Student Theses and Dissertations

Date of Award

2021

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Brivanlou Laboratory

Abstract

The specification of germ cells during embryonic development is vital not only for the development of an organism, but quite literally for the survival and propagation of its entire species. Recent work has demonstrated that several aspects of human primordial germ cell (hPGC) development are specific to primates, necessitating model systems and in vivo validation that is also species specific. In this work, a synthesis of in vitro and in vivo techniques is used to investigate hPGC specification within a human embryonic stem cell model of a gastrulating embryo known as a gastruloid. An hPGC transcriptomic signature that indicates migratory potential via canonical and novel mechanisms is indicated, raising several potential candidates for further investigation into the under-studied migratory phase of germ cell development. We seek to generate validation assays of hPGC function in an embryonic context by investigating migratory potential in grafts to the chick embryo, and demonstrate that despite significant differences between chick and human routes of migration in vivo, human in vitro-derived PGCs are in fact able to demonstrate migratory behavior in the chick, following chick migratory patterns and demonstrating a specific homing towards the chick mesonephros and gonad. These experiments not only provide a functional validation for in vitro-derived hPGCs that is complementary to molecular and epigenetic analysis, but also hint at the elements of hPGC development that are conserved throughout evolution. The gastruloid system is then used for further investigation into the hPGC niche. The power of this model system relies upon self-organization due to endogenous signaling in response to an exogenous BMP4 initiating signal, in a manner analogous to that found in the human embryo. We harness this power, using single cell image quantification and genetic tools including CRISPR-Cas9 to probe further into the signaling environment of the hPGC niche in the gastruloid model. We elucidate the role of each leg of the BMP4 – WNT – ACTIVIN/NODAL signaling cascade in development of this niche, which determines gastrulation events, in hPGC specification. These experiments not only demonstrate how BMP4 acts independently in addition to this cascade to directly specify hPGCs, but also how all three pathways work in harmony to generate self-organization of hPGCs within their gastruloid niche. By probing further into the ontogeny of hPGC specification, we find that upregulation of BLIMP1 alone, a canonical regulator of PGCs, is sufficient to induce later markers of hPGC fate, and surprisingly is also sufficient to downregulate SOX2 (a marker of epiblast and early ectodermal fate) and upregulate SOX17, which has been previously placed upstream of BLIMP1. Finally, we propose the marmoset as a good model of primate reproduction and embryogenesis, in an attempt to describe a non-human primate (NHP) system for validation of hPGC characteristics observed in vitro. We systematically investigate marmoset embryonic development in vivo using serial, high temporal and spatial resolution ultrasound imaging. We describe the morphological characteristics of implantation, gastrulation, neurulation, and organogenesis, as well as the curious marmoset phenotype of an elongated peri-gastrulation window, incorporating slowed embryonic growth and rapid extraembryonic development. In doing so, we generate an annotated ultrasound atlas of marmoset embryogenesis, and train models to identify developmental stages and predict fertilization ages from a single frame. In addition, we suggest that the extended peri-gastrulation window in the marmoset will provide a unique opportunity to perform in utero genetic editing, lineage tracing, and even allogenic transplantation to complement in vitro studies of hPGC development. This body of work provides a synthesis of culture techniques, genetic and molecular tools, and imaging systems that will provide a foundation for the exploration of not only human PGC development, but also hopefully a generalizable roadmap for translation between in vitro and in vivo studies early primate development.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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