Bystrom, B. | Search bioRxiv

bioRxiv2021

Aniridia-related Keratopathy Relevant Cell Signaling Pathways in Human Fetal Corneas

BackgroundTo study aniridia-related keratopathy (ARK) relevant cell signaling pathways (Notch1, Wnt/{beta}-catenin, Sonic hedgehog (SHH) and mTOR) in normal human fetal corneas in comparison with normal human adult corneas. Results20 wg fetal and normal adult corneas showed similar staining patterns for Notch1, however 10-11 wg fetal corneas showed increased presence of Notch1. Numb and Dlk1 had an enhanced presence in the fetal corneas compared to the adult corneas. Fetal corneas showed stronger immunolabeling with antibodies against {beta}-catenin, Wnt5a and Wnt7a, Gli1, Hes1, p-rpS6, and mTOR when compared to the adult corneas. Gene expression of Notch1, Wnt5A, Wnt7A, {beta}-catenin, Hes1, mTOR and rps6 was higher in the 9-12 wg fetal corneas when compared to adult corneas. ConclusionsThe cell signaling pathway differences found between human fetal and adult corneas were similar to those previously found in ARK corneas with the exception of Notch1. Analogous profiles of cell signaling pathway activation between human fetal corneas and ARK corneas suggests that there is a less differentiated host milieu in ARK.

developmental biology↗

bioRxiv2021

Cell signaling pathways in human mutant PAX6 corneal cells: an in vitro model for aniridia-related keratopathy

PURPOSETo establish an in vitro model of aniridia-related keratopathy (ARK) using CRISPR/Cas9 engineered human keratocytes with mutations in the PAX6 gene, and to study the Notch Homolog 1, Translocation-Associated (Notch1), sonic hedgehog (SHH), mammalian target of rapamycin (mTOR), and Wnt/{beta}-catenin signaling pathways in the PAX6 mutant keratocytes. METHODSPrimary human keratocytes were isolated from healthy corneas. Keratocytes were transduced with Cas9 lentiviral particles in order to create cells stably expressing Cas9 nuclease. Lentiviral particles carrying PAX6 sgRNA were transduced into the Cas9 keratocytes creating mutants. Analysis of signaling pathways was assessed by RT-qPCR for gene expression and western blot for protein expression. RESULTSHuman keratocytes stably expressing Cas9 nuclease were created. Keratocytes carrying PAX6 gene mutation were successfully generated. PAX6 mutant keratocytes showed modified expression patterns of extracellular matrix components such as collagens and fibrotic markers. Analysis of the Notch1, SHH, mTOR, and Wnt/{beta}-catenin signaling pathways in the PAX6 mutant keratocytes revealed altered gene and protein expression of the key players involved in these pathways. CONCLUSIONSA properly functioning PAX6 gene in keratocytes is crucial for the regulation of signaling pathways important for cell fate determination, proliferation, and inflammation. Pax6 mutation in the in vitro settings leads to changes in these pathways which resemble those found in corneas of patients with ARK.

molecular biology↗