Student Theses and Dissertations
Date of Award
2020
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Eric D. Siggia
Keywords
human embryonic stem cells, gastrulation, Wnt signaling, Activin/Nodal pathway, epigenetic regulation, germ layer specification
Abstract
Development of a single cell, the fertilized egg, into an entire organism is a fascinating example of biological organization that gives rise to many forms of multicellular life. Cell-cell signaling is central to development, particularly in organisms such as vertebrates, where embryonic cells become progressively restricted in their potential through a hierarchy of decision points guided by the signals they receive. All cells contain the same genetic information in the form of DNA, and we now know that the DNA is modified epigenetically to impart distinct functions to each adult cell type. However, we are just beginning to uncover how the epigenome is modified over the course of development and how signaling pathways might direct these modifications. One of the earliest developmental decision points in bilaterian embryos, which includes vertebrates, occurs during gastrulation when cells are directed into one of three primary germ layers. Model systems, including frog, fish, chicken, and mouse, have been very useful for uncovering the identity and function of the key signaling networks driving vertebrate gastrulation. However, how these signals function together in time to modify cellular behavior has been difficult to tease apart in embryos. With the isolation of human embryonic stem cells (hESCs), many aspects of development can now be reconstituted and manipulated in culture, thus allowing us for the first time to investigate the early stages of our own development. We can now also address how mechanisms proposed from model systems function dynamically or in unanticipated ways. In this work, I used hESCs and a stem cell-based model of gastrulation (human gastruloids) to disentangle the function and timing of two key signaling pathways, namely Wnt and Activin/Nodal. From studies in model organisms both pathways are known to be required for initiating gastrulation and for the formation of mesoderm and endoderm, but it has been proposed that the level of Activin/Nodal signaling ultimately determines germ layer identity. In order to test these predictions in a model of human gastrulation, I first developed hESC lines with fluorescent reporters of Activin/Nodal signaling that facilitate quantitative measurements of the cellular response in real-time. With these lines I was able to establish that hESCs display a dose-dependent response to Activin ligands at the level of the intracellular effector Smad2. However, this response is transient, and the cells do not adopt germ layer identities even when the pathway response is transiently saturated. By manipulating the relative timing of Wnt and Activin ligands, I demonstrated that hESCs can record Wnt signals without differentiating and that this signaling memory makes cells competent to form mesoderm and endoderm in response to subsequently supplied Activin. My observations do not eliminate the co-requirement for Wnt and Activin in germ layer differentiation, which has been emphasized by previous studies of mouse and human ESCs, but rather they highlight the fact that instructive Wnt signals occur temporally up-stream of Activin signals to induce complete germ layer patterning. My initial efforts to uncover the mechanism of Wnt signaling memory identified a role for the bromodomain and extra-terminal domain (BET) family of epigenetic readers, which facilitate transcription through interactions with acetylated residues of histones and other proteins and are thought to promote tumor formation in the context of cancer. I showed that inhibition of BET protein function selectively eliminates mesoderm and endoderm in human gastruloids and produces phenotypes in frog embryos consistent with disruption of early Wnt signaling. The concept that Wnt signaling primes cells to differentiate without directing them to commit to a particular fate defines a new aspect to how this signaling pathway functions. These observations also establish an experimental context to further investigate the link between developmental signaling pathways and the epigenetic landscape. As I hope to demonstrate throughout this thesis, hESCs are a powerful means to uncover both conserved and potentially human-specific mechanisms of vertebrate development. In the context of Wnt and Activin signaling my results highlight the importance of the temporal interaction between Wnt and Activin signaling in patterning the vertebrate embryo and suggests an evolutionarily conversed mechanism of Wnt signaling memory that could explain how signals are integrated to direct cellular behavior in both development and disease.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Yoney, Anna Kresovich, "Cell Memory and Fate in Human Development" (2020). Student Theses and Dissertations. 595.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/595
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy