Biology & Pre-Medical Studies
Biology
Biology
The vertebrate lens is a transparent, avascular structure essential for focusing light onto the retina. Damage or loss of the lens, as seen in cataracts or injury, results in significant visual impairment (REF). In humans, lens regeneration is extremely limited, requiring surgical intervention. However, certain amphibians, including Pleurodeles waltl, possess the remarkable ability to regenerate a fully functional lens following removal.
P. waltl serves as an ideal model organism for studying regeneration due to its robust capacity for tissue regeneration (Elewa et al., 2017). Following lentectomy, lens regeneration occurs specifically from the dorsal iris pigment epithelial cells (iPECs) as they re-enter the cell cycle at 4 days post-lentectomy (dpl), which transdifferentiate into lens cells. In contrast, ventral iPECs fail to initiate this regenerative process despite being anatomically similar and phenotypically identical.
A key question in regenerative biology is why dorsal iPECs are competent for regeneration while ventral iPECs are not. This asymmetry suggests the presence of molecular pathways that either promote regeneration dorsally or inhibit it ventrally. Understanding these differences is critical for identifying mechanisms that could potentially unlock regenerative capacity in otherwise non-regenerative tissues.
The Ephrin pathway entails 14 receptors and ligands divided into two classes, A and B, with bidirectional signaling (Arvanitis & Davy, 2008). This bidirectional signaling of the Ephrin pathway allows forward signaling to proceed through the receptor tyrosine kinase domain and reverse signaling to proceed through cytoplasmic domains and adaptor proteins or associated kinases (Arvanitis & Davy, 2008). Ephrin-A ligands are GPI-anchored, while Ephrin-B ligands are located in the transmembrane with PDZ-binding motifs (Arvanitis & Davy, 2008). Ephrin-B1, Ephrin-B2, Ephrin-A5, EphB2, EphB3, EphA5, and EphA7 are expressed in the intact newt eye and during early stages of lens regeneration (Tsissios, 2022).
Activation of the Ephrin signaling pathway requires receptor-ligand clustering into higher-order arrays, allowing the signals to guide boundary formation and cellular positioning (Arvanitis & Davy, 2008; Taylor et al., 2017). Specifically in the retina, the Ephrin-B2:EphB4 axis is central for arterial-venous specification in retinal vasculature (Kaczmarek et al., 2021). Meanwhile, Ephrin-A:EphA gradients help to shape retinal ganglion cell projections to the superior colliculus to ensure proper retinotopy (Kaczmarek et al., 2021), showing the importance of this pathway in eye physiology. With the development of small molecules 123C4 and UniPR1331, we are able to target activation and inhibition of the Ephrin pathway. 123C4 is an agonist (activator) that binds EphA4 and induces its phosphorylation (Wu et al., 2017, Dennys et al., 2022). UniPR1331 is a pan-Eph:Ephrin protein-protein interaction inhibitor that binds to the extracellular region of the Eph receptors, allowing it to block ligand binding (Rusnati et al., 2022, Festuccia et al., 2018). This also allows us to inhibit forward and reverse signaling with the use of small molecule UniPR1331 (Festuccia et al., 2018). Therefore, the use of 123C4 and UniPR1331 will help us to understand the role of the Ephrin pathway in lens regeneration.
While Ephrin signaling is implicated in developmental processes, its role in lens regeneration, particularly in restricting ventral iPEC plasticity, remains unclear. Determining whether this pathway actively suppresses regeneration in ventral tissue is essential for understanding the molecular basis of regenerative asymmetry. We hypothesize that Ephrin signaling inhibits lens regeneration in ventral iPECs, and that modulation of this pathway will alter their regenerative potential.
The primary goal of this project is to determine whether the manipulation of the Ephrin signaling pathway through the use of small molecules 123C4 and UniPR1331 can induce lens regeneration from the ventral iris of P. waltl. Specifically, this study seeks to investigate whether inhibition or activation of this pathway influences the regenerative capacity of ventral iPECs, which typically lack the ability to reprogram into lens cells. To accomplish this, the Ephrin pathway changes will first be characterized using WB analysis to identify the presence of Eph receptors and their phosphorylated (activated) forms using specific antibodies in PW13 cells and embryo heads. Following successful characterization, the effects of the pathway activation and inhibition will be assessed through in vivo injections of 123C4 and UniPR1331 in newt lentectomized eyes. This approach will allow for a comprehensive evaluation of how modulation of the Ephrin pathway impacts cellular reprogramming and regenerative outcomes in the newt lens.
In vitro Protein Extraction
Cell culture of PW13 cells grown in A1 media was utilized for characterization. The PW13 cells were split every 5-7 days at a 1:2 ratio to maintain the culture. The PW13 cells were treated with small molecules UniPR1331 and 123C4 and stored in a -80℃ freezer until used for protein extraction. Treated PW13 cells were then processed through protein extraction by cell lysis via sonication and centrifugation. The supernatant was collected and quantified via Bradford Assay to determine the appropriate amount of protein needed for a 30µg concentration. The control (DMSO) and treated (UniPR1331 and 123C4) samples were prepared for Western Blots.
In vivo Protein Extraction
Embryos at 11 days post fertilization (dpf) were treated with small molecules 123C4 and UniPR1331 in their water for 24 hours. [Include how many embryos are in each sample per treatment at the respective concentrations.] The heads of the embryos were collected at 12 dpf per IACUC protocol and processed through protein extraction. The treated 12 dpf embryo heads were used in protein extraction by cell lysis via sonication and centrifugation. The supernatant was collected to be used in quantification via Bradford Assay. This quantification was used to determine the appropriate amount of protein needed for a 30 µg concentration. The control (DMSO) and treated (5µM UniPR1331, 25µM UniPR1331, 1µM 123C4, and 10µM 123C4) samples were prepared for Western Blots.
Western Blot Analysis
The proteins from the above-mentioned protein extractions were separated by running a Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) at 120 Volts for 1 hour and 15 minutes. Once electrophoresis is complete, the proteins were transferred from the polyacrylamide onto a PVDF membrane. This membrane was incubated in blocking buffer for 10 minutes before incubating with the specific primary antibodies (EPH Total, pEPH, ERK Total, pERK, and Actin). The membrane was then washed with TBST 1X before incubating with the secondary antibody (Anti-rabbit HRP and Anti-mouse 700) for at least 2 hours. The membrane was then imaged using BioRad ChemiDoc. Through protein purification of the 12 dpf embryo heads and the use of WB, we are able to characterize the status of the Ephrin receptor activity (phosphorylation presence).
In vivo Lentecomy Model
Eighteen 3-month-old newts underwent lens removal (lentectomy) per IACUC protocol and were treated with the small molecules described above to observe the small molecule activity in ventral iris regeneration. Six 3-mo lentectomized newts per experimental condition were treated with 123C4 and UniPR1331 small molecules at 5µM concentrations each. This treatment lasted 12 days post-lentectomy with the small molecules in the water.
Histological Analysis
The eyes of these 3-mo lentectomized newts were collected per IACUC protocol for paraffin embedding and sectioned for Hematoxylin and Eosin staining. The H&E staining was utilized for histological analysis of these experimental conditions to observe possible ventral iris regeneration.
From our countless studies, we can conclude that the 123C4 pharmaceutical treatment was effective in increasing the accumulation of pERK. We can also see that in one of the twelve samples, there is a double dorsal lens produced in the EphA5-FC recombinant treatment. After a second study done to confirm the production of a double dorsal lens, there were only singular dorsal lenses observed upon regeneration.
The KDRT lab will continue this research and expand on the pharmaceutical treatments via intraocular injections with hopes of producing double dorsal lens or ventral lens regeneration.
I am thankful to have been given the opportunity to complete this project with funding from the Hughes Scholars Program and the Undergraduate Research Award Program. I would also like to thank my mentors, Drs. Katia Del Rio-Tsonis and José Raúl Pérez-Estrada for their endless support and guidance throughout this project. Finally, I would like to thank the KDRT lab for providing me with such a wonderful lab environment to complete this research project. Thank you to all of the individuals who have helped me along the way and made this project possible.
I have enhanced my critical thinking skills as well as my professionalism in the KDRT Lab. I have also been able to lead others and improve my teamwork skills.
I adhered to the IACUC protocols during this research.