Pluripotency and patterning in nonhuman primate embryogenesis

Pluripotent cells of the embryo have the remarkable ability to form all somatic tissues and the germ line. Mice have long provided a framework for our understanding of this property of the early embryo. However, primate embryogenesis differs in anatomical architecture, developmental timing, and molecular configuration. Obvious ethical considerations limit human embryo research, thus nonhuman primates offer an opportunity to unravel primate-specific features of pluripotency. This thesis examines common marmoset (Callithrix jacchus) development to explore the molecular cues that govern (1) naïve pluripotency in the preimplantation epiblast, and (2) primed pluripotency in the embryonic disc (EmDisc) in the context of anterior-posterior axis patterning. First, imaging and virtual reconstruction of implanted marmoset embryos generated genome-wide, 3D expression patterns before and after primitive streak formation. Spatial transcriptome analysis revealed that pluripotency is sustained to the anterior EmDisc, in contrast to the mouse. Integration of RNA-seq datasets of marmoset preimplantation embryos generated a blueprint from zygote to gastrulation in the marmoset. I used this transcriptional reference map to determine the developmental state of marmoset pluripotent stem cells (PSCs) by single-cell RNA-seq. Spatial identity mapping identified that primed marmoset PSCs correspond to the anterior EmDisc. Resetting marmoset PSCs to an earlier developmental state induced characteristic changes associated with naïve identity, remarkably including transcriptional correspondence to the preimplantation epiblast. In contrast to human, marmoset PSCs could be reset without histone deacetylase inhibition and did not require aPKC inhibition. Instead, marmoset naïve PSCs relied on exogenous ActA for long-term propagation. To functionally interrogate the signals controlling amniogenesis and gastrulation from the pluripotent EmDisc, I leveraged PSC-based embryo models. Encapsulation of marmoset PSCs into agarose microgels yielded a 3D culture platform for lineage entry into EmDisc or amnion fate. Primed PSCs gave rise to amnion spheroids, in contrast to naïve PSCs. Pathway modulation revealed that Activin/Nodal and FGF signalling blocked amniogenesis, while BMP promoted amnion lineage entry. Investigating the combinatorial roles of these pathways in EmDisc patterning, I found that joint BMP, Activin/Nodal, and FGF signalling emulated the emergence of primitive streak and amnion formation in micropatterned gastruloids, similar to human. WNT was critically essential for initiation of the primitive streak, while Hedgehog did not exhibit patterning effects. To explore the transcriptional circuitry of anterior-posterior patterning, I screened 1000 developmentally relevant genes for a role in self-renewal. I identified 120 positive and 28 negative regulators of pluripotency. Intersecting these hits with the spatial transcriptome dataset, I performed a candidate-knockdown screen in micropatterned gastruloids and discovered CLIP3, PDZD4, and HAND1 as essential regulators of the pluripotent compartment. Together, this study puts forward a molecular framework for maintenance of the embryonic pluripotent compartment throughout periimplantation development in a nonhuman primate species.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2022

Author dc:creator

• Slatery, Erin

Advisor dc:contributor.advisor

• Boroviak, Thorsten

• Pluripotency
• Nonhuman primate

Licence

• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

Author Identifier

0000-0002-9097-6618

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/346190