During early human placental development, the conceptus attaches itself to the uterus through cytotrophoblast invasion. Invasive cytotrophoblast cells differentiate from precursor villous cytotrophoblasts, but the essential regulating factors in this process are unknown. Basic helix-loop-helix (bHLH) transcription factor dimers are essential regulators of mouse trophoblast development. We therefore examined the importance of this family of factors in the human placenta. In many cell lineages, bHLH factors are sequestered by members of the Id family, HLH proteins that lack the basic DNA binding domain (Inhibitor of DNA binding proteins (Id-1 to Id-4)). During differentiation of some tissues, Id expression declines, allowing bHLH factors to dimerize, bind DNA and trans-activate lineage-specific genes. To begin to study the role of bHLH transcription factors in human placental development, we first characterized Id expression in cytotrophoblast cells. The cells expressed Id-3 constitutively; Id-2 was downregulated, at the mRNA and protein levels, as the cells differentiated in culture and in situ, respectively. In cases when cytotrophoblast differentiation was compromised (in placentas from women with preeclampsia, or in cells grown under hypoxic conditions in culture), Id-2 expression was maintained. To assess the functional relevance of these correlations, we used an adenovirus vector to maintain Id-2 protein expression in cultured cytotrophoblasts. Compared to control (lacZ-expressing) cells, cytotrophoblasts transduced to constitutively express Id-2 retained characteristics of undifferentiated cells: (alpha)1 integrin expression was low and cyclin B expression was retained. Furthermore, invasion through Matrigel was partially inhibited and migration was strikingly enhanced in Id-2-expressing cells. These results suggest that Id-2 and the bHLH factors that it partners play important roles in human cytotrophoblast development.

Arnold
H. H.
,
Winter
B.
(
1998
)
Muscle differentiation: more complexity to the network of myogenic regulators.
Curr. Opin. Gen. Dev
8
,
539
544
Basyuk
E.
,
Cross
J. C.
,
Corbin
J.
,
Nakayama
H.
,
Hunter
P.
,
Nait-Oumesmar
B.
,
Lazzarini
R. A.
(
1999
)
Murine Gcm1 gene is expressed in a subset of placental trophoblast cells.
Dev. Dynam
214
,
303
311
Benezra
R.
,
Davis
R. L.
,
Lockshon
D.
,
Turner
D. L.
,
Weintraub
H.
(
1990
)
The protein Id: a negative regulator of helix-loop-helix DNA binding proteins.
Cell
61
,
49
59
Biggs
J.
,
Murphy
E. V.
,
Israel
M. A.
(
1992
)
A human Id-like helix-loop-helix protein expressed during early development.
Proc. Natl. Acad. Sci. USA
89
,
1512
1516
Cepko
C. L.
(
1999
)
The roles of intrinsic and extrinsic cues and bHLH genes in the determination of retinal cell fates.
Curr. Opin. Neurobiol
9
,
37
46
Chomczynski
P.
,
Sacchi
N.
(
1987
)
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem
162
,
156
159
Cross
J. C.
,
Werb
Z.
,
Fisher
S. J.
(
1994
)
Implantation and the placenta: key pieces of the development puzzle.
Science
266
,
1508
1518
Cross
J. C.
,
Flannery
M. L.
,
Blanar
M. A.
,
Steingrimsson
E.
,
Jenkins
N. A.
,
Copeland
N. G.
,
Rutter
W. J.
,
Werb
Z.
(
1995
)
Hxt encodes a basic helix-loop-helix transcription factor that regulates trophoblast cell development.
Development
121
,
2513
2523
Damsky
C. H.
,
Fisher
S. J.
(
1998
)
Trophoblast pseudo-vasculogenesis: faking it with endothelial adhesion receptors.
Curr. Opin. Cell. Biol
10
,
660
666
Damsky
C. H.
,
Fitzgerald
M. L.
,
Fisher
S. J.
(
1992
)
Distribution patterns of extracellular matrix components and adhesion receptors are intricately modulated during first trimester cytotrophoblast differentiation along the invasive pathway, in vivo.
J. Clin. Invest
89
,
210
222
Damsky
C. H.
,
Librach
C.
,
Lim
K. H.
,
Fitzgerald
M. L.
,
McMaster
M. T.
,
Janatpour
M.
,
Zhou
Y.
,
Logan
S. K.
,
Fisher
S. J.
(
1994
)
Integrin switching regulates normal trophoblast invasion.
Development
120
,
3657
3666
Ellis
S. A.
,
Palmer
M. S.
,
McMichael
A. J.
(
1990
)
Human trophoblast and the choriocarcinoma cell line BeWo express a truncated HLA Class I molecule.
J. Immunol
144
,
731
735
Firulli
A. B.
,
McFadden
D. G.
,
Lin
Q.
,
Srivastava
D.
,
Olson
E. N.
(
1998
)
Heart and extra-embryonic mesodermal defects in mouse embryos lacking the bHLH transcription factor Hand1.
Nat. Genet
18
,
266
270
Fisher
S. J.
,
Cui
T. Y.
,
Zhang
L.
,
Hartman
L.
,
Grahl
K.
,
Zhang
G. Y.
,
Tarpey
J.
,
Damsky
C. H.
(
1989
)
Adhesive and degradative properties of human placental cytotrophoblast cells in vitro.
J. Cell. Biol
109
,
891
902
Genbacev
O.
,
Joslin
R.
,
Damsky
C. H.
,
Polliotti
B. M.
,
Fisher
S. J.
(
1996
)
Hypoxia alters early gestation human cytotrophoblast differentiation/invasion in vitro and models the placental defects that occur in preeclampsia.
J. Clin. Invest
97
,
540
550
Genbacev
O.
,
Zhou
Y.
,
Ludlow
J. W.
,
Fisher
S. J.
(
1997
)
Regulation of human placental development by oxygen tension.
Science
277
,
1669
1672
Guillemot
F.
,
Nagy
A.
,
Auerbach
A.
,
Rossant
J.
,
Joyner
A. L.
(
1994
)
Essential role of Mash-2 in extraembryonic development.
Nature
371
,
333
336
Iavarone
A.
,
Garg
P.
,
Lasorella
A.
,
Hsu
J.
,
Israel
M. A.
(
1994
)
The helix-loop-helix protein Id-2 enhances cell proliferation and binds to the retinoblastoma protein.
Genes Dev
8
,
1270
1284
Janatpour
M. J.
,
Utset
M. F.
,
Cross
J. C.
,
Rossant
J.
,
Dong
J.
,
Israel
M. A.
,
Fisher
S. J.
(
1999
)
A repertoire of differentially expressed transcription factors that offers insight into mechanisms of human cytotrophoblast differentiation.
Dev. Genet
25
,
146
157
Jen
Y.
,
Manova
K.
,
Benezra
R.
(
1997
)
Each member of the Id gene family exhibits a unique expression pattern in mouse gastrulation and neurogenesis.
Dev. Dynam
208
,
92
106
Kageyama
R.
,
Sasai
Y.
,
Akazawa
C.
,
Ishibashi
M.
,
Takebayashi
K.
,
Shimizu
C.
,
Tomita
K.
,
Nakanishi
S.
(
1995
)
Regulation of mammalian neural development by helix-loop-helix transcription factors.
Crit. Rev. Neurobiol
9
,
177
188
Knöfler
M.
,
Meinhardt
G.
,
Vasicek
R.
,
Husslein
P.
,
Egarter
C.
(
1998
)
Molecular cloning of the human Hand1 gene/cDNA and its tissue-restricted expression in cytotrophoblastic cells and heart.
Gene
224
,
77
86
Kovats
S.
,
Main
E. K.
,
Librach
C.
,
Stubblebine
M.
,
Fisher
S. J.
,
DeMars
R.
(
1990
)
A class I antigen, HLA-G, expressed in human trophoblasts.
Science
248
,
220
223
Librach
C. L.
,
Werb
Z.
,
Fitzgerald
M. L.
,
Chiu
K.
,
Corwin
N. M.
,
Esteves
R. A.
,
Grobelny
D.
,
Galardy
R.
,
Damsky
C. H.
,
Fisher
S. J.
(
1991
)
92-kD type IV collagenase mediates invasion of human cytotrophoblasts.
J. Cell Biol
113
,
437
449
Lim
K.
,
Chae
C. B.
(
1989
)
A simple assay for DNA transfection byincubation of the cells in culture dishes with substrates for beta-galactosidase.
Biotechniques
7
,
576
579
Lim
K. H.
,
Zhou
Y.
,
Janatpour
M.
,
McMaster
M.
,
Bass
K.
,
Chun
S. H.
,
Fisher
S. J.
(
1997
)
Human cytotrophoblast differentiation/invasion is abnormal in pre-eclampsia.
Am. J. Pathol
151
,
1809
1818
Ludolph
D. C.
,
Konieczny
S. F.
(
1995
)
Transcription factor families: muscling in on the myogenic program.
FASEB J
9
,
1595
1604
McMaster
M. T.
,
Librach
C. L.
,
Zhou
Y.
,
Lim
K. H.
,
Janatpour
M. J.
,
DeMars
R.
,
Kovats
S.
,
Damsky
C.
,
Fisher
S. J.
(
1995
)
Human placental HLA-G expression is restricted to differentiated cytotrophoblasts.
J. Immunol
154
,
3771
3778
McMaster
M.
,
Zhou
Y.
,
Shorter
S.
,
Kapasi
K.
,
Geraghty
D.
,
Lim
K. H.
,
Fisher
S.
(
1998
)
HLA-G isoforms produced by placental cytotrophoblasts and found in amniotic fluid are due to unusual glycosylation.
J. Immunol
160
,
5922
5928
Molkentin
J. D.
,
Olson
E. N.
(
1996
)
Defining the regulatory networks for muscle development.
Curr. Opin. Genet. Dev
6
,
445
453
Murre
C.
,
Bain
G.
,
van Dijk
M. A.
,
Engel
I.
,
Furnari
B. A.
,
Massari
M. E.
,
Matthews
J. R.
,
Quong
M. W.
,
Rivera
R. R.
,
Stuiver
M. H.
(
1994
)
Structure and function of helix-loop-helix proteins.
Biochim. Biophys. Acta
1218
,
129
135
Nakayama
H.
,
Liu
Y.
,
Stifani
S.
,
Cross
J. C.
(
1997
)
Developmental restriction of Mash-2 expression in trophoblast correlates with potential activation of the notch-2 pathway.
Dev. Genet
21
,
21
30
Orkin
S. H.
(
1995
)
Transcription factors and hematopoietic development.
J. Biol. Chem
270
,
4955
4958
Orkin
S. H.
(
1996
)
Development of the hematopoietic system.
Curr. Opin. Genet. Dev
6
,
597
602
Prabhu
S.
,
Ignatova
A.
,
Park
S. T.
,
Sun
X. H.
(
1997
)
Regulation of the expression of cyclin-dependent kinase inhibitor p21 by E2A and Id proteins.
Mol. Cell. Biol
17
,
5888
5896
Ramsey
E. M.
,
Houston
M. L.
,
Harris
J. W.
(
1976
)
Interactions of the trophoblast and maternal tissues in three closely related primate species.
Am. J. Obstet. Gynecol
124
,
647
652
Riley
P.
,
Anson-Cartwright
L.
,
Cross
J. C.
(
1998
)
The Hand1 bHLH transcription factor is essential for placentation and cardiac morphogenesis.
Nat. Genet
18
,
271
275
Rinkenberger
J. L.
,
Cross
J. C.
,
Werb
Z.
(
1997
)
Molecular genetics of implantation in the mouse.
Dev. Genet
21
,
6
20
Roth
I.
,
Corry
D. B.
,
Locksley
R. M.
,
Abrams
J. S.
,
Litton
M. J.
,
Fisher
S. J.
(
1996
)
Human placental cytotrophoblasts produce the immunosuppressive cytokine interleukin 10.
J. Exp. Med
184
,
539
548
Saga
Y.
,
Hata
N.
,
Kobayashi
S.
,
Magnuson
T.
,
Seldin
M. F.
,
Taketo
M. M.
(
1996
)
MesP1: a novel basic helix-loop-helix protein expressed in the nascent mesodermal cells during mouse gastrulation.
Development
122
,
2769
2778
Sun
X. H.
,
Copeland
N. G.
,
Jenkins
N. A.
,
Baltimore
D.
(
1991
)
Id proteins Id1 and Id2 selectively inhibit DNA binding by one class of helix-loop-helix proteins.
Mol. Cell. Biol
11
,
5603
5611
Sutherland
A. E.
,
Calarco
P. G.
,
Damsky
C. H.
(
1993
)
Developmental regulation of integrin expression at the time of implantation in the mouse embryo.
Development
119
,
1175
1186
Tanaka
M.
,
Gertsenstein
M.
,
Rossant
J.
,
Nagy
A.
(
1997
)
Mash2 acts cell autonomously in mouse spongiotrophoblast development.
Dev. Biol
190
,
55
65
Werb
Z.
,
Alexander
C. M.
,
Adler
R. R.
(
1992
)
Expression and function of matrix metalloproteinases in development.
Matrix. Suppl
1
,
337
343
Yokota
Y.
,
Mansouri
A.
,
Mori
S.
,
Sugawara
S.
,
Adachi
S.
,
Nishikawa
S.
,
Gruss
P.
(
1999
)
Development of peripheral lymphoid organs and natural killer cells depends on the helix-loop-helix inhibitor Id2.
Nature
397
,
702
706
Zhou
Y.
,
Damsky
C. H.
,
Chiu
K.
,
Roberts
J. M.
,
Fisher
S. J.
(
1993
)
Preeclampsia is associated with abnormal expression of adhesion molecules by invasive cytotrophoblasts.
J. Clin. Invest
91
,
950
960
Zhou
Y.
,
Damsky
C. H.
,
Fisher
S. J.
(
1997
)
Preeclampsia is associated with failure of human cytotrophoblasts to mimic a vascular adhesion phenotype. One cause of defective endovascular invasion in this syndrome? [see comments].
J. Clin. Invest
99
,
2152
2164
Zhou
Y.
,
Fisher
S. J.
,
Janatpour
M.
,
Genbacev
O.
,
Dejana
E.
,
Wheelock
M.
,
Damsky
C. H.
(
1997
)
Human cytotrophoblasts adopt a vascular phenotype as they differentiate. A strategy for successful endovascular invasion? [see comments].
J. Clin. Invest
99
,
2139
2151
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