ANALISIS DE LA DEFENSA DE LA NATURALEZA APLICANDO LA

In summary, overexpression of Numb alone does not produce the expected phenotype of an excess of hair cells at the expense of supporting cells. This may be because Numb is unable to completely block Notch activity: for example, the Numb that was expressed lacks the sequence shown in mouse to localise Numb to the membrane and thus enable it to act on Notch (Dho et al., 1999). Numb may not be able to act alone: it may be necessary but not sufficient to cause a cell to differentiate into a hair cell, because other factors are required in conjunction with it for hair cell differentiation. There is also the possibility that it is not required at all, as suggested by the absence of Numb in cells of the basilar papilla undergoing terminal mitosis. Loss-of-function experiments would need to be conducted to completely rule out a central role of Numb in hair cell production. This could be achieved by mis-expressing a dominant negative c-Numb that only contained the PTB domain (Verdi et al., 1996).

On the other hand, there is evidence to suggest that, in some cases at least. Numb overexpression does alter cell fates in the otic epithelium. This, and the close similarity between results of Numb overexpression and Delta 1^" overexpression, suggests that appearances might not be quite as they seem. More careful analysis of infected regions within sensory patches, using a new series of specimens stained in different ways, might reveal that there are, after all, effects on the developmental choice between a hair-cell fate and supporting-cell fate.

Chapter 6

Discussion

6.1 Notch signaling is central to hair cell production, but the manner in which it operates is complex

Although the evidence from a wide variety of vertebrates points to a central role for Notch signaling in the differentiation of hair cells, the way in which it operates remains elusive. As demonstrated in this study, it is clearly not as the original lateral inhibition model supposed. The complexity corresponds to the multiplicity of Notch ligands expressed in the ear.

At least three Notch ligands are expressed during the development of the chick inner ear: Serratel is initially expressed in all cells of the developing sensory patch, and persists in supporting cells after hair cell differentiation. Deltal is expressed in scattered cells in two phases, firstly in the delaminating neuroblasts and later in the presumptive hair cells, and it is downregulated soon after hair cell differentiation. Only Serrate2

appears to be expressed in differentiated hair cells and persists in them until at least E12. Notchi is expressed throughout the otic epithelium. A similar pattern of expression of Notch and its ligands is seen in other vertebrates, such as mice, rats and fish

(Morrison et al., 1999; Lanford et al., 1999; Zine et al., 2000; Haddon et al., 1998a). The expression pattern of Serratel suggests a mechanism of lateral induction, whereby Notch activation up-regulates Serratel expression. Indeed, inhibition of Notch signaling, by expression of a dominant negative form of D e lta l, down-regulates the

expression of S erratel, supporting the hypothesis that Serratel expression is normally stimulated by Notch activation.

It is possible that different Notch receptors respond selectively to the different Notch ligands, providing a simple way in which the different Notch ligands could exert different effects. However, all three Notch ligands are likely to activate Notchi - the only known receptor to be expressed in chick otic epithelium (Shimizu et al., 2000). This suggests that all sensory patch cells initially contain activated Notch during their development, a hypothesis supported by the fact that several days before hair cell differentiation, all cells persistently express Serratel, which requires Notch activation. This early Notch activation may define the domain of the sensory patch. In such circumstances, it is difficult to see how a cell expressing Deltal could influence a neighbour by activating Notch, since Notch is already active in the neighbour because of the continual presence of Serratel on other adjacent cells. Indeed, Deltal expression in the nascent hair cell does not inhibit adjacent cells from differentiating as hair cells: artificial overexpression of Deltal in a patch of sensory cells does not prevent or even reduce the production of hair cells. Thus the simple model of lateral inhibition with feedback, mediated by Delta-Notch signaling, does not explain how hair cell production is controlled.

Serratel seems to have a more clearly definable role in sensory patch

development. In rats Jaggedi (Serratel) antisense oligonucleotides produce an excess of hair cells (Zine et al., 2000). It is tempting to propose on this basis, in the light of the gene expression data, that all the cells of a sensory patch are Notch active before the time of hair cell differentiation and that this inhibits commitment to a hair-cell fate, preventing premature differentiation. A cell must escape the state of Notch activation in order to differentiate as a hair cell. The question then is - how does a nascent hair cell escape Notch activation?

An answer is suggested by the observation that the Notch antagonist Numb is expressed in mature hair cells. However, overexpression of Numb in a sensory patch does not appear to affect the number of hair cells produced. As argued in previous chapters, it may be that only a total absence of the Notch signal will allow a cell to adopt the hair cell fate, and Numb alone may not be sufficient to completely block Notch activation. A similar hypothesis might also explain the absence of a hair cell phenotype with RCAS-Deital"".

I have presented evidence, on the other hand, that the RCAS-Deltat'’" and RCAS-Numb constructs used in this study do, in some cases, have effects on hair-cell commitment. I have also argued that the phenotype in other cases may have been subtle and may have escaped detection by the method of analysis used. Indeed, in the

Jagged2 and Hes1 knockout mice (Lanford et al., 1999; Zheng et al., 2000) the

phenotype is relatively mild, and could quite possibly be missed by taking transverse sections through the cochlea, instead of an en face-view. Interestingly, the hair cell phenotype of both these mutants was restricted to inner hair cells of the auditory epithelium, perhaps reflecting a further refinement and specialisation of the mechanism according to the type of hair cell being produced.