Poster E7 - Wound healing in Xenopus laevis embryonic tissues under tension

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Yingli Wang

Undergrad Researcher
University of Pittsburgh
Pittsburgh, Pennsylvania, United States

Co-Author(s)

JY

Jing Yang, Phd Candidate

Graduate Researcher
University of Pittsburgh, United States

Last Author(s)

LD

Lance Davidson

Principal Investigator
University of Pittsburgh, United States

Introduction: Epithelial wounds in the skin of embryos exhibit perfect healing (Metcalfe and Ferguson, 2007) (Davidson et al., 2002). Wound healing is a complex process influenced by mechanobiology; its success is crucial to restoring tissue integrity and function (Nodder and Martin, 1997) (Ly et al., 2024). These processes coordinate cell movement and cell signaling to restore tissue function as quickly as possible. The mechanical microenvironment in the tissue is thought to play an equally important role in the healing process, as cells and tissues can respond differently under external forces, yet the healing process in tissues under tension is not well studied. In this project, we used laser ablation techniques to create a small wound in the Xenopus laevis embryonic epithelium while under an applied uniaxial tension. The edge of the wound contracts until the wound is closed, causing surrounding cells to be strained locally. This project investigates how embryonic epithelial sheets heal under high tensile strain to better understand the mechanisms of biomechanical regulation in the wound healing process, offering insights into embryonic development and potential treatments for wound healing.

Materials and

Methods: We obtain embryos using standard methods (Kay and Peng, 1991) and culture them to Stage 9.5 (Nieuwkoop and Faber, 1967). Embryos are transferred to DFA media prior to microsurgical isolation of the animal cap explant (Keller, 1980). The explant is placed on a fibronectin-coated cassette for our custom stretcher (Fig. 1). The explant is attached to the underside of a polydimethylsiloxane (PDMS) strip gluing to the cassette. The cassette is then fitted into a custom, lightweight stretcher customized for the microscope stage. Two closed-loop linear motors precisely control the stretching rate and extent (Fig. 1). The stretcher exerts external strain on the explant, allowing for the investigation of how epithelia heal under tension, compared to the control group without tension.
As embryos reach Stage 13, we perform targeted laser ablations and observe with a confocal microscope. A single cell-cell junction is targeted and ablated each time under unstretched or stretched conditions to observe differences in wound healing (Fig. 2).
Image acquisition captures both rapid and relatively long-term responses after laser ablation. We collect images at intervals of 0.5 seconds to capture rapid changes in the early wounding process over the first 30 seconds and change the interval to 5 seconds for the following 9.5 minutes, recording the healing process. To conduct a detailed analysis, we performed strain analysis on 13 representative images selected at equal intervals to ensure the coverage throughout the entire process.

Results, Conclusions, and Discussions: This study analyzes the overall area strain in both unstretched and stretched conditions to understand the mechanism of embryonic epithelial tissues during wound healing. For more precise observation, we set four circular areas at approximately one cell around the wound: two perpendicular to the direction of the applied force, and two parallel to the direction of the applied tension (Fig. 2B and 2C). The choice of these sites allows us to evaluate the strain in regions surrounding the wound and compare strain perpendicular to the stretch axes. Preliminary data shows that the area along the direction of stretch is strained more than the area perpendicular to the stretch axis during wound closure, suggesting the wound closed asymmetrically by straining more along the stretch axis. We expected the tissue under tension would take longer to close than without external tension. Surprisingly, we did not observe a significant delay in the wound closure on the tissues under tension. This suggests that single wounded junctions close even with anisotropic external tension applied. Alternatively, a small delay in wound healing may be undetectable by our current method. Future experiments include ablating 3-5 cells in a row to create a larger wound and test if the size of the wound would affect the closure rate with or without external tension.
In conclusion, we investigate the wound healing process in Xenopus laevis embryonic epithelial tissues under uniaxial tension by laser ablating cell junctions, where we find the area along the stretch axis strains more than the area perpendicular to the stretch axis, with no significant delay in overall wound closure.

Acknowledgements (Optional):