cProx1 and Supporting Cells
The differential immunoreactivity of cProx1 detected across mitotically quiescent supporting cells in the basilar papilla is intriguing because there are few morphological or molecular features that define subsets of supporting cells in the avian auditory epithelium. Auditory supporting cells have many functions: they help to maintain the ionic integrity of the inner ear, they provide mechanical support within the sensory epithelium, and they generate the tectorial membrane in developing and mature ears (Cotanche, 1987; Shiel and Cotanche, 1990; Epstein and Cotanche, 1995). Furthermore, supporting cells serve as progenitors to new hair cells in mature birds (reviewed in Stone and Rubel, 2000a). It is unclear, however, if all supporting cells, or only a subset, serve these different functions. In fact, the term “supporting cell” is used widely to describe any cell in avian sensory epithelia that lacks hair cell features. Several markers have been reported to label all supporting cells within the basilar papilla (for review see Stone and Rubel, 2000a). By contrast, very few proteins are expressed in subsets of supporting cells (e.g., proliferating cell nuclear antigen [Bhave et al., 1995], fibroblast growth factor receptor 3 [Bermingham-McDonogh et al., 2001]). The distribution of these proteins does not appear to correlate with the spatial pattern of cProx1-positive supporting cells that we have observed.
In response to gentamicin treatment, hair cells in the proximal basilar papilla die and supporting cells in that region undergo mitosis. The greatest number of supporting cells enters the cell cycle at 3 days after gentamicin treatment (Bhave et al., 1995; Stone et al., 1999). At this time, there is a large increase in nuclear cProx1 immunoreactivity in the damaged area relative to the corresponding proximal region in untreated birds and relative to the apical, undamaged area in treated birds. Strong cProx1 labeling is seen in only half of the dividing cells at this time; the other half is unlabeled or only weakly labeled. As discussed in the previous section, levels of nuclear cProx1 immunoreactivity are also variable in resting supporting cells. It is not clear if these relative levels are preserved in supporting cells as they transition from a resting state to a mitotic state. In contrast to the drug-damaged basilar papilla, all S-phase supporting cells in the undamaged or damaged utricle show little or no cProx1 immunoreactivity in their nuclei. Therefore, levels of nuclear cProx1 immunolabel are highly variable in dividing supporting cells across and within sensory epithelia of the mature avian inner ear.
The roles of cProx1 in mitotically quiescent and dividing supporting cells during postembryonic hair cell production are not clear. Studies in other tissues and species demonstrate cProx1's homologues promote terminal mitosis. In the fruit fly CNS, neuroblasts divide to give rise to a ganglion mother cell (GMC), which is a progenitor for neurons and glia, and another neuroblast. After this division, Pros protein is elevated in the nucleus of the GMC, which becomes terminally mitotic, but is absent from the nucleus of the neuroblast, which continues to divide. In the GMC, Pros suppresses transcription of positive regulators of the cell cycle and thereby diminishes the cell's capacity for further mitotic activity (Li and Vaessin, 2000; Myster and Duronio, 2000). Examination of Prox1 function in the murine lens suggests that it regulates mitotic activity there as well. Lens progenitors in Prox1 knockout mice show delayed expression of negative regulators of the cell cycle, such as p27kip1, and undergo terminal mitosis at later-than-normal times (Wigle et al., 1999).
This knowledge leads to the hypothesis that nuclear cProx1 immunoreactivity in the mature auditory and vestibular epithelia delineates progenitor cells with different capacities for cell proliferation. Supporting cells with a low level of cProx1 may act like stem cells, having a relatively unlimited capacity for cell division, while supporting cells with a high level of cProx1, like GMCs in fruit flies, may be on the verge of undergoing terminal mitosis. Accordingly, proliferative supporting cells in the normal utricle, which express very low levels of or no cProx1 in S phase, would have the capacity to divide continuously. This interpretation is compatible with the finding that hair cells are regenerated on a continual basis in vestibular epithelia and that each supporting cell division leads to its self-renewal (Jørgensen and Mathiesen, 1988; Roberson et al., 1992; Kil et al., 1997; Stone and Rubel, 1999; Stone et al., 1999).
It is notable that cProx1 is also expressed in sensory and neural progenitors and precursors during development of the avian inner ear (Stone et al., 2003). Nuclear cProx1 immunoreactivity is elevated in the sensory primordial patches around the time when these regions are first detectable in the otocyst. Levels remain elevated in all progenitor cells during periods of intense proliferation. Some time after hair cells and supporting cells have differentiated many of their mature features, nuclear cProx1 immunoreactivity is largely down-regulated. Thus, hair cell regeneration clearly involves re-expression of a developmentally relevant gene. However, the temporal pattern of cProx1 expression in the sensory primordial patches does not support a function for the protein in directing terminal mitosis during development.
cProx1 and Regenerated Cells
As described above, dividing progenitor cells in the damaged basilar papilla and in the control utricle display differing levels of nuclear cProx1 immunoreactivity, and cProx1 protein appears to be inherited symmetrically. Consistent with these findings, pairs of sibling cells show a range of levels of cProx1 immunolabeling shortly after mitosis. In the regenerating basilar papilla, where dividing cells are either strongly cProx1-positive or cProx1-negative, very young sibling pairs (at 8 hr post-BrdU) display either symmetrically high or symmetrically low levels of nuclear cProx1 immunoreactivity. By contrast, in the regenerating utricle, where all dividing cells are cProx1-negative, all young sibling pairs at 8 hr post-BrdU are symmetrically cProx1-negative. Sibling pairs with asymmetric levels of cProx1 protein emerge in both tissues by 17 hr post-BrdU.
By 3 days post-BrdU in the basilar papilla, there is near-equal likelihood that differentiating siblings display symmetrically high, symmetrically low, or asymmetric levels of cProx1 immunoreactivity. This distribution is reflective of the near-equal probability in this tissue that any given supporting cell division will lead to the production of two hair cells, two supporting cells, or one of each cell type (Stone and Rubel, 2000). By contrast, in the undamaged utricle, most (76%) sibling pairs show asymmetric levels of cProx1 immunoreactivity at 3 days post-BrdU, reflecting that the majority (∼70%) of supporting cell divisions lead to the formation of a hair cell and a supporting cell in this tissue (Stone and Rubel, 1999; Stone et al., 1999). Thus, in auditory and vestibular epithelia, the distribution of cProx1 labeling across sibling pairs at 3 days post-BrdU mirrors the manner in which they differentiate (i.e., symmetrically vs. asymmetrically), but earlier patterns do not. The relative level of nuclear cProx1 in a postmitotic cell correlates with its fate; cells with a high degree of nuclear cProx1 immunoreactivity differentiate as hair cells, while cells with low or undetectable levels become supporting cells. Based on our data, we suspect that cProx1 protein moves from the nucleus to the cytoplasm in most regenerated hair cells between 8 and 14 days postgentamicin, which would correspond to between 5 and 11 days after they are generated.
In fruit flies, Pros is also expressed asymmetrically in sibling cells that acquire distinct fates (Hirata et al., 1995; Knoblich et al., 1995; Spana and Doe, 1995). This asymmetry is necessary to produce distinct cell types and/or to direct their differentiation (Chu-Lagraff et al., 1991; Doe et al., 1991; Vaessin et al., 1991; Matsuzaki et al., 1992; Manning and Doe, 1999; Reddy and Rodrigues, 1999). The mechanisms used to establish asymmetric Pros expression between sibling cells vary developmentally. For example, external sensory (ES) organs are composed of four different cells whose common progenitor is the sensory organ progenitor cell (SOP). ES cells are generated in adult flies as well as during embryogenesis. In both cases, the SOP divides and forms two daughter cells, the SOPIIa and the SOPIIb. The IIa cell divides and generates a hair cell and a socket cell, and the IIb cell divides to form a neuron and a sheath cell (but see Gho et al., 1999). In embryonic flies, each SOPII cell inherits different levels of Pros protein and/or mRNA during mitosis from the SOP cell (Knoblich et al., 1995; Spana and Doe, 1995). By contrast, in adult flies, Pros protein is not detected in the adult SOP. Instead, asymmetric levels of the protein are established in the two SOPII cells shortly after mitosis (Manning and Doe, 1999; Reddy and Rodrigues, 1999; but see Gho et al., 1999). Our results here demonstrate a similarity in Pros/Prox expression patterns between cell formation in the mature fly external sense organ and in the mature chicken hearing and balance organs. Previous studies have acknowledged the similarities in structure, function, and developmental regulation of sense-organ development across fruit flies and vertebrates (Adam et al., 1998; Eddison et al., 2000; Fritzsch et al., 2000; Hassan and Bellen, 2000). While postmitotically established asymmetry in Pros activity is critical for proper cell fate acquisition in the external sense organs of mature flies (Manning and Doe, 1999; Reddy and Rodrigues, 1999), it remains to be tested if this is also the case for cProx1 in the inner ear of mature chickens or other vertebrates.
What might be the role of cProx1 during hair cell differentiation? Because Prox1 knockout mice die before hair cells are born (Wigle et al., 1999), it has not been possible to characterize the gene's role in sensory development in the inner ear. Our finding that cProx1 immunoreactivity is elevated in hair cells but absent from supporting cells suggests the transcription factor promotes acquisition of the hair cell fate and/or directs the differentiation of committed hair cells. This proposed function is supported by studies that show Prospero and Prox1 control cellular differentiation in other species (see Introduction section). Variation in cProx1 expression among mitotic supporting cells may endow daughter cells with varying differentiative potentials (Fig. 9). Because cProx1 protein is not segregated asymmetrically in mitotic supporting cells, both sibling cells generated by a supporting cell with a relatively high level of cProx1 will inherit substantial protein. By contrast, mitotic supporting cells with low levels of cProx1 will generate two sibling cells with even lower cProx1 levels. Assuming cProx1 functions to drive specification or differentiation of hair cells, different levels of cProx1 in mitotic supporting cells would endow them with an initial bias toward generating two cell types of similar fate, with the fate dependent upon the relative level. The very low expression of cProx1 in utricular progenitors would favor the formation of sibling pairs in which both cells differentiate as supporting cells. This mechanism could confer some assurance that hair cells are not easily overproduced in the normal utricle, which has a nearly full contingent of hair cells at any given time. By contrast, in the drug-damaged basilar papilla in which there is complete hair cell loss in the area of regeneration and progenitors are either cProx1-positive or -negative, this mechanism would confer an equal likelihood of forming daughter cell pairs composed of two hair cells or two supporting cells.
Figure 9. Summary of cProx1 immunoreactivity during hair cell regeneration. This model depicts four scenarios that interpret our observations on cProx1 immunoreactivity during avian hair cell regeneration. A,B: The model contends that mitotically quiescent supporting cells (SCs, or progenitor cells) with weakly cProx1-positive nuclei (light gray) become highly cProx1-positive (dark gray) while dividing. A: Some of these cells generate two siblings, both of which have cProx1-positive nuclei and differentiate as hair cells (HCs, stippled). B: Additional cProx1-positive SCs form siblings that differentiate asymmetrically, as an HC and an SC. In these pairs, nuclear cProx1 immunoreactivity is down-regulated in the cell that acquires the SC fate. C,D: Quiescent SCs that are cProx1-negative remain so while dividing. D: Some of these cells generate two siblings, both of which have cProx1-negative nuclei and differentiate into SCs. C: Additional cProx1-negative SCs form siblings that differentiate asymmetrically. In these pairs, immunoreactivity for nuclear cProx1 is up-regulated in the cell that becomes an HC. Dividing SCs that are cProx1-positive are more likely to generate HCs than are cProx1-negative SCs. A–D: In the drug-damaged basilar papilla, all mechanisms of cell production shown here may be used. C: In the undamaged utricle; however, the mechanism used in the majority of cell divisions is shown.
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If cell fate outcomes during avian hair cell regeneration were to depend solely upon cProx1 levels in progenitor cells, there would be no evidence of asymmetric differentiation between sibling cells. In fact, 33% of sibling pairs generated in the basilar papilla at 3 days postgentamicin differentiate as a hair cell and a supporting cell (Stone and Rubel, 2000b), and approximately 70% of sibling pairs generated in the undamaged utricle do so as well (Stone and Rubel, 1999; Stone et al., 1999). In our model, therefore, asymmetric differentiation must result from additional signals that modify gene expression in postmitotic sibling cells, despite the early bias conferred upon them by maternal cProx1 expression. These signals may be derived from the cellular environment, which as discussed above is quite different between the drug-damaged basilar papilla and the undamaged utricle.
In fruit flies, two extracellular signaling pathways are believed to influence Pros transcription: one by means of the tyrosine kinase receptors sev and DER, and one by means of the Notch receptor (Manning and Doe, 1999; Reddy and Rodrigues, 1999; Xu et al., 2000). In mice, signaling through FGF receptors appears to be required for Prox1 expression in the CNS (Govindarajan and Overbeek, 2001). Multiple studies have implicated tyrosine kinase receptors or Notch receptors in the regulation of hair cell production in vertebrates, either during embryogenesis or regeneration (reviewed in Oesterle and Hume, 1999; Eddison et al., 2000; Stone and Rubel, 2000a; Fekete and Wu, 2002; Bermingham-McDonogh and Rubel, 2003), but it is not known if activation of these receptors modulates Prox1 transcription in vertebrates.
It is also possible that sensory epithelial progenitors throughout the mature avian inner ear are inherently different from one another, in that some are biased toward generating unlike cell types. Some supporting cells, such as those in the utricle and some in the basilar papilla, may contain cell fate determinants that are asymmetrically distributed in their cytoplasm before mitosis and passed on differentially to daughter cells, thereby ensuring that cells differentiate asymmetrically (reviewed in Jan and Jan, 1998). One such protein, Numb, has been shown recently to be asymmetrically distributed in progenitor cells in the developing otocyst (Eddison et al., 2000) and regenerating basilar papilla (J.S. Stone, unpublished observations), but its function in hair cell specification remains to be proven.
It is important to acknowledge that, in this study, we have only examined immunoreactivity for cProx1 protein and not protein levels. Therefore, it is not clear at this point whether changes in immunoreactivity reflect different levels of the protein or different degrees of its antigenicity. Changes in cProx1 immunoreactivity associated with cellular proliferation or differentiation have been described in the developing vertebrate lens (Duncan et al., 2002), hippocampus (Pleasure et al., 2000), and retina (Dyer et al., 2003), but how these changes are controlled has not been explored. Changes in protein levels can be achieved by altering protein transcription and translation, degradation, or subcellular localization, all of which have been postulated to regulate Pros protein levels in fruit flies (Hirata et al., 1995; Ikeshima-Kataoka et al., 1997; Li et al., 1997; Shen et al., 1997; Schuldt et al., 1998; Srinivasan et al., 1998; Manning and Doe, 1999; Reddy and Rodrigues, 1999; Ceron et al., 2001; Demidenko et al., 2001). Changes in antigenicity can reflect posttranslational changes in the protein structure (e.g., phosphorylation) or masking of the antigen site by binding of other molecules to the transcription factor. In fact, there are several phosphoisoforms of Pros (Srinivasan et al., 1998), and domains of Pros and Prox1 have been shown to bind to other proteins, such as Miranda and Pax6, respectively (e.g., Shen et al., 1998, Mikkola et al., 2001). The cellular mechanisms leading to the postmitotic changes in nuclear cProx1 immunoreactivity in the chicken's inner ear sensory epithelia require further exploration.