Retina regeneration in the chick embryo is not induced byspontaneous Mitf downregulation but requires FGF/FGFR/MEK/Erk dependentupregulation of Pax6

[摘要] Purpose: To elucidate the early cellular events that take placeduring induction of retina regeneration in the embryonic chick, focusingon the relationship between fibroblast growth factor (FGF) signaling andthe regulation of Pax6 and Mitf.Methods: The retina of embryonic day 4 (E4) chicks was removed and aheparin coated bead soaked in fibroblast growth factor 2 (FGF2) wasplaced into the optic cup. The pharmacological inhibitor PD173074 wasused to inhibit FGF receptors, PD98059 was used to inhibit MAPkinase-kinase/extracellular signal-regulated kinase (MEK/Erk) signaling.Retroviral constructs for paired box 6 (Pax6), MEK, and microphthalmia(Mitf) were also used in overexpression studies. Immunohistochemistrywas used to examine pErk, Pax6, Mitf, and melanosomal matrix protein 115(MMP115) immunoreactivity and bromodeoxyuridine (BrdU) incorporation atdifferent time points after removing the retina.Results: The embryonic chick has the ability to regenerate a newretina by the process of transdifferentiation of the retinal pigmentepithelium (RPE). We observed that during the induction oftransdifferentiation, downregulation of Mitf was not sufficient toinduce transdifferentiation at E4 and that FGF2 was required to drivePax6 protein expression and cell proliferation, both of which arenecessary for transdifferentiation. Furthermore, we show that FGF2 worksthrough the FGFR/MEK/Erk signaling cascade to increase Pax6 expressionand proliferation. Ectopic Mitf expression was able to inhibittransdifferentiation by acting downstream of FGFR/MEK/Erk signaling,likely by inhibiting the increase in Pax6 protein in the RPE.Conclusions: FGF2 stimulates Pax6 expression during induction oftransdifferentiation of the RPE through FGFR/MEK/Erk signaling cascade.This Pax6 expression is accompanied by an increase in BrdUincorporation. In addition, we show that Mitf is spontaneouslydownregulated after removal of the retina even in the absence of FGF2.This Mitf downregulation is not accompanied by Pax6 upregulation,demonstrating that FGF2 stimulated Pax6 upregulation is required fortransdifferentiation of the RPE. Furthermore, we show that ectopic Mitfexpression is able to protect the RPE from FGF2 inducedtransdifferentiation by inhibiting Pax6 upregulation.

[学科分类] 生物化学/生物物理