ABSTRACT
The goal of this study was the search for and structure-function analysis of the regulatory genes specific for pluripotent embryonic stem cells (ESCs). This was the first study in which PCR was used to obtain DNA fragments with primers constructed on the basis of OCT4 and NANOG mRNA. cDNA synthesized on mRNA isolated from human embryonic eye in the 9.5th week of development was used as a template in PCR analysis. PCR fragment DNA was sequenced. A comparative analysis of the nucleotide sequences demonstrated their 100% homology with the OCT4-pg1 retrogene and NANOG gene. Expression of the genes of interest was reliably detected in the cornea, crystalline lens, retina, and eye tunics in the 10.5th week of development. The nuclear localization of the products of the NANOG gene and OCT4-pg1 retrogene indicates that these proteins are classified with transcription factors. The role of the OCT4-pg1 retrogene and NANOG gene in self-renewal and differentiation of pluripotent cells in a developing eye is discussed.
Subject(s)
Embryonic Stem Cells/metabolism , Eye Proteins/biosynthesis , Eye/embryology , Gene Expression Regulation, Developmental/physiology , Homeodomain Proteins/biosynthesis , Octamer Transcription Factor-3/biosynthesis , Pluripotent Stem Cells/metabolism , Cell Differentiation/physiology , Cell Nucleus/genetics , Cell Nucleus/metabolism , Embryonic Stem Cells/cytology , Eye/cytology , Eye Proteins/genetics , Homeodomain Proteins/genetics , Humans , Nanog Homeobox Protein , Octamer Transcription Factor-3/genetics , Pluripotent Stem Cells/cytology , RNA, Messenger/biosynthesis , RNA, Messenger/geneticsABSTRACT
The pattern of the PITX2 gene expression was studied in the cornea, lens, retina, iridocorneal complex (ICC), and eye coats of human fetuses at weeks 9.5-22 of intrauterine development. Using the PCR method, PITX2 expression in all these tissues was revealed already at the earliest stage studied (9.5 weeks), being especially strong in the anterior eye complex (the cornea and lens) and weaker in the retina and sclera. The level of PITX2 expression in all eye tissues slightly decreased by week 15, increased to a high level in the ICC on week 18, and further decreased in all tissues by week 22. Using cDNA derived from the whole eyes of 8-, 9-, 10.5-, and 11-week fetuses, the expression of two PITX2 isoforms specific for eye tissues (A and B) was revealed. By means of in situ hybridization, the PITX2 mRNA was localized in the eye tissues of ectodermal and neuroectodermal origin.
