Pax-6 is expressed early in the differentiation of a corneal epithelial model system.
Pax-6 is a regulatory gene with a major role during visual system development, but its association with corneal epithelial differentiation is not clearly established. Using the RCE1-(5T5) cell line, which mimics corneal epithelial differentiation, we analyzed Pax-6 biological role. Immunostaining of proliferating colonies and confluent sheets showed that Pax-6-positive cells were also K3 keratin-positive, suggesting that Pax-6 is expressed in differentiating cells. Pax-6 mRNA was barely expressed in early cell cultures; but after confluence, its levels raised up to fivefold as demonstrated by Northern blot and RT-qPCR. The raise in Pax-6 expression preceded for 9 h the increase in LDH-H and LDH-M mRNAs, previously shown as early markers of corneal epithelial cell differentiation. The full-length mRNAs encoding for the two major Pax-6 isoforms were found at very low levels in proliferating cells, and abundantly expressed in the confluent stratified epithelia; Pax-6 mRNA was 2- to 2.5-fold more abundant than Pax-6(5a) mRNA. The ectopic expression of Pax-6 or Pax-6(5a) decreased proliferative ability leading to the formation of abortive, non-proliferative colonies. In contrast, culture conditions that delay or block corneal epithelial cell differentiation reduced or inhibited the expression of Pax-6. Collectively, results show that Pax-6 is the earlier differentiation marker expressed by corneal epithelial cells, and open the possibility for a major role of Pax-6 as the main driver of the differentiation of corneal epithelial cells.
García-Villegas
Refugio
R
Department of Physiology, Biophysics and Neurosciences, Centro de Investigación y de Estudios Avanzados del IPN, México City, Mexico.
Escamilla
Juan
J
Sánchez-Guzmán
Erika
E
Pastén
Angela
A
Hernández-Quintero
Miriam
M
Gómez-Flores
Eber
E
Castro-Muñozledo
Federico
F
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002103:Cadaverine / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D003315:Cornea / Q000166:cytology / Q000502:physiology; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology*; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D053558:Keratin-3 / Q000235:genetics / Q000378:metabolism; D045022:Lactate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D011817:Rabbits; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*
D015415:Biomarkers; D005136:Eye Proteins; D018398:Homeodomain Proteins; D053558:Keratin-3; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; D045022:Lactate Dehydrogenases; D002103:Cadaverine