Adaptive changes in the transcription factor
HoxA-11 are essential for the evolution
of pregnancy in mammals
Vincent J. Lynch*†, Andrea Tanzer*‡, Yajun Wang§, Frederick C. Leung§, Birgit Gellersen¶, Deena Emera*,
and Gunter P. Wagner*
*Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT 06511;§School of Biological Sciences, Kardoorie Biological Science
Building, Department of Zoology, University of Hong Kong, Pokfulam Road, Hong Kong, China;‡Institute of Theoretical Chemistry, University of Vienna,
Wa ¨hringerstrasse, 1090 Vienna, Austria; and¶Endokrinologikum Hamburg, Falkenried 88, 20251 Hamburg, Germany
Edited by Morris Goodman, Wayne State University School of Medicine, Detroit, MI, and approved August 19, 2008 (received for review March 8, 2008)
Evolutionary change in gene regulation can result from changes in
cis-regulatory elements, leading to differences in the temporal and
spatial expression of genes or in the coding region of transcription
factors leading to novel functions or both. Although there is a
growing body of evidence supporting the importance of cis-
regulatory evolution, examples of protein-mediated evolution of
novel developmental pathways have not been demonstrated.
Here, we investigate the evolution of prolactin (PRL) expression in
endometrial cells, which is essential for placentation/pregnancy in
eutherian mammals and is a direct regulatory target of the tran-
scription factor HoxA-11. Here, we show that (i) endometrial PRL
expression is a derived feature of placental mammals, (ii) the PRL
regulatory gene HoxA-11 experienced a period of strong positive
selection in the stem-lineage of eutherian mammals, and (iii) only
HoxA-11 proteins from placental mammals, including the recon-
the promoter used in endometrial cells. In contrast, HoxA-11 from
the reconstructed therian ancestor, opossum, platypus, and
chicken are unable to up-regulate PRL expression. These results
demonstrate that the evolution of novel gene expression domains
is not only mediated by the evolution of cis-regulatory elements
but can also require evolutionary changes of transcription factor
evolution of development ? functional divergence ? molecular evolution ?
rise to phenotypic novelty and the evolution of gene regulation
(1, 2). Mechanistically, embryonic development is governed by
the precise temporal and spatial deployment of gene regulatory
networks (1, 2). Thus, the evolution of development is funda-
mentally a question of the origin and evolution of gene regula-
tion and regulatory networks. An emerging principle from
evolutionary developmental studies is that adaptive mutations
with phenotypic effects are much more likely to occur in
cis-regulatory elements than in protein-coding genes (3–6).
Indeed, there is ample evidence for the importance of cis-
regulatory evolution (4–6), but it has also been shown that
transcription factors do not remain functionally equivalent dur-
ing evolution (7–9). This finding suggests that the evolution of
transcription factors themselves may play an active role in the
evolution of development and the origin of morphological
innovations (10, 11). So far, however, no detailed evolutionary
scenario has been elucidated that explains the role of transcrip-
tion factor evolution. Here, we reconstruct some of the events
that lead to the origin of pregnancy in eutherian mammals and
demonstrate that adaptive changes in a transcription factor
(HoxA-11) protein play an essential role.
One of the key evolutionary innovations of eutherian (pla-
cental) mammals is prolonged internal gestation and an invasive
evelopmental evolutionary studies have played an impor-
tant role in elucidating the molecular mechanisms that give
embryo (pregnancy), adaptations made possible by the evolution
of uterus and placenta. Knockout and knockdown studies have
identified many genes important for pregnancy, including tran-
scription factors, growth factors, and cell signaling molecules,
such as prolactin (PRL) (12). In humans, PRL is one of the most
dramatically induced genes in decidualized (differentiated) en-
dometrial stromal cells, and decidua-derived PRL is one of the
most abundant secretory products in the amniotic fluid (13–17).
Decidually expressed PRL has numerous functions, including
regulation of uterine epithelial cell differentiation, trophoblast
A particularly important role for decidual PRL is the suppres-
sion of genes detrimental to pregnancy, such as interleukin 6
(IL-6) and 20?-hydroxysteroid dehydrogenase (20?-HSD),
which promote inflammation and catabolize progesterone, re-
spectively (13, 18, 19). Thus, the proper regulation of decidual
PRL is essential for implantation and pregnancy.
Although the evolutionary mechanisms and timing of PRL
recruitment into uterine expression are unclear, the molecular
mechanisms leading to decidual PRL activation are well under-
stood. Several genes have been identified that influence endo-
metrial PRL expression, such as C/EBP?, FOXO1A, ETS1 and
HoxA-11 is required is to activate expression of PRL (V.J.L.,
B.G., and G.P.W., unpublished data). In addition to its role in
PRL regulation, HoxA-11 is essential for the pre- and postnatal
development of the female reproductive tract and is required for
successful implantation of the mammalian blastocyst (25–28).
Remarkably, a previous molecular evolutionary analysis of
HoxA-11 suggested that an episode of positive Darwinian se-
lection occurred in the stem-lineage of placental mammals,
coincident with the origin of the uterus and pregnancy (29). This
result suggests that adaptive evolution in HoxA-11 may have
generated a novel PRL regulatory ability, possibly through the
origin of new cofactor associations required for PRL expression
To clarify the evolutionary mechanisms that gave rise to
uterine PRL expression, we have examined in detail the molec-
ular and functional evolution of the PRL regulatory transcrip-
tion factor HoxA-11. We show that endometrial PRL expression
Author contributions: V.J.L. and G.P.W. designed research; V.J.L., A.T., Y.W., and D.E.
performed research; Y.W., F.C.L., and B.G. contributed new reagents/analytic tools; V.J.L.
analyzed data; and V.J.L. and G.P.W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
†To whom correspondence should be addressed at: OML 327, Department of Ecology and
Evolutionary Biology, Yale University, New Haven, CT 06511. E-mail: vincent.j.lynch@
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
September 30, 2008 ?
vol. 105 ?
no. 39 www.pnas.org?cgi?doi?10.1073?pnas.0802355105
is a derived feature of placental mammals and that the episode
of positive selection in HoxA-11 occurred coincident with the
origin of its ability to interact functionally with another tran-
scription factor protein (FOXO1A) and thereby up-regulate
PRL from the promoter used in endometrial cells. In contrast,
HoxA-11 from opossum, the reconstructed therian ancestor,
platypus, and chicken is unable to up-regulate PRL expression.
Remarkably, although PRL is not expressed in the uterus of the
opossum (or chicken), genes normally silenced in the uterus
during pregnancy (IL-6 and 20?-HSD) are expressed in the
uterus of the pregnant opossum. These results identify some of
the initial stages in the evolution of a novel gene regulatory
network and demonstrate that gene recruitment is not only
mediated by the evolution of cis-regulatory elements but also
requires evolution of transcription factor proteins themselves.
Results and Discussion
the uterus and placenta of placental mammals (13–17), PRL has
not been detected in the oviduct of fish, amphibians, and squamate
reptiles (30–32). However, the PRLR is expressed in the oviducts
of these species (30–32). Similarly, the PRLR is expressed in the
uterus of marsupials (33), but the expression of PRL in the uterine
tissues of marsupials has not been directly tested. We assayed PRL
and PRLR expression by RT-PCR in the female reproductive
tissues and placenta of a gestating marsupial, the gray short-tailed
opossum (Monodelphis domestica), and found that PRL was not
stage 32 fetus in this species [supporting information (SI) Fig. S1],
although the RT-PCR primers efficiently amplify targets when
paired with primers targeting introns and using genomic DNA as
template (Fig. 1A). We also assayed PRL expression in chicken
oviduct and pituitary and found that although PRL was expressed
in pituitary it was not expressed in oviducts of egg-laying hens (Fig.
1B). To determine whether PRL is also expressed the female
reproductive tract in basal mammalian lineages, we assayed PRL
expression in the placental tissue of the African elephant (Lox-
odonta africana) by RACE and found that PRL expression is
expressed in this tissue (Fig. S2). Thus, we conclude that PRL
expression in the uterus is a derived feature of placental mammals.
An important role of PRL expression in the uterus of pla-
cental mammal is to silence the expression of genes that are
detrimental to pregnancy, such as IL-6 and 20?-HSD, which
(13, 18, 19). The coincident origin in the stem-lineage of
placental mammals of pregnancy and PRL expression in the
female reproductive tract suggests that some of the functions of
test this hypothesis we assayed IL-6 and 20?-HSD expression by
RT-PCR in the female reproductive tissues of a gestating
opossum (M. domestica) and found that both genes were ex-
pressed in the endometrium, ovary, and stage 32 fetus (Fig. S3).
This result suggests that a major direct effect of recruitment of
PRL into uterine expression may have been silencing the ex-
pression of IL-6 and 20?-HSD, allowing a more invasive embryo
and prolonged gestation without eliciting a maternal immune
response that was detrimental to the developing embryo.
Our finding that PRL expression in the uterus is an innovation
of placental mammals is consistent with the recent finding that
the enhancer driving decidual PRL (dPRL) expression in human
endometrial stromal cells is a eutherian-specific transposable
element (MER20) (34). We have identified this element from all
major mammalian lineages by searching mammalian genome
databases. Using a Bayesian relaxed-clock method, we dated the
insertion of the MER20 element upstream of PRL to ?166–155
175 MYA (35, 36) and coincident with a reported period of rapid
evolution in HoxA-11 (29), suggesting that the earliest time that
PRL could have been expressed in the uterus of placental
mammals was shortly after their origin.
To test directly for an episode of positive Darwinian selection
in HoxA-11 in the stem lineage of placental mammals, we used
likelihood models of dN/dSratio variation among lineages and
sites (37–39) on a large HoxA-11 dataset. Under the simplest
model of rate variation, which constrains all lineages in the
phylogeny to have the same dN/dSratio, the dN/dSratio was 0.146
(Table S1). To test for an episode of accelerated evolution in the
stem-lineage of placental mammals we used a two-ratios model
that estimated the rate of the eutherian stem-lineage (?E)
separately from all other lineages (?0). Under this model, the
dN/dS ratio in eutherian stem-lineage was significantly higher
than the background rate (P ? 0.011) (Table S1). A three-ratio
model that estimates a separate dN/dS ratio for the eutherian
stem-lineage, crown group eutherians (?CE) and all other ani-
mals found that although the eutherian stem-lineage evolved
significantly faster than nonplacentals, the intensity of purifying
selection has increased moderately within the crown group
eutherians (P ? 0.027) (Table S1). We also tested for amino acid
sites that were positively selected in the eutherian stem-lineage
by comparing branch-site model A, which allows for a class of
codons to have dN/dS?1, with the null model (M1a), which does
is a significantly better fit to the data than the model that does
not allow for selected sites (P ? 0.005) (Table S1), identified 10
sites under positive selection (? ? 1.51–2.41) in the stem-lineage
of placental mammals (Fig. 2). Although these sites are under
relatively weak negative selection in nonplacental mammals
(? ? 0.44), they are under extremely strong selective constraints
within the extant placental mammals (? ? 0.08) (Fig. 3).
tracts. (A) Expression of PRL in tissues of the opossum female reproductive
are the number of cycles used in PCRs. M, 100-bp ladder; NTC, no template
RT-PCR primers are functional. (B) Expression of PRL in the oviducts of two
egg-laying hens. Note that PRL is only expressed in chicken pituitary.
Lynch et al.
September 30, 2008 ?
vol. 105 ?
no. 39 ?
Together, these findings suggest that the HoxA-11 protein has
acquired an additional functional constraint during the origin of
To test whether HoxA-11 genes in the Eutheria evolve under
different selective constraints than in other vertebrate lineages,
we estimated the coefficient of functional divergence (?), which
measures the difference in evolutionary rate at amino acid sites
between two clades. Rejection of the null hypothesis (? ? 0) is
evidence for altered functional constraints between clades (40,
41). We found significant evidence of type I functional diver-
gence between eutherians and sauropsids (?I? 0.46 ? 0.18, P ?
0.01; Fig. 1) and significant but weak evidence of type II
functional divergence (?II? 0.09 ? 0.05, P ? 0.05; Fig. 1). Type
I sites (40) are amino acids that are conserved within the
Eutheria but variable in the sauropsids, indicating they evolved
whereas type II sites (41) are amino acids that are fixed in
eutherians and sauropsids for different amino acids, indicating a
functional switch. The greater degree of type I (?I? 0.46) than
type II (?II? 0.09) functional divergence suggests that selection
recruited weakly constrained amino acids into a novel function
rather than amino acids that were under strong preexisting
functional constraints. These data suggest that selection acted
within existing structural and functional constraints on proteins
to generate novel functions (Fig. 3).
The episode of adaptive evolution in HoxA-11 in the stem-
lineage of eutherians and the absence of PRL expression in the
uterus and oviduct of nonplacental mammals suggests that the
ability to regulate PRL expression in the decidua might be a
novel function of HoxA-11 in eutherians. We initially tested for
functional differences between human and chicken HoxA-11
genes by using an in vivo knockdown/xenogene rescue system in
cultured human endometrial stromal cells (hESCs). Briefly,
hESCs were induced to decidualize with physiological levels of
17?-estradiol and progestin while intrinsic HoxA-11 expression
was knocked down with a morpholino, followed by rescue with
either mouse or chicken HoxA-11. Eight days after differentia-
tion, genes known to be expressed in decidualized stroma, but
not expressed in undifferentiated cells, were highly expressed in
cells treated with both random-sequence control morpholino
(Fig. 3) and untreated cells (data not shown). However, decidu-
alized cells treated with the HoxA-11-specific morpholino failed
to express PRL and TGF-?. To test whether HoxA-11 knock-
down was reversible, we rescued the knockdown with mouse
HoxA-11, which recovered both PRL and TGF-? expression
(Fig. 3). We next rescued the knockdown with chicken HoxA-11,
which does not have the derived amino acid replacements of
placental mammals, and we recovered TGF-? expression but
failed to rescue PRL expression (Fig. 4). These results suggest
that the HoxA-11 protein underwent amino acid substitutions
necessary for the derived ability to regulate PRL but that the
mammals. Eutherians are shown in dark red, with the stem-lineage of euth-
erians in red. Ten sites were identified under positive selection with ?E?
1.5–2.4 depending on how codon frequencies were modeled (equal or em-
pirical). Type I (?I) and type II (?II) functional divergence was estimated be-
tween eutherians and sauropsids (shown in green). ?I, P ? 0.01; ?II, P ? 0.05.
Branch lengths are proportional to the number of nonsynonymous substitu-
tions per codon. The red filled and white filled circles indicate the location of
the ancestral eutherian and therian sequences recreated for functional tests.
Positive selection on HoxA-11 exon 1 in the stem-lineage of placental
exon 1. The black bar represents exon 1 of HoxA-11, red marks indicate the
relative locations of sites that were substituted in the stem-lineage of euth-
erians. The two sequence logos show site variation within eutherians (Top)
and the noneutherians (Bottom). The inferred amino acids at these sites is
shown for the reconstructed therian ancestor (Middle). Amino acids are color
?I,type I site; ?II, type II site. The rate of evolution of these sites is shown for
eutherians and noneutherians.
Location of positively selected and functionally divergent sites in
HoxA-11 genes in endometrial cells. The expression of GAPDH (endogenous
control) and marker genes of decidualized stromal cells (PRL; PC6, proprotein
were assayed by RT-PCR after hormone-induced differentiation and morpho-
lino treatment. Treatments are shown on the left. H/rMO, hormone and
random morpholino. H/MO, hormone and HoxA-11-specific morpholino.
H/MO/Mmu, hormone and HoxA-11-specific morpholino rescued with mouse
HoxA-11. H/MO/Gga, hormone and HoxA-11-specific morpholino rescued
PCR is shown next to the treatments.
Partial functional nonequivalence of human, mouse, and chicken
www.pnas.org?cgi?doi?10.1073?pnas.0802355105Lynch et al.
ability to regulate TGF-? is ancestral for the amniote HoxA-11
The knockdown/rescue approach described above is a power-
ful technique to test for functional divergence, particularly in
nonmodel organisms, but it can be complicated by complex auto-
and cross-regulatory interactions that can interfere with the
knockdown, the rescue, or both. Therefore, it is desirable to test
functional divergence in more controlled assays, particularly
outside of the cell type of interest, once functional differences
are identified. To this end, we used a luciferase reporter assay in
HeLa cells to test specifically the ability of HoxA-11 genes from
the major amniote lineages to activate transcription from the
decidual PRL enhancer. Transfection with HoxA-11 alone re-
pressed reporter gene expression, consistent with previous stud-
ies that found that the HoxA-11 protein acts as an intrinsic
repressor (42). Cotransfection of mouse or human HoxA-11 with
FOXO1A led to dramatic activation from the PRL enhancer
(Fig. 5), consistent with the rescue results in hESCs. Next, we
tested whether the opossum, platypus, and chicken HoxA-11,
which lack the derived placental-specific adaptive amino acid
substitutions, activated luciferase expression from the decidual
the orthologs from opossum, platypus, and chicken, species that
lack PRL expression in uterus and oviducts, repressed luciferase
expression when cotransfected with FOXO1A (Fig. 4). Finally,
we tested whether opossum HoxA-11 could activate expression
from the dPRL enhancer when paired with the opossum
FOXO1A gene. Similarly to the opossum HoxA-11 paired with
human FOXO1A, opossum HoxA-11/opossum FOXO1A failed
to activate luciferase expression (Fig. 5).
To test specifically whether the amino acids identified from
the statistical analysis as under positive selection and/or func-
tional divergence were sufficient to confer PRL regulatory
ability on a HoxA-11 gene that lacks PRL regulatory functions,
we reconstructed the ancestral therian (i.e., the protein from the
last common ancestor of humans and opossum) HoxA-11 gene
expression from the decidual PRL enhancer. As expected, the
ancestral therian HoxA-11 gene, which lacks the selected and
functionally divergent amino acid sites, was unable to cooperate
functionally with FOXO1A to up-regulate PRL expression (Fig.
5). However, the ancestral eutherian HoxA-11 gene, which has
the selected and functionally divergent amino acid sites and was
reconstructed without ambiguity (Bayesian posterior probability
of 1.0), did have PRL activation ability (Fig. 5). These results
show that at least some of the amino acid substitutions in the
stem-lineage of placental mammals generated a novel functional
interaction with FOXO1A that was used to recruit PRL expres-
sion into the uterus of placental mammals.
Although protein-mediated evolution of developmental path-
ways is rarely excluded as a means of gene regulatory evolution,
the contribution of protein change to the origin of novel gene
regulatory networks is generally not considered to play a major
role in evolution. The primary argument against protein-
mediated evolution of gene regulatory networks is the negative
pleiotropic effects of mutations that are ascribed to changes in
protein-coding genes (3, 4). For example, given the multiple
functions of HoxA-11 in blood cell differentiation (43) and
and male (27, 28) and female reproductive systems (25, 27, 28),
it seems unlikely that a novel function could emerge in endo-
metrial cells without simultaneously having deleterious effects in
these other contexts. However, our data indicate that selection
acted to maintain ancestral functions during the emergence of a
novel function by recruiting amino acid sites that were previously
under weak functional constraints and thus free to acquire novel
functions. The identification of an episode of adaptive evolution
in HoxA-11 coincident with the origin of a novel function
demonstrates a clear link between adaptive protein evolution
and the emergence of a novel function. This and other examples
of functional divergence among transcription factors (7–9) in-
dicate that the evolution of proteins themselves actively contrib-
utes to the evolution of development.
HoxA-11 alone (HsaHoxA11) repressed reporter gene expression. Transfection with human FOXO1A alone (HsaFOXO1A) had no effect on reporter gene
expression. Cotransfection of human FOXO1A with human HoxA-11 (human A11) or mouse HoxA-11 (mouse A11) activated reporter gene expression.
gene expression. Cotransfection of the reconstructed ancestral eutherian HoxA-11 gene (AncEutherian A11) with the human FOXO1A gene activated reporter
gene expression, conversely cotransfection of the reconstructed ancestral therian HoxA-11 gene (AncTherian A11) with the human FOXO1A did not lead to
activation of reporter gene expression. Expression levels are shown as fold changes relative to luciferase expression in cells transfected with the reporter gene
(d332/luc3) and empty vector (pcDNA3.1). n ? 6, mean ? SEM.
Functional divergence in amniote HoxA-11 genes ability to up-regulate expression from the decidual PRL enhancer. The ability of HoxA-11 genes to
Lynch et al.
September 30, 2008 ?
vol. 105 ?
no. 39 ?
Materials and Methods
Gene Expression Surveys in Elephant, Opossum, and Chicken. Expression of PRL,
PRLR, FOXO1A, IL-6, and 20?-HSD in the pregnant (stage 32) opossum (M.
domestica) endometrium, myometrium, placenta, ovary, and fetus was as-
sayed by RT-PCR. Similarly, the expression of PRL in sexually mature (egg-
elephant (L. africana) was assayed by RT-PCR. Details are provided in the SI
Molecular Evolutionary Analysis of HoxA-11. HoxA-11 genes were identified
from BLAST searches of whole-genome databases at National Center for
Biotechnology Information, BLAT University of California at Santa Cruz, and
BLAST ENSEMBL; see ST Text for a list of included species. We used codon-
based maximum-likelihood models of coding sequence evolution imple-
mented in CODEML in the PAML 4 package of programs (49) to test for
lineages and amino acid sites under positive selection. Sites were classified as
Bayes (BEB) method with a posterior probability of ?0.90 under model A.
model (M0) and used in further analyses. To ensure convergence of the
maximum-likelihood estimates generated under the branch-specific and
branch-site models we altered the starting values of the dN/dS ratio, the
and reanalyzed the data. Difference in selective pressure between putatively
averaging the sitewise dN/dS values from those sites with the site-specific
model M3 with three rate categories. Ancestral sequences were inferred with
estimate ancestral character states, and the phylogeny shown in Fig. 2. The
Bayesian posterior probability of the reconstructed therian ancestral se-
quence (AncTherian) was 0.96, and the probability of the eutherian (AncEu-
therian) ancestral sequence was 1.0. Functional divergence was tested with
DIVERGE 2 (50). A limitation of functional divergence analyses is the require-
ment of at least four taxa per clade for estimation of the coefficient of
functional divergence (?). Given this limitation, we only tested for functional
divergence between placental mammals and birds/reptiles.
HoxA-11 Expression Vector Construction. We amplified axon 1 of HoxA-11 by
opossum (Didelphis virginiana), platypus (Ornithorynchus anatinus), and
chicken (G. gallus) by using previously described degenerate primers (29).
the human exon 2 sequence to exon 1 of the above species by using primer
overlap PCR mutagenesis; C-terminal His6tags were added by PCR mutagen-
esis. His-tagged PCR products were cloned into the mammalian expression
vector pcDNA3.1(?) (Invitrogen) and verified by sequencing. Proper expres-
sion of tagged HoxA-11 genes was checked in HeLa cells with immunocyto-
chemistry using an anti-His antibody. The ancestral therian (AncTherian) and
ancestral eutherian (AncEutherian) genes were synthesized by GeneScript
Corp. with human optimized codon usage and ligated into pcDNA3.1.
In Vivo HoxA-11 Knockdown/Xenogene Rescue. Tests of functional nonequiva-
lence were performed in a telomerase-immortalized human endometrial
stromal cell line (CRL-4003; American Type Culture Collection) treated either
with an antisense morpholino (GeneTools) generated against the human
HoxA-11 5?-UTR or a random sequence morpholino. Morpholinos were deliv-
ered by using EndoPorter (GeneTools) according to the manufacturer’s pro-
tocols. Twelve hours after morpholino treatment, cells were differentiated
with 10?9M 17?-estradiol and 10?8M medroxyprogesterone acetate, and
was rescued with either a mouse HoxA-11 or chicken HoxA-11 expression
vector. Efficacy of morpholino knockdown and rescue was determined by
and the expression of the housekeeping gene GAPDH by RT-PCR after RNA
extraction (RNeasy; Qiagen) and cDNA preparation (High-Capacity Reverse
Transcriptase kit, Applied Biosystems). RT-PCR primers were designed to span
at least one intron.
Transient Transfection and Luciferase Reporter System. HeLa cells were grown
in 24-well culture plates in DMEM supplemented with 10% FBS. At 80%
ing to the manufacturer’s instructions by using 100 ng of the dPRL luciferase
reporter construct dPRL-332/Luc3, which contains the wild-type dPRL pro-
moter sequence ?332 to ?65 relative to the human dPRL transcriptional start
of empty pcDNA3.1(?) (vehicle control) or 100 ng of the FOXO1A expression
vector pcDNA3.1(?)-FOXO1A (Addgene plasmid 13507) and 100 ng of one of
the HoxA-11 expression vectors described above. In addition, the effect of
both FOXO1A alone and HoxA-11 alone on reporter gene expression was
FOXO1A/100 ng of empty pcDNA3.1(?), or 100 ng of pcDNA3.1(?)-hHoxA-
11/100 ng of empty vector. Forty-eight hours after transfections, total mRNA
was harvested, and cDNA was prepared by using a High-Capacity Reverse
TaqMan primer/probe pair (Applied Biosystems). Each experiment was re-
peated twice, with three replicates per experiment. qRT-PCRs were also
performed in triplicate.
ACKNOWLEDGMENTS. We thank Dr. J. J. Roth for assistance in making
expression constructs and tissue culture and Dr. T. Williams for help with
immunocytochemistry. We also thank Dr. K. Smith (Duke University, Durham,
NC) for providing pregnant opossum uterus samples and Dr. D. Wildman
(Wayne State University, Detroit, MI) for providing RACE ready cDNA from
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