Retina regeneration in the chick embryo is not induced by spontaneous Mitf downregulation but requires FGF/FGFR/MEK/Erk dependent upregulation of Pax6.
OBJECTIVE
To elucidate the early cellular events that take place during induction of retina regeneration in the embryonic chick, focusing on the relationship between fibroblast growth factor (FGF) signaling and the regulation of Pax6 and Mitf.


METHODS
The retina of embryonic day 4 (E4) chicks was removed and a heparin coated bead soaked in fibroblast growth factor 2 (FGF2) was placed into the optic cup. The pharmacological inhibitor PD173074 was used to inhibit FGF receptors, PD98059 was used to inhibit MAP kinase-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. Immunohistochemistry was used to examine pErk, Pax6, Mitf, and melanosomal matrix protein 115 (MMP115) immunoreactivity and bromodeoxyuridine (BrdU) incorporation at different time points after removing the retina.


RESULTS
The embryonic chick has the ability to regenerate a new retina by the process of transdifferentiation of the retinal pigment epithelium (RPE). We observed that during the induction of transdifferentiation, downregulation of Mitf was not sufficient to induce transdifferentiation at E4 and that FGF2 was required to drive Pax6 protein expression and cell proliferation, both of which are necessary for transdifferentiation. Furthermore, we show that FGF2 works through the FGFR/MEK/Erk signaling cascade to increase Pax6 expression and proliferation. Ectopic Mitf expression was able to inhibit transdifferentiation 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 of transdifferentiation of the RPE through FGFR/MEK/Erk signaling cascade. This Pax6 expression is accompanied by an increase in BrdU incorporation. In addition, we show that Mitf is spontaneously downregulated 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 for transdifferentiation of the RPE. Furthermore, we show that ectopic Mitf expression is able to protect the RPE from FGF2 induced transdifferentiation by inhibiting Pax6 upregulation.
Spence
Jason R
JR
Department of Zoology, Miami University, Oxford, Ohio 45056, USA. Jason.Spence@cchmc.org <Jason.Spence@cchmc.org>
Madhavan
Mayur
M
Aycinena
Juan-Carlos
JC
Del Rio-Tsonis
Katia
K
D000818:Animals; D030161:Avian Proteins / Q000378:metabolism; D049109:Cell Proliferation; D002642:Chick Embryo / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D015536:Down-Regulation; D047108:Embryonic Development; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D051739:Microphthalmia-Associated Transcription Factor / Q000378:metabolism* / Q000502:physiology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000378:metabolism*; D010857:Pigment Epithelium of Eye / Q000196:embryology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D012038:Regeneration / Q000502:physiology*; D012097:Repressor Proteins / Q000378:metabolism*; D012160:Retina / Q000196:embryology*; D012190:Retroviridae / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D015854:Up-Regulation
D030161:Avian Proteins; D005136:Eye Proteins; D018398:Homeodomain Proteins; D036341:Intercellular Signaling Peptides and Proteins; D051739:Microphthalmia-Associated Transcription Factor; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D017468:Receptors, Fibroblast Growth Factor; D012097:Repressor Proteins; D016222:Fibroblast Growth Factor 2; C071193:MMP115 protein, Gallus gallus; D017346:Protein Serine-Threonine Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases