The Eye: Our Window to the World
The eye is one of the major sensory organs, allowing us to see the world around us. When light hits the eye, it first encounters the cornea, the frontal dome-shaped part of the eye. As light passes through the cornea, it reaches the lens, which focuses light on the retina at the back of the eye. The retina contains the light-sensing cells of the eye — the rods and cones —which translate light into a message that is sent to the brain through the optic nerve. The colored part of the eye is called the iris and contains muscles that contract and dilate the pupil (the black part of the eye) to control the amount of light that enters the eye. Finally, the sclera is the white outermost part of the eye, which becomes continuous with the cornea at the front of the eye.
Vision is a significant part of our everyday lives, and a variety of human vision conditions are due to abnormalities in the eye. For example, near-sightedness and far-sightedness occur when light does not focus on the retina (either in front of or behind, respectively). A more serious ocular disease is retinitis pigmentosa, a degenerative eye disease that occurs when the epithelial layer of the retina (the rods and cones) dies over time, resulting in progressive blindness.
Lifeline® is excited to announce the release of our NEW human scleral fibroblasts! Scleral fibroblasts produce the majority of the extracellular matrix in the sclera of the eye and may play a role in development of myopia, or near-sighted vision. Lifeline® scleral fibroblasts are quality-tested to ensure growth and morphology over 15 population doublings. They are never exposed to antimicrobials or phenol red and are optimized for growth in FibroLifeS2 ® medium. Scleral fibroblasts join the ocular cells in the Lifeline® catalog, which also includes human corneal epithelial cells, optimized for growth in our OcuLife™ optimized medium.
Recent Research Using Lifeline® Human Corneal Epithelial Cells to Study Transcriptional Regulation
Transcriptional programs are a key regulators of tissue development. Transcription factor binding to enhancer regions activates genes distantly located in the genome, and identification of these enhancer regions increases our understanding of the transcriptional programs that regulate distinct differentiation processes. Using ChIP-Seq in Lifeline® normal human corneal epithelial cells, Klein and colleagues set out to define the enhancer landscape during corneal epithelial cell differentiation. Their ChIP-Seq used antibodies against the histone marks H3K4me3 (low levels indicate an active enhancer), H3K4me1 (high levels indicate an active enhancer), and H3K27ac (high levels indicate an active enhancer) and identified two types of enhancers: typical enhancers (TEs) and super enhancers (SEs). SEs are longer than TEs with a high density of bound transcription factors and activating histone marks.
Using this experimental setup, the researchers found 1,154 SEs and 12,424 TEs in Lifeline® corneal epithelial cells, many of which were located near genes known to be involved in development of the eye. When corneal epithelial TEs and SEs were compared with those from other cell types, the authors found that corneal epithelial TEs clustered with those from other epithelial cell types, but SEs from corneal epithelial cells were relatively unique, located close to genes important for the processes of sensory organ development and cell signaling.
To understand the specific transcription factors that might be important for activating these enhancer regions during corneal epithelial differentiation, the authors examined the transcription factor binding motifs in these regions and found an enrichment of KLF motifs in TEs and SEs. The KLF family is highly involved in corneal development and the authors identified a time-dependent expression pattern of KLF7 and KLF4, which have antagonistic expression patterns over the course of corneal development. Using tissue samples from mouse eyes, the researchers found that KLF7 was expressed during embryonic development, but absent 50 days after birth. In contrast, KLF4 expression was absent in the developing eye, but present after birth.
To understand the mechanistic basis for this, the authors performed loss-of-function experiments in Lifeline® corneal epithelial cells. Using microarray analysis to monitor global gene expression, the authors found that in proliferating corneal epithelial cells, KLF7 knockdown upregulated genes involved in corneal differentiation (Pax6 and Aldh3a1), suggesting that KLF7 normally suppresses the differentiation program to maintain a progenitor state. In contrast, many of the genes upregulated by KLF7 knockdown were actually downregulated by KLF4 knockdown, suggesting that KLF4 normally activates the differentiation program.
To evaluate whether these enhancer regions might play a role in corneal disease, the researchers compared their data with that of disease-associated single nucleotide polymorphisms (SNPs). Interestingly, they found that two TEs overlapped two SNPS, one associated with Stevens-Johnson syndrome and another with corneal astigmatism. To determine whether these SNPs had a functional role in each respective TE, the authors cloned each SNP and expressed them in Lifeline® corneal epithelial cells. They found that the SNP associated with Stevens-Johnson syndrome illustrated reduced enhancer activity, while the SNP associated with astigmatism had increased enhancer activity. Furthermore, they found that the enhancer associated with the astigmatism SNP had an Ets transcription factor motif; the authors then demonstrated that the Ets family member EHF bound to the wild-type enhancer and decreased enhancer activity, suggesting that the associated SNP might disrupt EHF binding and subsequently increase enhancer activity.
Together, the results of this study demonstrate that an elegant regulatory network of enhancers controls corneal epithelial differentiation and identifies two disease-related SNPs that affect enhancer activity, which may contribute to disease pathogenesis.
Our blog features new research every other week. Come back to check in with us to see how researchers around the world are using our cells!