| Description |
151 p. : ill. |
| Note |
Advisor: Daniel A. Rappolee. |
| Thesis |
Thesis (Ph.D.)--Wayne State University, 2013. |
| Summary |
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. |
| Subject |
Developmental biology
|
|
Cytology
|
| Added Title |
Wayne State University thesis (Ph.D.) : Physiology.
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| OCLC # |
862917684 |
|
