Hyperosmotic Stress Enzyme Signaling Modulates Oct4, Nanog, And Rex1 Expression And Induces Prioritized Differentiation Of Murine Embryonic Stem Cells

<p>HYPEROSMOTIC STRESS ENZYME SIGNALING MODULATES OCT4, NANOG,</p> <p>AND REX1 EXPRESSION AND INDUCES PRIORITIZED DIFFERENTIATION OF</p> <p>MURINE EMBRYONIC STEM CELLS</p> <p>by</p> <p>JILL SLATER</p> <p>MAY 2013</p> <p>Advisor: Daniel Rappolee, Ph.D.</p> <p>Major: Physiology</p> <p>Degree: Doctor of Philosophy</p> <p>Transcription factor expression and therefore lineage identity in the periimplantation</p> <p>embryo and its stem cells may be influenced by extracellular stresses,</p> <p>potentially affecting pregnancy outcome. Cellular stress forces cells to suppress some</p> <p>normal activities (such as protein synthesis and cell proliferation) in order to repair</p> <p>stress-damaged macromolecules and restore homeostasis. Therefore, any new</p> <p>activities that embryonic cells initiate while concurrently funding the demands of the</p> <p>stress response reveal the developmental priorities of these cells. Previous work</p> <p>showed that cultured multipotent trophoblast stem cells (TSC) initiated differentiation in</p> <p>response to hyperosmotic stress, favoring the development of the earliest functioning</p> <p>placental lineage (parietal trophoblast giant cells) while suppressing that of laterdifferentiating</p> <p>lineages (chorionic/syncytiotrophoblast).</p> <p>The studies described in this dissertation studied the stress response of the other</p> <p>extant lineage of the early blastocyst, cells derived from the inner cell mass, murine</p> <p>embryonic stem cells (mESC). Hyperosmotic stress slowed mESC accumulation due to</p> <p>slowing of the cell cycle, not apoptosis. PI3K signaling was responsible for cell survival</p> <p>136</p> <p>under stressed conditions. Stress initially triggered mESC differentiation through MEK1,</p> <p>JNK, and PI3K signaling, leading to proteasomal degradation of OCT4, NANOG, SOX2,</p> <p>and REX1 protein. Concurrent with this post-transcriptional effect was the degradation</p> <p>of their mRNA transcripts. As stress continued, cells adapted, cell cycle resumed, and</p> <p>OCT4 and NANOG mRNA and protein expression returned to near normal levels. The</p> <p>protein recovery was mediated by p38 and PI3K signaling, as well as by that of an</p> <p>unknown MEK1/2 target. REX1 expression, however, did not recover; its ongoing</p> <p>suppression was due to JNK signaling. mESC did not overtly differentiate during stress,</p> <p>but were primed to differentiate toward the extraembryonic lineages, upregulating</p> <p>markers of primitive endoderm and suppressing epiblast markers.</p> <p>The studies were continued in the peri-implantation model, embryoid bodies</p> <p>(EBs), in which differentiation is allowed rather than actively suppressed. Unstressed</p> <p>EB culture recapitulated the lineage inductions of in vivo embryos. EBs were only able</p> <p>to be cultured in the presence of low levels of hyperosmotic stress (10mM sorbitol);</p> <p>higher levels led to a failure of mESC to aggregate. Aggregation and subsequent</p> <p>embryoid body formation was rescued when either JNK or p38 MAPKs were inhibited</p> <p>during mESC culture. Low levels of osmotic stress increased the magnitude of primitive</p> <p>endoderm markers, Lrp2 and Dab2. Transient, sub-lethal stress delivered prior to the</p> <p>start of hanging drop culture was remembered by mESC, suppressing differentiation</p> <p>events slated to occur from 1-6d later. Mesoderm marker, Brachyury, and anterior</p> <p>visceral endoderm marker, Goosecoid, expression was suppressed. The timing of</p> <p>stress delivery was very significant in determining its outcome. Hyperosmotic stress</p> <p>delivered at the onset of differentiation induced a prioritized differentiation of mESC,</p> <p>inducing the earlier-developing primitive endoderm, and strongly suppressing later137</p> <p>developing mesoderm and anterior visceral endoderm.</p>

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Open Access Dissertation

Discipline thesis:degree_discipline

Physiology

Year dc:date.available

2012

Author dc:creator

• Slater, Jill A.

Contributors dc:contributor

• Daniel A. Rappolee

• hyperosmotic stress
• mouse embryonic stem cells
• prioritized differentiation
• Cell Biology
• Developmental Biology

Repository record dc:identifier

https://digitalcommons.wayne.edu/oa_dissertations/700

OAI identifier oai:identifier

oai:digitalcommons.wayne.edu:oa_dissertations-1699