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.
","abstract_html":"<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>","abstract_has_math":false,"creators":["Slater, Jill A."],"institution":null,"degree_name":"Ph.D.","degree_level":"Open Access Dissertation","degree_discipline":"Physiology","degree_department":null,"school":null,"contributors":["Daniel A. Rappolee"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2012,"date_issued":"2012-01-01T08:00:00Z","date_published":"2012-01-01T08:00:00Z","updated_at":"2026-07-24T05:59:19Z","subjects":["hyperosmotic stress","mouse embryonic stem cells","prioritized differentiation","Cell Biology","Developmental Biology"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://digitalcommons.wayne.edu/oa_dissertations/700","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Daniel A. Rappolee"]},{"key":"dc:creator","label":"Author","values":["Slater, Jill A."]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2013-01-01T08:00:00Z"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Physiology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Open Access Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["hyperosmotic stress","mouse embryonic stem cells","prioritized differentiation","Cell Biology","Developmental Biology"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://digitalcommons.wayne.edu/oa_dissertations/700"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.
"]},{"key":"dc:title","label":"Title","values":["Hyperosmotic Stress Enzyme Signaling Modulates Oct4, Nanog, And Rex1 Expression And Induces Prioritized Differentiation Of Murine Embryonic Stem Cells"]}]}],"canonical_facts":{"dc:contributor":["Daniel A. Rappolee"],"dc:creator":["Slater, Jill A."],"dc:date.available":["2013-01-01T08:00:00Z"],"dc:description.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.
"],"dc:identifier":["https://digitalcommons.wayne.edu/oa_dissertations/700"],"dc:subject":["hyperosmotic stress","mouse embryonic stem cells","prioritized differentiation","Cell Biology","Developmental Biology"],"dc:title":["Hyperosmotic Stress Enzyme Signaling Modulates Oct4, Nanog, And Rex1 Expression And Induces Prioritized Differentiation Of Murine Embryonic Stem Cells"],"thesis:degree_discipline":["Physiology"],"thesis:degree_level":["Open Access Dissertation"],"thesis:degree_name":["Ph.D."]},"updated_at":"2026-07-24T05:59:19Z"}