Mechanical mapping of lineage boundaries in the developing Xenopus laevis embryo

Stem cells are capable of both self-renewal and differentiation into a range of other cell types and must regularly make decisions between these fates. While we have a good understanding of how the chemical environment influences these decisions, there is mounting evidence that stem cells integrate mechanical and chemical signals *in vitro* when making fate decisions. Specifically, substrate stiffness has been shown to be an important regulator of stem cell fate, though we currently know little about the contribution of viscosity, and *in vivo* nvestigations are still missing. In this project I used atomic force microscopy (AFM) to investigate the viscoelasticity of the developing *Xenopus laevis* embryo and compared mechanical maps to fate maps of the developing embryo found in literature, along with my own lineage tracing. First, I determined that the presence of the vitelline membrane obscures the mechanical properties of the embryo below, and so must be removed, and that the AFM could reliably measure embryonic tissue up to an angle of 21° from horizontal. I then measured the stiffness and viscosity of three distinct embryonic regions: the vegetal pole, the animal pole, and the equator, during early gastrulation. I found the vegetal pole to be the stiffest and most viscous region, followed by the equator and then the animal pole. The vegetal pole also had the largest viscous component contribution, followed by the animal pole and then the equator. I found that the embryo stiffness is increasing over time, while the viscosity remains constant, allowing me to apply a correction to past and future measurements that remove the effect of embryonic aging on the stiffness. To determine the role of the mechanical environment in stem cell lineage commitment during development, I labelled two single cells with fluorescent dextrans at the 8-cell stage, then tracked their descendants and measured mechanical maps across the lineage boundaries. I found that primarily endoderm cell regions were both stiffer and more viscous than ectoderm cell regions, and that this endoderm region was more elastic dominated than the ectoderm region. I found a clear correlation between local mechanical properties and stem cell fate choice/lineage restriction during embryonic development *in vivo*, and my future work will focus on expanding these investigations to look for a causal relationship. I also found evidence that the viscous component of biological tissue should be considered when investigating the impact of the mechanical environment on stem cells. A better understanding of how stem cell decisions are regulated *in vivo* could lead to advances in stem cell treatments for disease or illness, as well as increased control over stem cells in the laboratory.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• McGinn, Ross

Advisor dc:contributor.advisor

• Franze, Kristian

• AFM
• Atomic Force Microscopy
• Biophysics
• Developmental biology
• Mechanobiology
• Stem cells
• Xenopus laevis

Licence

• https://www.repository.cam.ac.uk/bitstreams/1f0cbe29-a16a-45e2-8547-b7bf332fae32/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.109450

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/369775