טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentNir Eshkar
SubjectMIR450 regulates Pax6 and Sox2 in eye morphogenesis
and corneal homeostasis
DepartmentDepartment of Medicine
Supervisor ? Ruby Shalom-Feuerste
Full Thesis textFull thesis text - English Version


Abstract

Embryonic development of the eye depends on timely action of transcription factors and inductive signals ensuring the correct formation of the different tissue components during morphogenesis. In adulthood, the cornea serves as a physical barrier preventing the entry of deleterious agents into the intraocular surface. It is widely accepted that the extraordinary dynamic replenishment of the corneal epithelium relays on stem cells located at the limbus. Although limbal stem cell-based therapy was successfully applied to cure blindness of thousands of patients worldwide, stem cell features and self-renewal mechanisms remain poorly understood.  Elucidating the molecular circuitry of corneal epithelial development and stem cell regulation is required for understanding the etiology of numerous corneal diseases and may pave the way for efficient regenerative medicine.

Paired Box Protein 6 (Pax6) and SRY-related high-mobility-group (HMG)-box protein-2 (Sox2) are key regulators of eye development while studies illustrate that mice lacking PAX6 or SOX2 genes display an early failure in lens placode formation. A precise dosage of Pax6 and Sox2 proteins is required for normal eye development and integrity while their miss-expression results in various eye abnormalities and blindness. The overall objective of this study was to test the molecular circuitry by which Pax6 and Sox2 are regulated.  In a previous study in our lab it was found that miR-450b-5p (referred hereafter miR-450b) can bind to the 3’-untranslated region (UTR) of PAX6, and functioning as a repressor of Pax6.

Here, we show that miR-450b, along with its cluster member miR-450a-5p (referred hereafter miR-450a), can bind to 3’UTR of SOX2 and repress the expression of Sox2 protein. In-situ hybridization and immunofluorescent staining revealed that miR-450b and Pax6/Sox2 are reciprocally expressed during lens development. Similarly, in sharp contrast to Pax6/Sox2, miR-450b was not expressed by limbal stem cells but highly expressed in differentiated layers of the cornea. In line, over-expression of miR-450a/b induced a down regulation of Pax6 and Sox2 during adult limbal stem cell differentiation suggesting that miR-450a/b directly represses Pax6 and Sox2 to regulate corneal maintenance. Interestingly, clonogenicity test on primary limbal epithelial cell culture reveled that miR-450b interferes with the ability of stem cells to produce large proliferative colonies that are known to have therapeutic features, indicating that miR-450b plays a role in limbal stem cell differentiation. Moreover, specific silencing RNA against Pax6 and Sox2 significantly attenuated the clonogenic potential of limbal epithelial stem cells. These data indicate that Pax6 and Sox2 are important regulators of limbal stem cell self-renewal and/or differentiation and that miR-450b represses stemness through inhibition of Pax6/Sox2. Interestingly, bone morphogenic protein 4 (BMP4), which is an essential morphogen for eye embryogenesis and corneal maintenance up-regulated miR-450b and concomitantly repressed Pax6 during corneal epithelial commitment of human embryonic stem cells (hESCs) and also in mouse embryos isolated ex vivo during early lens morphogenesis. Altogether, these data suggest a new mechanism of Pax6 regulation by BMP4-mediated microRNA that may have significance for corneal/lens development, homeostasis and diseases.