First published online 29 June 2005
doi: 10.1242/dev.01923
Development 132, 3393-3403 (2005)
Published by The Company of Biologists 2005
Hypoxia-inducible factor-dependent histone deacetylase activity determines stem cell fate in the placenta
Emin Maltepe1,
Geoffrey W. Krampitz2,
Kelly M. Okazaki2,
Kristy Red-Horse2,
Winifred Mak3,
M. Celeste Simon4,5,6 and
Susan J. Fisher2,7,8,9,*
1 Department of Pediatrics and Molecular Medicine Program, University of
California, Parnassus Avenue, San Francisco, CA 94143, USA
2 Department of Cell and Tissue Biology, University of California, Parnassus
Avenue, San Francisco, CA 94143, USA
3 Obstetrics and Gynecology, Cedars-Sinai Medical Center, 8700 Beverly
Boulevard, West Hollywood, CA 90048, USA
4 Howard Hughes Medical Institute, Abramson Family Cancer Research Institute,
University of Pennsylvania, Philadelphia, PA 19104, USA
5 Department of Cell and Developmental Biology, University of Pennsylvania,
Philadelphia, PA 19104, USA
6 Abramson Cancer Research Institute, University of Pennsylvania, Philadelphia,
PA 19104, USA
7 Obstetrics, Gynecology and Reproductive Sciences, University of California,
Parnassus Avenue. San Francisco, CA 94143, USA
8 Department of Pharmaceutical Chemistry, University of California, Parnassus
Avenue, San Francisco, CA 94143, USA
9 Department of Anatomy, University of California, Parnassus Avenue, San
Francisco, CA 94143, USA
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Fig. 1. Arnt-null TS cells differentiate into chorionic trophoblasts and
syncytiotrophoblasts. Undifferentiated wild-type (A) and Arnt-null
(B) TS cells grown on fibroblast feeders exhibited identical morphological
features. Differentiated wild-type cells (C) formed large trophoblast giant
cells and smaller spongiotrophoblasts. Differentiated Arnt-null TS
cells (D) produced large multinucleated syncytiotrophoblasts and smaller
chorionic trophoblasts. E-cadherin staining of differentiated wild-type cells
revealed one or occasionally two nuclei per cell (E), whereas
Arnt-null cells formed syncytia containing multiple nuclei (F). In
some cases, multinucleated cells with peripherally distributed nuclei (G,
arrowheads) and central clearing appeared to form epithelial-lined spaces. (H)
RNA FISH analysis of Xist RNA revealed a single inactive X chromosome per
nucleus in differentiated Arnt-null TS cells, additional evidence for
lack of endoreduplication. (I) Analysis of lineage-specific markers by
northern blot hybridization and RNase protection assays confirmed these
findings.
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Fig. 3. Oxygen-independent modulation of HIF expression during TS cell
differentiation. (A) Levels of mRNAs encoding Arnt, HIF1 and
HIF2 were assayed by Northern blot hybridization after differentiation
under standard tissue culture conditions for 1 week. (B) Protein levels of the
HIFs and related factors were assayed by immunoblot. ARNT protein was solely
detected in the nuclei of wild-type TS cells and levels did not change with
differentiation. In normoxia (20% O2), HIF1 expression was
strongly induced in wild-type but not mutant TS cells. With regard to
HIF2, high cytoplasmic expression was observed only after
differentiation of Arnt-null TS cells, with comparable levels of
nuclear expression in both wild-type and mutant cells after FGF4 and heparin
withdrawal. pVHL levels, in both the cytoplasmic and nuclear fractions, were
induced during differentiation. HSP90ß expression, which was confined to
the nucleus, did not change significantly during either wild-type or mutant TS
cell differentiation. (C) The effects of hypoxia (2% O2) on ARNT,
HIF1 and HSP90ß expression in undifferentiated wild-type and
mutant TS cells mirrored the effects of differentiation. (D) As expected, mRNA
levels of the HIF1 target genes VEGF and GLUT1 were upregulated when wild
type, but not mutant TS cells were cultured under hypoxic conditions. PGK1,
which was upregulated by hypoxia in wild-type TS cells, was expressed only at
low levels in the Arnt-null mutants.
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Fig. 4. The hypoxia-normoxia transition promotes spongiotrophoblast expansion.
Hypoxia fails to redirect wild-type or mutant TS cell fate in vitro. In
contrast, hypoxia followed by reoxygenation (2*) led to an expansion of the
spongiotrophoblast lineage, as shown by the dramatic enhancement of 4311
expression in wild-type TS cells.
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Fig. 5. HDAC activity is altered in Arnt-null TS cells. (A)
Differentiation of wild-type TS cells was associated with increased levels of
acetylated H4 (Ac-H4). A similar increase was observed during differentiation
of the Arnt-null TS cells, although baseline levels in the
undifferentiated state were much higher than those observed in wild-type
cells. HDAC1 expression, which does not change during differentiation, was
used as a loading control. (B) Compensatory upregulation of multiple HDACs was
observed in whole-cell lysates prepared from undifferentiated
Arnt-null TS cells. (C) Analysis of nuclear extracts revealed altered
translocation of multiple HDACs in mutant TS cells.
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Fig. 6. HDAC inhibition phenocopies ARNT deletion with respect to HIF1 target gene
expression and TS cell differentiation. (A) Undifferentiated wild-type TS
cells were cultured with (+) or without (–) 100 nM trichostatin A (TSA)
or 100 nM geldanamycin A (GA). Then Pgk1 gene expression was analyzed
by northern blot hybridization. Both compounds significantly reduced
Pgk1 mRNA levels, but to a lesser extent than ARNT deletion. (B)
Wild-type TS cells were differentiated in the presence or absence of TSA or GA
and the expression of mRNAs encoding lineage-markers was analyzed by northern
blot hybridization. Both compounds inhibited Pl-I expression, suggesting
impaired trophoblast giant cell differentiation. Interestingly, TSA but not
GA, inhibited 4311 expression, suggesting differential effects of these
compounds on spongiotrophoblast differentiation. Enhanced expression of Tfeb
suggested that TS cell differentiation was redirected to chorionic trophoblast
formation. (C) Phase-contrast microscopy of differentiated wild-type TS cells
cultured with or without 100 nM TSA for the duration of the differentiation
period. Although many large trophoblast giant cells and clusters of
spongiotrophoblasts were seen in untreated cultures, TSA treatment promoted
syncytialization (broken circles indicated by arrows) along with cytoplasmic
vacuolization and membrane breakdown (arrowheads) as seen with differentiating
Arnt-null cells.
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Fig. 7. Model of trophoblast stem cell differentiation: the oxygen-independent role
of HIF-regulated HDAC activity. In the presence of FGF4, TS cells are
maintained in a replicative pluripotent state. Upon growth factor withdrawal,
wild-type TS cells differentiate along the pathway that leads to giant cell
formation. Although portrayed as a linear sequence, it is also possible that
this differentiation pathway bifurcates, with either spongiotrophoblasts or
trophoblast giant cells being produced. HIF activity suppresses generation of
chorionic trophoblasts and syncytiotrophoblasts, whereas HDAC and HSP90
activity is required for trophoblast giant cell formation in vitro. In support
of this model, Arnt-null TS cells predominantly differentiated into
chorionic trophoblasts and syncytiotrophoblasts, with the HDAC inhibitor TSA
having similar effects. Interestingly, the HSP90 inhibitor geldanamycin
selectively inhibited trophoblast giant cell differentiation.
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© The Company of Biologists Ltd 2005
