitometry, CNBr cleavage, and V8 protease digestion. This biochemical expression of the cartilage phenotype preceded morphologic recognition of otic capsular cartilage by almost 2 days. Type II collagen synthesis continued to increase and predominate through Day 16 of gestation by which time the otic labyrinth was surrounded by mature cartilage. The minor cartilage collagen chains, 1 alpha, 2 alpha, and 3 alpha, first appeared on different days of gestation. The 1 alpha, and 3 alpha chains were synthesized by explants from 11-day embryos while the 2 alpha chain appeared during Day 13, just before overt differentiation of mature cartilage. These results suggested that the 1 alpha, 2 alpha, and 3 alpha chains may not form heterotrimers containing all three chains and that synthesis of the 2 alpha chain may be associated with stabilization of the cartilaginous matrix. Comparison of these data with the patterns of collagen production by mutant, diseased, or experimentally manipulated inner ear tissues may provide insights into the molecular basis of chondrogenic tissue interactions.     

A fate map of chick otic cup closure reveals lineage boundaries in the dorsal otocyst

The vertebrate inner ear is structurally complex, consisting of fluid-filled tubules and sensory organs that subserve the functions of hearing and balance. The epithelial parts of the inner ear are derived from the otic placode, which deepens to form a cup before closing to form the otic vesicle. We fate-mapped the rim of the otic cup to monitor the cellular movements associated with otocyst formation and to aid in interpreting the changing gene expression patterns of the early otic field. Twelve sites around the rim, defined as positions of a clock face, were targeted by iontophoretic injection of fluorescent, lipophilic dye. Labeled cells were imaged 24 and 48 h after injection. The data show that the entire dorsal rim of the otic cup becomes the endolymphatic duct (ED), while the posteroventral rim becomes the lateral otocyst wall. Two intersecting boundaries of lineage restriction were identified near the dorsal pole: one bisecting the ED into anterior and posterior halves and the other defining its lateral edge. We hypothesize that signaling across compartment boundaries may play a critical role in duct specification. This model is discussed in the context of mouse mutants that are defective in both hindbrain development and ED outgrowth.     

Molecular mechanisms underlying inner ear patterning defects in kreisler mutants

Prior studies have shown that kreisler mutants display early inner ear defects that are related to abnormal hindbrain development and signaling. These defects in kreisler mice have been linked to mutation of the kr/mafB gene. To investigate potential relevance of kr/mafB and abnormal hindbrain development in inner ear patterning, we analyzed the ear morphogenesis in kreisler mice using a paint-fill technique. We also examined the expression patterns of a battery of genes important for normal inner ear patterning and development. Our results indicate that the loss of dorsal otic structures such as the endolymphatic duct and sac is attributable to the downregulation of Gbx2, Dlx5 and Wnt2b in the dorsal region of the otocyst. In contrast, the expanded expression domain of Otx2 in the ventral otic region likely contributes to the cochlear phenotype seen in kreisler mutants. Sensory organ development is also markedly disrupted in kreisler mutants. This pattern of defects and gene expression changes is remarkably similar to that observed in Gbx2 mutants. Taken together, the data show an important role for hindbrain cues, and indirectly, kr/mafB, in guiding inner ear morphogenesis. The data also identify Gbx2, Dlx5, Wnt2b and Otx2 as key otic genes ultimately affected by perturbation of the kr/mafB-hindbrain pathway.     

Epithelial control of periotic mesenchyme chondrogenesis

Morphogenesis of the cartilaginous otic capsule is directed by interactions between the epithelial anlage of the membranous labyrinth (otocyst) and its associated periotic mesenchyme. Utilizing a developmental series of high-density (micromass) cultures of periotic mesenchyme to model capsule chondrogenesis, we have shown that the early influence of otic epithelium in cultures of 10.5- or 14-gestation day (gd) periotic mesenchyme results in initiation or suppression of chondrogenesis, respectively. Furthermore, we have shown that introduction of otic epithelium at two distinct times during in vitro development to cultures of 10.5-gd mesenchyme cells results first in an initiation and then in an inhibition of their chondrogenic response. These influences of epithelial tissue on chondrogenic differentiation by periotic mesenchyme are not tissue specific but are characterized by temporal selectivity. The ability of otic epithelium to influence chondrogenesis and the competence of the periotic mesenchyme to respond to its signals are dependent upon the developmental stage of both tissues. This study provides conclusive evidence that otic epithelium acts as a developmental "switch" during otic capsule morphogenesis, signaling first the turning on and then the turning off of chondrogenic programs in the responding cephalic mesenchyme.