Cell Division and Maintenance of Epithelial Integrity in the Deafened Auditory Epithelium

Quiescence is among the hallmarks of the sensory epithelium of the cochlea. When auditory sensory cells (hair cells) degenerate they are not replaced, and therefore hearing loss is permanent. Cochlear hair cells are susceptible to several types of lesions, including aminoglycoside antibiotics. The application of the aminoglycoside neomycin in the inner ear mimics cases of severe hair cell loss and leads to collapse of the cochlear epithelium. We now report that in mature guinea pig cochleae injected with neomycin, the remaining non-sensory cells undergo a robust proliferative response. p27Kip1, an inhibitor of cell cycle in the cochlea, was present in non-dividing cells and absent during mitosis. Dividing cells retained their tight junction complexes and maintained the structural confluence of the auditory epithelium during cell division. The plane of mitosis was invariably parallel to the luminal surface. These results indicate that the flat epithelium of the cochlea can down-regulate p27Kip1 and divide after a severe lesion and suggest that the cell divisions assist in maintaining the epithelial confluence throughout the cochlea. Presence of mitosis in the tissue presents therapeutic opportunities for gene transfer and stem cells therapies.     

New mechanism of melanosomes transport into hair of lambs of different breeds and genotypes.

By light microscopic investigation of skin and wool specimens of newborn lambs, we discovered a previously unknown mechanism for melanosomes transport in the process of dermal papilla melanocytes regular mitosis and migration into the hair shaft. This mechanism plays a great role in hair pigmentation especially in dominant (ED/ED) and recessive (Aa/Aa) black lambs of all investigated breeds. The rate of pigment cell mitosis, proliferation, and migration differs greatly in lambs of investigated color genotypes. In black genotypes the rate of melanocyte mitosis is very high and is approximately the same as in the hair bulb matrix cells, whereas in brown and red genotypes this rate is much lower. Melanocyte mitosis in the light red and tan groups was not found.     

Drosophila Pins-binding protein Mud regulates spindle-polarity coupling and centrosome organization

The orientation of the mitotic spindle relative to the cell axis determines whether polarized cells undergo symmetric or asymmetric divisions. Drosophila epithelial cells and neuroblasts provide an ideal pair of cells to study the regulatory mechanisms involved. Epithelial cells divide symmetrically, perpendicular to the apical-basal axis. In the asymmetric divisions of neuroblasts, by contrast, the spindle reorients parallel to that axis, leading to the unequal distribution of cell-fate determinants to one daughter cell. Receptor-independent G-protein signalling involving the GoLoco protein Pins is essential for spindle orientation in both cell types. Here, we identify Mushroom body defect (Mud) as a downstream effector in this pathway. Mud directly associates and colocalizes with Pins at the cell cortex overlying the spindle pole(s) in both neuroblasts and epithelial cells. The cortical Mud protein is essential for proper spindle orientation in the two different division modes. Moreover, Mud localizes to centrosomes during mitosis independently of Pins to regulate centrosomal organization. We propose that Drosophila Mud, vertebrate NuMA and Caenorhabditis elegans Lin-5 (refs 5, 6) have conserved roles in the mechanism by which G-proteins regulate the mitotic spindle.     

Survival of bundleless hair cells and subsequent bundle replacement in the bullfrog's saccule.

Our senses of hearing and balance depend upon hair cells, the sensory receptors of the inner ear. Millions of people suffer from hearing and balance deficits caused by damage to hair cells as a result of exposure to noise, aminoglycoside antibiotics, and antitumor drugs. In some species such damage can be reversed through the production of new cells. This proliferative response is limited in mammals but it has been hypothesized that damaged hair cells might survive and undergo intracellular repair. We examined the fate of bullfrog saccular hair cells after exposure to a low dose of the aminoglycoside antibiotic gentamicin to determine whether hair cells could survive such treatment and subsequently be repaired. In organ cultures of the bullfrog saccule a combination of time-lapse video microscopy, two-photon microscopy, electron microscopy, and immunocytochemistry showed that hair cells can lose their hair bundle and survive as bundleless cells for at least 1 week. Time-lapse and electron microscopy revealed stages in the separation of the bundle from the cell body. Scanning electron microscopy (SEM) of cultures fixed 2, 4, and 7 days after antibiotic treatment showed that numerous new hair bundles were produced between 4 and 7 days of culture. Further examination revealed hair cells with small repaired hair bundles alongside damaged remnants of larger surviving bundles. The results indicate that sensory hair cells can undergo intracellular self-repair in the absence of mitosis, offering new possibilities for functional hair cell recovery and an explanation for non-proliferative recovery.     

Forced expression of a dominant-negative chimeric tropomyosin causes abnormal motile behavior during cell division

Forced expression of the chimeric human fibroblast tropomyosin 5/3 (hTM5/3) in CHO cell was previously shown to affect cytokinesis [Warren et al., 1995: J. Cell Biol. 129:697-708]. To further investigate the phenotypic consequences of misexpression, we have compared mitotic spindle organization and dynamic 2D and 3D shape changes during mitosis in normal cells and in a hTM5/3 misexpressing (mutant) cell line. Immunofluorescence microscopy of wild type and mutant cells stained with monoclonal anti-tubulin antibody revealed that the overall structures of mitotic spindles were not significantly different. However, the axis of the mitotic spindle in mutant cells was more frequently misaligned with the long axis of the cell than that of wild type cells. To assess behavioral differences during mitosis, wild type and mutant cells were reconstructed in 2D and 3D and motion analyzed with the computer-assisted 2D and 3D Dynamic Image Analysis Systems (2D-DIAS, 3D-DIAS). Mutant cells abnormally formed large numbers of blebs during the later stages of mitosis and took longer to proceed from the start of anaphase to the start of cytokinesis. Furthermore, each mutant cell undergoing mitosis exhibited greater shape complexity than wild type cells, and in every case lifted one of the two evolving daughter cells off the substratum and abnormally twisted. These results demonstrate that misexpression of hTM5/3 in CHO cells leads to morphological instability during mitosis. Misexpression of hTM5/3 interferes with normal tropomyosin function, suggesting in turn that tropomyosin plays a role through its interaction with actin microfilaments in the regulation of the contractile ring, in the localized suppression of blebbing, in the maintenance of polarity and spatial symmetry during cytokinesis, and in cell spreading after cytokinesis is complete.     

New Mechanism of Melanosomes Transport into Hair of Lambs of Different Breeds and Genotypes

By light microscopic investigation of skin and wool specimens of newborn lambs, we discovered a previously unknown mechanism for melanosomes transport in the process of dermal papilla melanocytes regular mitosis and migration into the hair shaft. This mechanism plays a great role in hair pigmentation especially in dominant (E^D/E^D) and recessive (A^a/A^a) black lambs of all investigated breeds. The rate of pigment cell mitosis, proliferation, and migration differs greatly in lambs of investigated color genotypes. In black genotypes the rate of melanocyte mitosis is very high and is approximately the same as in the hair bulb matrix cells, whereas in brown and red genotypes this rate is much lower. Melanocyte mitosis in the light red and tan groups was not found.     

Survival of bundleless hair cells and subsequent bundle replacement in the bullfrog's saccule

Our senses of hearing and balance depend upon hair cells, the sensory receptors of the inner ear. Millions of people suffer from hearing and balance deficits caused by damage to hair cells as a result of exposure to noise, aminoglycoside antibiotics, and antitumor drugs. In some species such damage can be reversed through the production of new cells. This proliferative response is limited in mammals but it has been hypothesized that damaged hair cells might survive and undergo intracellular repair. We examined the fate of bullfrog saccular hair cells after exposure to a low dose of the aminoglycoside antibiotic gentamicin to determine whether hair cells could survive such treatment and subsequently be repaired. In organ cultures of the bullfrog saccule a combination of time‐lapse video microscopy, two‐photon microscopy, electron microscopy, and immunocytochemistry showed that hair cells can lose their hair bundle and survive as bundleless cells for at least 1 week. Time‐lapse and electron microscopy revealed stages in the separation of the bundle from the cell body. Scanning electron microscopy (SEM) of cultures fixed 2, 4, and 7 days after antibiotic treatment showed that numerous new hair bundles were produced between 4 and 7 days of culture. Further examination revealed hair cells with small repaired hair bundles alongside damaged remnants of larger surviving bundles. The results indicate that sensory hair cells can undergo intracellular self‐repair in the absence of mitosis, offering new possibilities for functional hair cell recovery and an explanation for non‐proliferative recovery. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 81–92, 2002; DOI 10.1002/neu.10002     

Mosaic arrangement of SCPb-, FMRFamide-, and histamine-like immunoreactive sensory hair cells in the statocyst of the gastropod mollusc Pleurobranchaea japonica

A pair of statocysts are located in the periganglionic connective tissue of the pedal ganglia of the opisthobranch mollusc Pleurobranchaea japonica. Light- and electron-microscopic observations show that the sensory epithelium of the statocyst consists of 13 disk-shaped hair cells. Each hair cell sends a single axon to the cerebral ganglion through the static nerve. Neurotransmitters in the hair cells were examined by means of immunocytochemistry. Our results show that the 13 sensory hair cells include two SCPB-, three FMRFamide-, and eight histamine-like immunoreactive cells. One hair cell contains a transmitter substance other than SCPB-, FMRFamide, histamine, serotonin, or GABA. One of the two SCPB-like immunoreactive cells, located in the ventral region of the statocyst, is the largest cell in the statocyst. The other, located in the anterodorsal region, shows co-immunoreactivity to both SCPB and FMRFamide antisera. Among the three FMRFamide-like immunoreactive hair cells, one is located in the posteroventral region, separated from the other two, which are adjacent to each other in the anterodorsal region. All the eight histamine-like immunoreactive hair cells are adjacent to one another, occupying the remainder of a triangular pyramid-shaped region. These immunoreactive cells are symmetrically placed in the right and left statocysts. This mosaic arrangement was identical among specimens. Thus the static nerve may code information about position or movement of the statoliths, with the use of different transmitters in the mosaic arrangement of the hair cells.     

CELL DIVISION IN BULBOCHAETE. II. HAIR CELL FORMATION1

Most vegetative cells of Bulbochaete, and all those of Oedogonium, possess an apical, circular discontinuity in the structure of their secondary wall. Rupture of the wall at this precise site permits expansion of the ring during cell division and release of the zoospore following zoosporogenesis. Certain cells of Bulbochaete (always the apical daughter cell of a division pair) lack this type of discontinuity. Instead, the apical wall is thinned out on one side, so that the cell bulges asymmetrically. In the middle of the bulge is a wall discontinuity which extends only part way around the cell. The wall will rupture here, too, for zoospore release, but if a cell having such a wall, divides, it invariably does so asymmetrically, with one pole of the spindle located in the bulge. Cytokinesis then cuts off a small, colorless daughter cell. The wall ruptures at the discontinuity, and this daughter cell emerges through the slit and differentiates into a hair. The creation of hairs in such cells commences with the deposition of a pad of primary wall lining the bulge. Golgi bodies are involved in its secretion, but not in that of a secondary wall layer which forms next in the premitotic cell and covers the primary wall. The cell becomes polarized; the nucleus migrates toward this region as the chloroplast moves aside. After the asymmetric mitosis, a curved phycoplast cuts off the hair cell nucleus and prevents the chloroplast from moving back into the future hair, whose cytoplasm soon loses much of its affinity for heavy metal stains. Following rupture of the parental wall, the phycoplast moves some distance past the limits of the newly deposited secondary wall layer and then forms a cross wall under the hair. The secondary wall of the hair is not continuous with the secondary wall structure of the parental cell; the circular discontinuity that arises around the base of the bulging parental wall is then perpetuated and accentuated as the hair's secondary wall thickens. This wall weakening becomes the dislocation that will predetermine the site of the ring and consequently the direction of cell expansion in the next normal division of the cell subtending the hair. Abnormal ring formation and the creation of terminal twin hairs have also been examined. The lip of the growing hair contains a characteristic organization of membranes and other components which may be related to the organization of the hair's numerous longitudinally oriented microtubules. These results are discussed in terms of the morphology of the wall in the Oedogionales generally. The creation of the special wall morphology that leads to hair cell formation is considered to be ontogenetically related to a similar wall morphology that is involved in formation of the fertilization pore of the oogonium.     

Pre-axonogenesis migration of afferent pioneer cells in the grasshopper embryo

In insects, afferent neurons arise primarily from the ectodermal epithelium in the periphery and differentiate at the site of their precursor mitosis. Here we describe ectodermally derived cells that migrate away from their site of origin and initiate axonogenesis at a distant location. In embryonic grasshopper limb buds, the first two pairs of afferents to differentiate are the pair of Ti1 pioneers at the limb tip and the pair of Cx1 cells found at the base of the limb. While the Ti1 pioneers arise from the mitosis of a pioneer mother cell at the limb tip, the Cx1 cells are shown to emerge from the epithelium at circumferential positions that are approximately 150 degrees apart and that belong to different embryonic compartments. The cells migrate into contact with each other before initiating axonogenesis, and their axons then extend in a new direction that is orthogonal to the route of cell migration.     

Transdifferentiation of corneal epithelium: evidence for a linkage between the segregation of epidermal stem cells and the induction of hair follicles during embryogenesis

Corneal epithelium transdifferentiation into a hair-bearing epidermis provides a particularly useful system for studying the possibility that transient amplifying (TA) cells are able to activate different genetic programs in response to a change in their fibroblast environment, as well as to follow the different steps of rebuilding an epidermis from induced stem cells. Corneal stem and TA cells are found in different locations - stem cells at the periphery, in the limbus, and TA cells more central. Moreover, the TA cells already express the differentiating corneal-type keratin pair K3/K12, whereas the limbal keratinocytes express the basal keratin pair K5/K14. In contrast, suprabasal epidermal keratinocytes express keratin pair K1-2/K10, and basal keratinocytes the keratin pair K5/K14. The results of tissue recombination experiments show that adult central corneal cells are able to respond to specific information originating from embryonic dermis. First, the cells located at the base of the corneal epithelium show a decrease in expression of K12 keratin, followed by an increase in K5 expression; they then proliferate and form hair follicles. The first K10 expressing cells appear at the junction of the new hair follicles and the covering corneal epithelium. Their expansion finally gives rise to epidermal strata, which displace the corneal suprabasal keratinocytes. Corneal TA cells can thus be reprogrammed to form epidermal cells, first by reverting to a basal epithelial-type, then to hair pegs and probably concomitantly to hair stem cells. This confirms the role of the hair as the main reservoir of epidermal stem cells and raises the question of the nature of the dermal messages which are both involved in hair induction and stem cell specification.     

Synthesis of unique low molecular weight proteins during late G2 and mitosis

Changes in protein synthesis were examined during the cell cycle of Chinese hamster ovary cells by labeling synchronized cells at various times with [35S]methionine and separating the proteins on two-dimensional polyacrylamide gels. Several proteins, including tubulin, showed marked differences in their relative rates of synthesis during the cell cycle. A few proteins were found to be synthesized at a specific time during the cycle. In particular, a pair of proteins of approximately 21,000 daltons and isoelectric point of 5.5 were found to be synthesized only in late G2 and mitotic cells. Cells that were labeled during mitosis and then allowed to divide showed no trace of these proteins, indicating that their presence is transient and that they are likely involved in mitosis.     

KIF18B is a cell type–specific regulator of spindle orientation in the epidermis

Proper spindle orientation is required for asymmetric cell division and the establishment of complex tissue architecture. In the developing epidermis, spindle orientation requires a conserved cortical protein complex of LGN/NuMA/dynein-dynactin. However, how microtubule dynamics are regulated to interact with this machinery and properly position the mitotic spindle is not fully understood. Furthermore, our understanding of the processes that link spindle orientation during asymmetric cell division to cell fate specification in distinct tissue contexts remains incomplete. We report a role for the microtubule catastrophe factor KIF18B in regulating microtubule dynamics to promote spindle orientation in keratinocytes. During mitosis, KIF18B accumulates at the cell cortex, colocalizing with the conserved spindle orientation machinery. In vivo we find that KIF18B is required for oriented cell divisions within the hair placode, the first stage of hair follicle morphogenesis, but is not essential in the interfollicular epidermis. Disrupting spindle orientation in the placode, using mutations in either KIF18B or NuMA, results in aberrant cell fate marker expression of hair follicle progenitor cells. These data functionally link spindle orientation to cell fate decisions during hair follicle morphogenesis. Taken together, our data demonstrate a role for regulated microtubule dynamics in spindle orientation in epidermal cells. This work also highlights the importance of spindle orientation during asymmetric cell division to dictate cell fate specification.     

The protein kinase Kin4 inhibits exit from mitosis in response to spindle position defects

Accurate nuclear position is essential for each daughter cell to receive one DNA complement. In budding yeast, a surveillance mechanism known as the spindle position checkpoint ensures that exit from mitosis only occurs when the anaphase nucleus is positioned along the mother-bud axis. We identified the protein kinase Kin4 as a component of the spindle position checkpoint. KIN4 prevents exit from mitosis in cells with mispositioned nuclei by inhibiting the mitotic exit network (MEN), a GTPase signaling cascade that promotes exit from mitosis. Kin4 is active in cells with mispositioned nuclei and predominantly localizes to mother cells, where it is ideally situated to inhibit MEN signaling at spindle pole bodies (SPBs) when anaphase spindle elongation occurs within the mother cell.     

Retinoic acid signaling is necessary for the development of the organ of Corti.

The cellular mosaic of the mammalian organ of Corti represents one of the most highly ordered structures in any vertebrate system. A single row of inner hair cells and three or four rows of outer hair cells extend along the basal-to-apical axis of the cochlea. The factors that play a role in the development of specific cell types within the cochlea are largely unknown; however, the results of previous studies have strongly suggested that retinoic acid plays a role in the development of cells as hair cells. To determine whether cochlear progenitor cells can respond directly to retinoic acid, the expression patterns for each of the RAR and RXR receptors within the embryonic cochlear duct were determined by in situ hybridization. Results indicate that RARalpha, RXRalpha, and RXRgamma are initially expressed throughout the cochlear duct. As development continues, the expression of each receptor becomes more intense in cells that will develop as hair cells. At the same time, receptor expression is down-regulated in cells that will develop as nonsensory cell types. To determine the effects of retinoic acid signaling during the development of the organ of Corti, activation of retinoid receptors was blocked in cultures of the embryonic cochlea through receptor-specific antagonism or inhibition of retinoic acid synthesis. Results indicate that inhibition of retinoic acid signaling induces a significant decrease in the number of cells that develop as hair cells and a disruption in the development of the organ of Corti. These results demonstrate that cells within the developing cochlea can respond to retinoic acid and that signaling by retinoic acid is necessary for the normal development of the organ of Corti.     

Mechano-electrical transducer currents in hair cells of the cultured neonatal mouse cochlea.

The first step towards the generation of the receptor potential in hair cells is the gating of the transducer channels and subsequent flow of transducer current, induced by deflection of the stereocilia. We describe properties of the transducer current in outer hair cells of neonatal mice. Less extensive observations on inner hair cells suggest that their transducer currents have similar characteristics. The hair bundles were stimulated by force from a fluid jet. The transducer currents in outer hair cells are the largest found so far in any hair cell, with a chord conductance of up to 9.2 nS at -84 mV. The transfer function suggests that the channel has at least two closed states and one open state. The permeabilities for sodium, potassium and caesium are similar, consistent with the channel being a fairly non-selective cation channel. At negative potentials the currents adapt in most cells, although never as completely as in hair cells of lower vertebrates. If the unit conductance of the transducer channel is similar to that of the turtle's auditory hair cells (100 pS), then there are about 90 channels per hair bundle, or one channel between every pair of adjacent stereocilia in neighbouring rows.     

Spatial organization in the saccule and lagena of a teleost: hair cell pattern and innervation

The relationship between the hair cell orientation pattern and innervation in the saccule and lagena of the teleost Helostoma temmincki (the kissing gourami) was investigated with scanning electron microscopy and the Winkelmann-Schmitt silver impregnation technique. The hair cell pattern in the saccule consists of four orthogonally oriented groups. The anterior two groups are oriented along the animal's rostrocaudal axis, and the posterior two are oriented along its dorsoventral axis. The pattern of hair cell orientations in the lagena is a typical bidirectional one. Two divisions of the eighth nerve innervate the saccule. The anterior division innervates the horizontally oriented hair cell groups, and the posterior division innervates the dorsoventrally oriented groups. A single nerve innervates the lagena, with the majority of fibers innervating one or the other of the two lagenar hair cell groups. The segregated pattern of innervation according to hair cell orientation groups in the saccule was confirmed in other species. Individual types of axonal terminations appear to innervate hair cells of specific ciliary bundle types.     

Planar cell polarity in the inner ear: how do hair cells acquire their oriented structure?

Sensory hair cells in the ear and lateral line have an asymmetrical hair-bundle structure, essential for their function as directional mechanotransducers. We examine four questions: (1) how does the planar asymmetry of the individual hair cell originate? (2) How are the orientations of neighboring hair cells coordinated? (3) How is the orientation of a group of hair cells controlled in relation to the ear as a whole? (4) How does the initial cell asymmetry lead to creation of the asymmetrical hair bundle? Studies of the development of hairs and bristles in Drosophila, combined with genetic data from vertebrates, suggest that the answer to questions (1) and (2) lies in asymmetries that develop at the cell cortex and at cell-cell junctions, generated by products of a set of primary planar cell polarity genes, including the transmembrane receptor Frizzled. A separate and largely independent mechanism controls asymmmetric allocation of cell fate determinants such as Numb at mitosis, in Drosophila and possibly in the ear also. Little is known about long-range signals that might orient hair cells globally in the ear, but progress has been made in identifying a set of genes responsible for read-out of the primary polarity specification. These genes, in flies and vertebrates, provide a link to assembly of the polarized cytoskeleton; myosin VIIA appears to belong in this group. The mechanism creating the staircase pattern of stereocilium lengths is unknown, but could involve regulation of stereocilium growth by Ca(2+) ions entering via transduction channels.     

Topology of microtubules and actin in the life cycle of Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The morphology of budding and conjugating cells and associated changes in microtubules and actin distribution were studied in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) by phase-contrast and fluorescence microscopy. The non-budding interphase cell showed a nucleus situated in the central position and bundles of cytoplasmic microtubules either stretching parallel to the longitudinal cell axis or randomly distributed in the cell; none of these, however, had a character of astral microtubules. During mitosis, the nucleus divided in the daughter cell, cytoplasmic microtubules disappeared and were replaced by a spindle. The cytoplasmic microtubules reappeared after mitosis had finished. Actin patches were present both in the bud and the mother cell. Cells were induced to mate by transfer to ribitol- containing medium without nitrogen. Partner cells fused by conjugation projections where actin patches had been accumulated. Cell fusion resulted in a zygote that produced a basidium with parallel bundles of microtubules extended along its axis and with actin patches concentrated at the apex. The fused nucleus moved towards the tip of the basidium. During this movement, nuclear division was taking place; the nuclei were eventually distributed to basidiospores. Mitochondria appeared as vesicles of various sizes; their large amounts were found, often lying adjacent to microtubules, in the subcortical cytoplasm of both vegetative cells and zygotes.     

Synaptonemal Complex Components Promote Centromere Pairing in Pre-meiotic Germ Cells

Mitosis and meiosis are two distinct cell division programs. During mitosis, sister chromatids separate, whereas during the first meiotic division, homologous chromosomes pair and then segregate from each other. In most organisms, germ cells do both programs sequentially, as they first amplify through mitosis, before switching to meiosis to produce haploid gametes. Here, we show that autosomal chromosomes are unpaired at their centromeres in Drosophila germline stem cells, and become paired during the following four mitosis of the differentiating daughter cell. Surprisingly, we further demonstrate that components of the central region of the synaptonemal complex are already expressed in the mitotic region of the ovaries, localize close to centromeres, and promote de novo association of centromeres. Our results thus show that meiotic proteins and meiotic organization of centromeres, which are key features to ensure reductional segregation, are laid out in amplifying germ cells, before meiosis has started.