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FIGURE 5.16 Regeneration of hair cells in the chick basilar epithelium after antibiotic-induced injury. (A) A whole mount of untreated basilar epithelium immunostained for the hair cell marker calmodulin, showing the alternating pattern of hair cells (purple, arrow) and support cells (arrowhead). (B) Diagram showing arrangement of hair cells and supporting cells in transverse section. Hair cells (hc, purple) reside closer to the lumen (lu) and do not contact the basement membrane (bl), whereas support cells rest on the bl and span the epithelium. Arrow points to stereocilia. (C) Support cells divide near the lumen of the injured epithelium as shown by whole-mount BrdU staining. (D) Support cell nuclei migrate toward the lumen and the cell detaches from the basal lamina and undergoes mitosis into a pair of daughter cells, one of which differentiates into a hair cell, while the other remains a stem (support) cell. Reproduced with permission from Stone and Rubel, Delta 1 expression during avian hair cell regeneration. Development 126:961-973. Copyright 1999, The Company of Biologists.

sound and motion is transduced to electrical impulses by auditory sensory cells called hair cells, which reside in the sensory epithelium lining these structures (figure 5.16). Each hair cell is flask-shaped and is surrounded by support cells. The hair cells extend from the luminal surface part way toward the basement membrane of the epithelium, but do not contact it, whereas the support cells are columnar and span the whole distance from the basement membrane to the lumen. The mechanosensitive organelles of the hair cells are the stereocilia, or "hair bundles," which are supported by a dense core of actin filaments that are completely replaced via treadmilling over a 48-hr period (Schneider et al., 2002).

A major cause of hearing loss and balance problems is dysfunction of the inner ear caused by loss of hair cells due to noise trauma, toxic drug damage (for example, aminoglycoside antibiotics such as gentamy-

cin), infection, or age-related degeneration. Up to 120 million persons worldwide are estimated to suffer from hearing impairment (Lowenheim, 2000).

Adult birds are able to regenerate sensory cells in both the sensory epithelium of the cochlea and the utricle of the vestibular apparatus. In mature birds, the epithelial cells of the cochlea are mitotically quiescent. Sensory hair cells in the vestibular sensory epithelium, however, normally turn over by apoptosis and are replenished by maintenance regeneration (Kil et al., 1997; Stone and Rubel, 2000). Hair cells of both cochlea and utricle are able to regenerate after injury (Roberson and Rubel, 1995; Oesterle and Rubel, 1996; Kil et al., 1997; Stone and Rubel, 2000), with virtually complete recovery of hearing.

The stem cells responsible for both maintenance and injury-induced hair cell regeneration in birds are most likely the support cells, based on the size, shape, and location of the nuclei of cells that divide and differentiate into hair cells in the epithelium (figure 5.16). Support cell nuclei are small, are oval-shaped and lie near the basement membrane of the epithelium, whereas hair cell nuclei are large, are round, and lie closer to the lumen (Fekete, 1996; Fekete and Wu, 2002). The nuclei of the hair cell progenitors migrate from the basal lamina toward the lumen as they progress from S phase to M (Cotanche et al., 1994; Tsue et al., 1994; Kil et al., 1997), similar to the migration of dividing cells observed in the differentiation of the neural tube (Sauer, 1935), suggesting that they are derived from support cells. At the lumen, one member of the pair of cells generated by mitosis differentiates into a hair cell, the other into a support cell (Stone and Cotanche, 1994). A similar asymmetric division has also been reported in the neuromasts of the lateral line system in the salamander, which detects motion of the water (Jones and Corwin, 1996).

A number of markers are expressed by all support cells within the epithelium of the cochlea, but only the homeodomain transcription factor, c(hicken)Prox1 is differentially regulated in a way that suggests it might have a key role in determining hair cell fate and differentiation (Stone et al., 2004). Prox1 is required for cell cycle withdrawal in a number of vertebrate and invertebrate embryonic tissues. In the damaged auditory epithelium of the cochlea, some quiescent support cells do not express cProx1, but others exhibit a low level of expression. CProx1 is absent in half the activated support cells three days after gentamycin treatment, but present in the other half. Sibling pairs of cells following division exhibit three patterns of cProx1 expression, in approximately equivalent numbers: symmetrical low, symmetrical high, and asymmetric (figure 5.17). In the undamaged vestibular epithelium of the utricle, none of the quiescent support cells

FIGURE 5.17 Expression of cProxl (green) in sibling pairs of BrdU-labeled (red) cells derived by the division of support cells in the damaged basilar epithelium. Whole-mount immunostaining. (A) Arrowheads = asymmetric expression of cProxl. Short arrows = asymmetric low levels of cProxl expression. Long arrow = symmetric high levels of cProxl expression. (B-D) Higher magnification of symmetrical high (+/+), symmetrical low (-/-) and asymmetric (+/-) expression of cProxl at 10, 8 and 17 hr post-BrdU, respectively. Reproduced with permission from Stone et al., cProxl immunore-activity distinguishes progenitor cells and predicts hair cell fate during avian hair cell regeneration. Dev Dynam 230:597-6l4. Copyright 2004, Wiley-Liss.

FIGURE 5.17 Expression of cProxl (green) in sibling pairs of BrdU-labeled (red) cells derived by the division of support cells in the damaged basilar epithelium. Whole-mount immunostaining. (A) Arrowheads = asymmetric expression of cProxl. Short arrows = asymmetric low levels of cProxl expression. Long arrow = symmetric high levels of cProxl expression. (B-D) Higher magnification of symmetrical high (+/+), symmetrical low (-/-) and asymmetric (+/-) expression of cProxl at 10, 8 and 17 hr post-BrdU, respectively. Reproduced with permission from Stone et al., cProxl immunore-activity distinguishes progenitor cells and predicts hair cell fate during avian hair cell regeneration. Dev Dynam 230:597-6l4. Copyright 2004, Wiley-Liss.

expresses cProx. During maintenance regeneration, sibling pairs of cells are at first negative for cProxl, but then the majority of them exhibit asymmetric expression. The cell of the pair that expresses cProxl differentiates into a hair cell in both the cichlea and the utricle. figure 5.18 illustrates all the patterns of cProxl expression and their relationships to patterns of differentiation.

The pattern of one hair cell surrounded by several support cells is believed to be generated by lateral inhibition from one member of a mitotic pair to the other (Cotanche, 1987). The Delta/Notch signaling system has been implicated in the lateral inhibition of sensory cell differentiation of the zebrafish inner ear (Haddon et al., 1998a, b). Delta and Notch are at first equally expressed in both members of support cell mitotic pairs in the gentamycin-damaged chick vestibular epithelium; Delta is then downregulated in the cell that will become a support cell and upregulated in the hair cell (Stone and Rubel, 1999). The factors regulating this asymmetric regulation of Delta expression are unknown. In the chick cochlea, the FGF/FGFR3 signaling pathway has been implicated in maintaining the stemness of support cells in the normal basal epithelium (Bermingham-McDonogh et al., 2001). FGFR3 is highly expressed in the support cells of the epithelium.

FIGURE 5.18 Patterns of cProxl expression in sibling pairs of support cell progeny and hair/support cell differentiation during hair cell regeneration. Gray is high expression, white is cProxl-negative. (A) Production of two hair cells (HC/HC). (B, C) Production of a support cell and a hair cell (SC/HC). (D) Production of two support cells. A, B, D are all observed in the damaged basilar epithelium. C is the rule in the undamaged utricle. Reproduced with permission from Stone et al., cProxl immunoreactivity distinguishes progenitor cells and predicts hair cell fate during avian hair cell regeneration. Dev Dynam 230:597-614. Copyright 2004, Wiley-Liss.

FIGURE 5.18 Patterns of cProxl expression in sibling pairs of support cell progeny and hair/support cell differentiation during hair cell regeneration. Gray is high expression, white is cProxl-negative. (A) Production of two hair cells (HC/HC). (B, C) Production of a support cell and a hair cell (SC/HC). (D) Production of two support cells. A, B, D are all observed in the damaged basilar epithelium. C is the rule in the undamaged utricle. Reproduced with permission from Stone et al., cProxl immunoreactivity distinguishes progenitor cells and predicts hair cell fate during avian hair cell regeneration. Dev Dynam 230:597-614. Copyright 2004, Wiley-Liss.

The hypothesis is that adjacent hair cells secrete an FGF ligand that binds to a Trk on the support cells and prevents their differentiation. When hair cells are damaged, the ligand is no longer available, FGFR3 is downregulated in the support cells, and they enter the cell cycle. Once new support cells differentiate and production of ligand resumes, their expression of FGFR3 is upregulated and their proliferation is inhibited.

The hair cells of mammals normally do not regenerate and permanent hearing and balance deficits are the usual result of injury to the sensory epithelium. Nevertheless, there is in vivo and in vitro evidence that hair cells can regenerate to some extent in the vestibular apparatus of guinea pigs and humans. Scanning and transmission electron microscopy of gentamycin-treated utricular sensory epithelium in guinea pigs showed complete hair cell loss within a week after treatment. By four weeks, however, cells with organized bundles of stereocilia had reappeared at the luminal surface (Forge et al., 1993). Proliferating support cells were observed throughout guinea pig and human utricular sensory epithelium cultured in vitro with gentamycin and labeled with BrdU or 3H-thymidine. Some of the labeled cells developed into immature hair cells with small apical tufts of stereocilia (Warchol et al., 1993).

The developing mouse organ of Corti synthesizes retinoic acid (RA). Exogenous RA exerts a concentration-dependent stimulation of hair cell development in cultured mouse organs of Corti (Kelley et al., 1993). Lefebvre et al. (1993) have reported that RA and TGF-a stimulate the regeneration of hair cells in neomycin-treated rat organ of Corti sensory epithelium.

In mice, mitosis is normally completed in the sensory epithelial cells of the organ of Corti of mice by embryonic day 14. The negative regulator of cell cycle progression, the p27Kip1 protein, mediates their exit from the cell cycle, and they differentiate into hair cells and support cells (Parker et al., 1995; Nakayama et al., 1998). Disruption of the p27Kip1 gene allows cell division to continue in the sensory epithelium of the organ of Corti in postnatal and adult mice. However, p27Kip1 expression is downregulated in differentiating hair cells and is unlikely to maintain mitotic arrest in these cells (Lowenheim et al., 1999; Chen and Segil, 1999). Sage et al. (2005) identified the Rb gene in a microarray analysis as the regulator of mitotic arrest in hair cells (see also Taylor and Forge, 2005).

The effect of knocking out specific genes can be tested by transgenic Cre/lox techniques (Branda and Dymecki, 2004). Transgenic animals are created in which genes or portions of genes are flanked by loxP sequences that are the recognition sites for the bacte-riophage Cre recombinase. Such genes are said to be "floxed." The animals are also made transgenic for Cre under the control of a cell-specific promoter, or Cre or a Cre-promoter construct can be introduced into tissues or cells in vitro by a viral vector. Cre will be activated in those cells with transcription factors for the cell-specific promoter. Cre-recombinase binds to the loxP sites and excises the gene flanked by these sites.

Sage et al. (2005) tested the effect of knocking out Rb in utricles by culturing utricles from transgenic mice carrying Rb flanked with loxP sites (FIGURE 5.19). The utricles were transfected with adenovirus constitutively making Cre recombinase, which removed the Rb gene. The result was that hair cells re-entered the cell cycle and divided while still differentiated. That is, they produced more hair cells by compensatory hyperplasia. This means that it might be possible to treat deafness by transient inactivation of Rb; more broadly, it suggests that it might be possible to induce compensatory hyperplasia in other differentiated cell types as a mechanism of restoring normal numbers of such cells instead of relying only on resident stem cells or stem cells created by dedifferentiation.

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