PAX6 regulates human corneal epithelium cell identity.
PAX6, a paired box transcription factor, is necessary for eye development. However, how it regulates the cell identity of human corneal epithelial cells (CECs) is not well understood. We aimed to clarify the function of PAX6 in human CECs using gene knockout via the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein 9 (Cas9) system. We designed guide RNAs for different targets in PAX6. PAX6-depleted CECs maintained the epithelial morphology, but became larger. Global analyses using microarray revealed that down-regulated genes were primarily CEC-specific and included keratin 12, keratin 3, clusterin (CLU), aldehyde dehydrogenase 3 family member A1 (ALDH3A1), angiopoietin-like 7 (ANGPTL7) and transketolase (TKT), while up-regulated genes were primarily epidermis-related and included keratin 10, keratin 1, involucrin (IVL), filaggrin (FLG). These findings suggest that PAX6 maintains CEC identity by regulating differentiation.
Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan; Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Hirokoji-agaru, Kawaramachi-dori, Kamigyo-ku, Kyoto 602-0841, Japan; Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Koji
K
Kitazawa
Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
Takafusa
T
Hikichi
Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Hirokoji-agaru, Kawaramachi-dori, Kamigyo-ku, Kyoto 602-0841, Japan.
Takahiro
T
Nakamura
Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Chie
C
Sotozono
Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Hirokoji-agaru, Kawaramachi-dori, Kamigyo-ku, Kyoto 602-0841, Japan.
Shigeru
S
Kinoshita
Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan; CREST (Core Research for Evolutional Science and Technology), JST (Japan Science and Technology Agency), Honcho 4-1-8, Kawaguchi, Saitama, 332-0012, Japan. Electronic address: smasui@cira.kyoto-u.ac.jp.
Shinji
S
Masui
D015153:Blotting, Western; D002454:Cell Differentiation; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism*; D000091344:Filaggrin Proteins; D005786:Gene Expression Regulation; D055786:Gene Knockout Techniques; D006801:Humans; D007150:Immunohistochemistry; D046228:Microarray Analysis; D000071841:PAX6 Transcription Factor / Q000096:biosynthesis / Q000235:genetics*; D012313:RNA / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D012313:RNA
CRISPR/Cas9; Cornea; Corneal epithelial cell; Corneal epithelium; Keratin 12; Keratinization; PAX6; Transcription factor