The Study Of Gene Atoh1/Math1Induced Vestibular Hair Cell Regeneration Based On Transgenic Mouse

OBJECTIVE Previous in vitro studies have demonstrated that over-expression of Atohl/Mathl can produce new hair cells in rat’s utricle; however, in vivo study is still needed to serve for possible clinical application. Based on one special transgenic mouse line which can over express Atohl/Mathl at certain time point after birth in the whole inner ear, the current study is designed to answer the following questions,1whether over-expression of Atohl/Mathl can promote the generation of new vestibular hair cell in vivo;2which kind of cell has the ability to turn into hair cell under the induction of Atohl/Mathl;3whether this ability gradually disappear after birth;4whether Atohl/Mathl has the ability to promote proliferation when it over expresses in vivo;5whether it is possible to induce new generated hair cell by regulating other cell signal pathways.METHOD One transgenic mouse model which can over express Atohl/Mathl at certain time point and in certain tissues after the induction of doxycycline was used in this study. In this mouse model, the kinds of tissues where the Cre recombinase will express were defined by the gene Foxgl, so the Cre recombinase was specially expressed in the epithelium of inner ear. In the epithelium of inner ear, the Cre recombinase mediated excision of an upstream stop cassette of the rtTA gene. When rtTA is bound to doxycycline and worked at the TRE, the downstream Atoh1/Math1would express. In this study, we used food and water containing doxycycline to feed the mouse at certain time point after birth, and then observed whether there were new hair cells in the utricle epithelium. We also performed Brdu injections on some mouse during the doxycycline induction, and try to analysis the proliferation status by using the Brdu antibody. At last, by using the utricle whole mount culture technique, we tested the effects of inhibiting Notch, FGF and BMP path during the culture of P2utricle RESULTS The semi-quantitative RT PCR showed the mRNA level of Atohl/Mathl in the positive samples which had the genotype of pTM1+, foxg1cre+and rtTA+was much higher than the control which had a genotype of pTM1-, foxg1cre+and rtTA+. At P21which was the last time point in this study, we used the Atohl/Mathl-GFP gene as a reporter to check the Atohl/Mathl expression pattern. In the positive utricles, the GFP could be seen in both hair cell layer and supporting cell layer. In the control, the GFP mainly distributed in the hair cell layer, there were quite few cells expressing Atohl/Mathl-GFP in the supporting cell layer. In the current study, we checked the effect of over-expression of Atohl/Mathl in P0, P7, P14and P21stages. At P0, induced by doxycycline for4days could generate new hair cells in the striolar and juxtastriolar region as well as the nonsensory region. With the days increased after birth, the number of new generated hair cells decreased gradually. At P7stage, no new hair cell could be seen in the nonsensory region already. At P21stage, the new generated hair cell at the striolar and juxtastriolar region also disappeared. In the mitosis analysis, we found there was active mitosis during the generation of new hair cell especially in the striolar region. And some cells could be marked by both Brdu and Myo7a, indicating these hair cells differentiated from the mitosis daughter cell. In the utricle culture experiment, we found inhibiting the Notch and FGF path could also induce new generated hair cell in the striolar region; however, antagonizing the BMP path did not have any effect.CONCLUSION Over-expression of Atohl/Mathl can produce new generated vestibular hair cells in vivo; the supporting cell in the striolar and juxtastrolar region as well as the cell in the nonsensory region have the ability to turn into hair cell; Such ability gradually disappear after birth; Atohl/Mathl can promote proliferation during the generation of new hair cell and some hair cells differentiate from the mitosis daughter cell; inhibiting the Notch and FGF path can promote the generation of new hair cell, however inhibiting the BMP path do not have any effects.