Math1, a transcription factor, is one of the critical genes for the formation of hair cells (HC) in the inner ear. The importance of Math1 was first shown in Math1 knockout mice which lack HCs (Bermingham et. al., 1999) leading to the hypothesis that Math1 was essential and sufficient for the formation of HCs. However, our recent study demonstrated that Math1- null HCs are able to be generated and to properly differentiate in the Math1-null chimeric mice, in which Math1-null cells are intermingled with wild-type cells (Du et. al., 2007). The presence of Math1-null cells within the chimeric inner ear provides evidence both that 1) wild-type cells can rescue Math1-null HCs in the chimeric environment, and 2) that given the correct external cues, cells that lack Math1 remain able to differentiate as hair cells (Du et. al., 2007). These unique cells can now be used to further explore the role of Math1 in HC development by more extensively characterizing their long-term potential as well as identifying molecular candidates that may mediate this rescue. A limitation of the previous study was that the system used to mark the genotypically mutant HCs, ss-galactosidase (ss-gal) from the mutant construct, was decreasing in expression and thus examination of mutant cells could be followed only up to postnatal days 4.5. Moreover, the tissue processing needed for ss-gal visualization compromised the integrity of the RNA. In the present proposal, these limitations will be circumvented by several chimeric combinations including the use of Math1-null mice that constitutively express green fluorescent protein and the use of other marker systems in the wild-type mice (including ROSA26 transgenic mice).
Specific Aim 1 will begin to examine the long-term potential of these cells by testing the hypothesis that these HCs survive a normal lifespan and appear morphologically normal at all ages. Thus, the inner ears of chimeric mice will be examined at a range of ages using immunocytochemistry for HC markers. While the mechanism of this rescue is currently unknown, we hypothesize that at the time of differentiation of HCs, wild-type cells are able to activate downstream or parallel molecular pathways, allowing rescue of genotypically mutant HCs.
Specific Aim 2 will compare the expression profiles of several molecules (e.g. Delta1, Bdnf) in chimeric Math1- null cells with those from the controls to assess whether these are molecular candidates for mediating this rescue. This data will provide information about the role of Math1 in the expression of these molecules as well as provide insight into the role of other molecules in the rescue of the genotypically Math1-null cells in the chimeric environment. One of the potential means to cure congenital deafness or deafness that occurs later in life is through the generation of new hair cells in the inner ear and Math1 is one of the candidate molecules that has shown promise in being able to generate new hair cells. This proposal will provide insight into how Math1 functions in the generation of hair cells.
One of the potential means to cure congenital deafness or deafness that occurs later in life is through the generation of new hair cells in the inner ear and Math1 is one of the candidate molecules that has shown promise in being able to generate new hair cells. This proposal will provide insight into how Math1 functions in the generation of hair cells.
