Dean of Engineering and Co-Director of the Templeton Institute Union College, New York, United States
Introduction: The chick embryo is an excellent model organism to observe embryonic flexure and torsion. The system of Hamburger and Hamilton (HH) organized embryonic development into 46 stages [4]. Flexure describes the bending on the dorsal side (fig 1 panel A), while torsion describes twisting with respect to the cranio-caudal axis. Improper flexure can result in birth defects that impact several organs, including the heart. For instance, earlier studies have highlighted the significance of flexure in the process of cardiac looping [1]. However, the mechanisms causing flexion are not fully understood; so in this research, we investigated the role of differential cell proliferation. To suppress cell proliferation, aphidicolin – known to arrest the cell cycle in the early S phase – was used. Previous research done in our lab showed statistically significant difference of torsion progression between control and aphidicolin treatment group embryos [2]. In the prior research, global inhibition of cell proliferation was studied where embryos were incubated with growth media and aphidicolin solution. However, according to data from finite element analysis, the area highlighted in red (fig 1 panel B) contributes most to flexure formation. Hence, we aimed to study flexion of the embryos under localized inhibition of cell proliferation by injecting aphidicolin into the site suggested by computer simulations.
Materials and
Methods: Experiments consisted of harvesting chick embryos from incubated eggs, injection, and imaging. The harvesting technique outlined in [3] was used. Embryos were divided into control, sham, and aphidicolin injection groups with a Hamilton Company 10ul syringe used for injection. Sham and aphidicolin group embryos were injected with 0.5 ul of saline and 0.5 ul of 400uM aphidicolin solution respectively. We mainly focused on embryos around HH stage 12 and tracked their development. All injections were done within 2 seconds. Aphidicolin solution was made by dissolving powdered aphidicolin in dimethyl sulfoxide, and food coloring was added to all the solutions to enhance visualization. Imaging of embryos by light microscopy were done at 0, 7, 19, and 25 hours.
Results, Conclusions, and Discussions: Figure 1 top row shows control and bottom row shows aphidicolin-injected embryonic development. All controls (n=5) developed properly (please see panels A to D in fig 1). Sham injection embryos (n=5, results not shown) developed indistinguishable from control embryos, while some had minor deformations at the injection site. Aphidicolin injection embryos (please see panels A’ to D’ in fig 1) showed significantly deviated flexion. For example, while some showed improper flexion such as with sharp, abrupt corners, others did exhibit proper flexion but at a slower rate compared to controls. Our results are consistent with the research mentioned earlier. Specifically the changes in flexure observed in our study suggest the need for localized cell proliferation for proper flexure formation.
Acknowledgements (Optional): References
[1] Ramasubramanian, A., Chu-Lagraff, Q. B., Buma, T., Chico, K. T., Carnes, M. E., Burnett, K. R., ... & Gordon, S. S. (2013). On the role of intrinsic and extrinsic forces in early cardiac S-looping. Developmental Dynamics, 242(7), 801-816. [2] Wang, D., & Ramasubramanian, A. (2022, April). Does Cell Proliferation Affect Embryonic Flexure and Torsion?. In Experimental Biology Conference. [3] Voronov, D. A., & Taber, L. A. (2002). Cardiac looping in experimental conditions: effects of extraembryonic forces. Developmental dynamics: an official publication of the American Association of Anatomists, 224(4), 413-421. [4] Hamburger, V., & Hamilton, H. L. (1992). A series of normal stages in the development of the chick embryo. Developmental dynamics, 195(4), 231-272.
This material is based upon work supported by the National Science Foundation under Grant Number 1936733. We would like to thank Dr Kristen Fox, Professor of Chemistry at Union College for her assistance with chemical dilutions.
