Access Type

Open Access Dissertation

Date of Award

January 2012

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Physiology

First Advisor

Daniel A. Rappolee

Abstract

HYPEROSMOTIC STRESS ENZYME SIGNALING MODULATES OCT4, NANOG,

AND REX1 EXPRESSION AND INDUCES PRIORITIZED DIFFERENTIATION OF

MURINE EMBRYONIC STEM CELLS

by

JILL SLATER

MAY 2013

Advisor: Daniel Rappolee, Ph.D.

Major: Physiology

Degree: Doctor of Philosophy

Transcription factor expression and therefore lineage identity in the periimplantation

embryo and its stem cells may be influenced by extracellular stresses,

potentially affecting pregnancy outcome. Cellular stress forces cells to suppress some

normal activities (such as protein synthesis and cell proliferation) in order to repair

stress-damaged macromolecules and restore homeostasis. Therefore, any new

activities that embryonic cells initiate while concurrently funding the demands of the

stress response reveal the developmental priorities of these cells. Previous work

showed that cultured multipotent trophoblast stem cells (TSC) initiated differentiation in

response to hyperosmotic stress, favoring the development of the earliest functioning

placental lineage (parietal trophoblast giant cells) while suppressing that of laterdifferentiating

lineages (chorionic/syncytiotrophoblast).

The studies described in this dissertation studied the stress response of the other

extant lineage of the early blastocyst, cells derived from the inner cell mass, murine

embryonic stem cells (mESC). Hyperosmotic stress slowed mESC accumulation due to

slowing of the cell cycle, not apoptosis. PI3K signaling was responsible for cell survival

136

under stressed conditions. Stress initially triggered mESC differentiation through MEK1,

JNK, and PI3K signaling, leading to proteasomal degradation of OCT4, NANOG, SOX2,

and REX1 protein. Concurrent with this post-transcriptional effect was the degradation

of their mRNA transcripts. As stress continued, cells adapted, cell cycle resumed, and

OCT4 and NANOG mRNA and protein expression returned to near normal levels. The

protein recovery was mediated by p38 and PI3K signaling, as well as by that of an

unknown MEK1/2 target. REX1 expression, however, did not recover; its ongoing

suppression was due to JNK signaling. mESC did not overtly differentiate during stress,

but were primed to differentiate toward the extraembryonic lineages, upregulating

markers of primitive endoderm and suppressing epiblast markers.

The studies were continued in the peri-implantation model, embryoid bodies

(EBs), in which differentiation is allowed rather than actively suppressed. Unstressed

EB culture recapitulated the lineage inductions of in vivo embryos. EBs were only able

to be cultured in the presence of low levels of hyperosmotic stress (10mM sorbitol);

higher levels led to a failure of mESC to aggregate. Aggregation and subsequent

embryoid body formation was rescued when either JNK or p38 MAPKs were inhibited

during mESC culture. Low levels of osmotic stress increased the magnitude of primitive

endoderm markers, Lrp2 and Dab2. Transient, sub-lethal stress delivered prior to the

start of hanging drop culture was remembered by mESC, suppressing differentiation

events slated to occur from 1-6d later. Mesoderm marker, Brachyury, and anterior

visceral endoderm marker, Goosecoid, expression was suppressed. The timing of

stress delivery was very significant in determining its outcome. Hyperosmotic stress

delivered at the onset of differentiation induced a prioritized differentiation of mESC,

inducing the earlier-developing primitive endoderm, and strongly suppressing later137

developing mesoderm and anterior visceral endoderm.

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