(Received for publication, July 20, 1995; and in revised form, August 17, 1995)
From the
Adenosine deaminase (ADA, EC 3.5.4.4) is an essential enzyme of purine metabolism that is expressed at very high levels in the murine placenta where it accounts for over 95% of the ADA present at the fetal gestation site. We have recently shown that ADA-deficient fetuses, which also lack ADA in their adjoining placentas, die during late fetal development in association with profound purine metabolic disturbances and hepatocellular impairment. We have now investigated the potential importance of placental ADA by genetically restoring the enzyme to placentas of ADA-deficient fetuses. This genetic engineering strategy corrected most of the purine metabolic disturbances, prevented serious fetal liver damage, and rescued the fetuses from perinatal lethality. Our findings suggest that placental ADA is important for murine fetal development and illustrate a general strategy for the tissue specific correction of phenotypes associated with null mutations in mice.
During mammalian development the first differentiation event gives rise to the trophectoderm, which in turn provides lineages for specialized extraembryonic cells known as trophoblasts. These cells make the physical connection between the embryo and the maternal environment and play important roles in the implantation process and placental function(1) . Inadequate placental development is associated with a high incidence of early embryonic mortality (2, 3, 4) and serious pregnancy disorders such as preeclampsia(5) . Although the formation of a functional placenta is essential for mammalian embryogenisis and fetal development, relatively little is known about the molecular mechanisms that govern trophoblast differentiation and subsequent function. Determining what proteins are produced in trophoblasts and elucidating their physiological roles is an important part of understanding how this cell lineage contributes to the formation of a functional placenta.
One such protein that is highly abundant in the murine
placenta is adenosine deaminase (ADA). ()ADA is an essential
enzyme of purine metabolism that is expressed at very high levels in
trophoblasts of the murine placenta(6, 7) . The
physiological importance of ADA in trophoblasts of the placenta is not
known. However, recent evidence suggests that ADA is essential during
fetal stages of development. ADA-deficient fetuses, which also lacked
ADA in trophoblasts of their adjoining placentas, died perinatally in
association with profound purine metabolic disturbances and
hepatocellular impairment(8, 9) . Considering that
greater than 95% of ADA enzymatic activity found in the fetal gestation
site resides in trophoblasts of the placenta, it is likely that
placental ADA plays an essential role during fetal development. Here we
show that genetically restoring ADA to placentas of ADA-deficient
fetuses rescued them from perinatal lethality, thereby providing
compelling evidence for the importance of placental ADA for fetal
development.
To assess the importance of placental ADA in fetal development we devised a strategy to genetically restore ADA to placentas associated with ADA-deficient fetuses. The first step in this process was to design and construct an ADA minigene capable of targeting ADA expression to placentas of transgenic mice at levels comparable with that of endogenous ADA. This minigene was equipped with ADA placental gene regulatory elements (12) and modified to include a 36-bp deletion in the 5`-untranslated region (Fig. 1A), a feature that enabled us to distinguish the minigene transcript from native ADA transcripts. A number of transgenic lines were generated and all showed high levels of placental expression relative to expression in adjoining fetuses, a pattern of transgene expression consistent with previous results(12) . One particular transgenic line produced minigene transcripts in placentas at levels relatively close to that seen for native ADA transcripts (Fig. 1B). Consistent with this level of minigene expression, ADA enzymatic activity was doubled in these transgenic placentas (Fig. 1C). Minigene transcripts were not detected in the adjoining fetuses, nor was there any detectable increase in ADA enzymatic activity in transgenic fetuses. Thus, the ADA minigene locus carried in this line was well suited to test our hypothesis concerning the importance of placental ADA activity in fetal development.
Figure 1:
Structure
and expression of an ADA minigene locus in placentas of transgenic
mice. A, ADA minigene construct used in creating transgenic
mice. Important features of the construct include: the full-length
murine ADA cDNA (black boxes) containing the endogenous
polyadenylation sequences, intron 11, and 2 kb of 3`-untranslated
region and 3` flank from the murine Ada gene (shaded
boxes); the ADA promoter with a 36-bp deletion in the
5`-untranslated region (
) to distinguish native ADA transcripts
from ADA minigene transcripts; and 6.4 kb of 5`-flanking sequence from
the murine Ada gene containing regulatory elements capable of
targeting expression to the placenta prenatally and the forestomach
postnatally (open box)(12) . B, RNase
protections showing native ADA and ADA minigene transcript levels in
fetuses and placentas. Eight-hour exposures revealed a
300-bp
protected band corresponding to native ADA in nontransgenic and
transgenic (Tg) placentas and a
264-bp band corresponding
to ADA minigene transcripts only in Tg placentas. Overnight exposures
revealed low levels of native ADA transcripts in fetuses. C,
ADA enzymatic activity in nontransgenic and transgenic (Tg)
fetuses and placentas. Fetuses and placentas from the same mating
described in B were isolated and assayed for ADA enzymatic
activity.
To genetically restore ADA activity to placentas
associated with ADA-deficient fetuses, mice heterozygous for a null Ada allele (8) were intercrossed with mice hemizygous
for the ADA minigene locus. Among the progeny of such matings were
animals hemizygous for the ADA minigene locus and heterozygous for the
null Ada allele. When intercrossed, these animals served as a
source of fetuses that were homozygous for the null Ada allele (ada/ada
), some of which
carried the ADA minigene locus. To determine if ada
/ada
fetuses were
rescued by the presence of the ADA minigene locus, litters from these
matings were weaned at 3 weeks of age and genotyped (Fig. 2). As
a control, we also examined the progeny of intercrosses between animals
heterozygous for the null Ada allele and lacking a minigene.
Consistent with our previous results(8) , no ada
/ada
mice were present
at weaning; however, ada
/ada
mice carrying the ADA minigene locus were detected at a
percentage suggesting a 100% rescue of these animals (Table 1).
These data suggest that expression of the ADA minigene in the placentas
of ADA deficient fetuses is sufficient to rescue them from perinatal
lethality.
Figure 2:
Detection of the ADA minigene locus in
ADA-deficient mice. A, BamHI restriction enzyme map
of a region of murine Ada wild-type allele (Wt) and
mutant allele (Mt) encompassing exons 4 through 9 and the
murine ADA minigene (Tg). The hatched box indicates
the 3.5-kb internal probe used to track these alleles on Southern
blots. B, BamHI sites; neo, neomycin-resistant gene
inserted into exon 5(8) . B, Southern blot analysis of
the Ada locus and ADA minigene locus from an intercrossing
between animals hemizygous for the ADA minigene locus and heterozygous
for the null Ada allele. Genomic DNA was isolated from tails
at weaning, digested with BamHI, and hybridized with the
internal 3.5-kb probe shown in Fig. 2A. Hybridization
fragments were 4.7 (Wt), 6.3 (Mt), and 7.2 (Tg) kb in size. Genotypes included wild type mice with the
ADA minigene locus (Tg, +/+, lanes 2, 5, 7, and 8), mice heterozygous for the null Ada allele with (Tg, m1/+, lanes 1, 3, 6, 9, and 12) and without (m1/+, lane 11) the ADA minigene locus, and mice
homozygous for the null Ada allele (ada/ada
)
and containing the ADA minigene locus (Tg, m1/m1, lanes 4 and 10).
To verify that this genetic rescue was coupled to the
restoration of ADA enzymatic activity in the placenta, we measured ADA
activity in fetuses and placentas during late fetal development (Fig. 3). No detectable ADA activity was seen in ada/ada
fetuses or
placentas. In contrast, ada
/ada
placentas containing the ADA minigene locus showed high levels of
placental ADA activity, whereas little or no ADA activity was
associated with the adjoining fetuses. The small amounts of activity
found in some of these fetuses was likely to reside in the forestomach,
since activity was not found outside the gastrointestinal tract in
adult rescued mice. (
)This pattern of expression is
consistent with the regulatory elements used (12) as well as
observations that forestomach ADA expression increases at birth (16) . The high levels of ADA normally found in the placenta,
together with placental replacement and subsequent rescue of ada
/ada
fetuses, suggest
that placental ADA plays an important role in murine fetal development.
Figure 3: ADA enzymatic activity in fetuses and placentas on 17.5 dpc. Animals hemizygous for the ADA minigene locus and heterozygous for the null Ada allele were intercrossed. Pregnant females were sacrificed on 17.5 dpc, and individual fetuses and placentas were separated and assayed for ADA enzymatic activity. Measurements were made on fetuses and placentas that were heterozygous for the null Ada allele (m1/+, n = 3) and homozygous for the null Ada allele without (m1/m1, n = 2) or with (Tg, m1/m1, n = 2) the ADA minigene locus. Values are given as means specific activities ± S.E., N.D., not detected.
Previously, we found that profound disturbances in purine metabolism
occur in ada/ada
fetuses(8) . These disturbances include marked
accumulations of the ADA substrates adenosine and deoxyadenosine as
well as increases in the levels of dATP. To determine if these
disturbances were alleviated in genetically rescued ada
/ada
fetuses, we
measured the levels of ADA substrates and dATP. Although restoring ADA
activity to the placenta had little effect on the high levels of
adenosine in ada
/ada
fetuses (Fig. 4A), deoxyadenosine levels were
lowered over 30-fold (Fig. 4B). The high dATP levels
found in fetal blood of ada
/ada
fetuses (8) was prevented by expression of the ADA
minigene in the placenta (Fig. 4C). In ada
/ada
placentas
containing the ADA minigene locus, both adenosine and deoxyadenosine
were lowered to near normal levels (Fig. 4, D and E). These data suggest that restoring ADA to placentas of ada
/ada
fetuses corrects
most of the purine metabolic disturbances seen in ada
/ada
fetuses lacking
placental ADA.
Figure 4:
Levels of ADA substrates and dATP in
fetuses and placentas on 17.5 dpc. Fetuses and placentas from matings
described in the legend to Fig. 3were collected on 17.5 dpc,
and nucleosides and nucleotides were extracted and analyzed using
reversed phase HPLC. A, fetal adenosine levels; B,
fetal deoxyadenosine levels; C, dATP levels measured in fetal
blood; D, placental adenosine levels; E, placental
deoxyadenosine levels. Measurements were made on samples that were
heterozygous for the null Ada allele with the ADA minigene
locus (Tg, m1/+, n = 4) and samples homozygous for
the null Ada allele without (m1/m1, n = 2) or
with (Tg, m1/m1, n = 2) the ADA minigene locus. Tg,
m1/+ values are essentially the same as wild type values. Values
are given as means ± S.E.; N.D., not detected at a
lower limit of detection of 0.001 nmol/mg
protein.
The onset of the purine metabolic disturbances in ada/ada
fetuses corresponds
with progressive and severe hepatocellular damage(8) . This is
the only organ visibly damaged and may ultimately be responsible for
the death of these fetuses. Severe hepatocellular damage was not seen
in livers of ada
/ada
fetuses containing restored levels of placental ADA activity
(data not shown). Our findings suggest that the fetal liver is
sensitive to the purine metabolic disturbances resulting from ADA
deficiency (8) and that the prevention of these disturbances by
genetic replacement of ADA in the placenta is likely responsible for
the rescue of these fetuses.
In the current study we show that genetically restoring ADA enzymatic activity to placentas of ADA-deficient fetuses prevents most of the purine metabolic disturbances seen in ADA-deficient fetuses. This suggests that disturbances in purine metabolism may be responsible for the liver damage and perinatal lethality seen in ADA-deficient fetuses(8, 9) . The highest levels of ADA found during fetal stages of development are in the placenta, suggesting placental ADA is playing an important role during these stages of development. A major function of this enzyme in the placenta may be to prevent the accumulation of substrates that are potentially toxic to the developing embryo.
In attempting to understand the physiological importance of
ADA, attention is invariably focused on the metabolic impact of its
substrates, adenosine and deoxyadenosine(17) . Adenosine is an
extracellular signal that elicits a vast array of physiological
responses by engaging cell surface receptors(18) . Little,
however, is known with regard to the involvement of adenosine signaling
during mammalian development. Our results show that placental
correction of ADA enzymatic activity failed to prevent the accumulation
of adenosine in the fetus, suggesting that elevated adenosine levels
are not overtly detrimental to these fetuses. The other substrate,
deoxyadenosine, is a cytotoxic metabolite that kills target cells by
interfering with deoxynucleotide synthesis and/or disrupting cellular
transmethylation reactions(17) . Interference with
deoxynucleotide synthesis is mediated by the phosphorylation of
deoxyadenosine to dATP via nucleoside and nucleotide
kinases(19) . High concentrations of dATP inhibit
ribonucleotide reductase and disrupt deoxynucleotide synthesis needed
for DNA synthesis and repair(20, 21) . Another route
of deoxyadenosine cytotoxicity involves the inhibition S-adenosylhomocysteine (AdoHcy) hydrolase, leading to the
inhibition of transmethylation reactions critical to cellular
function(22, 23) . The most striking reversal in
purine metabolic disturbances seen in rescued fetuses pertained to
deoxyadenosine metabolism, with both deoxyadenosine and dATP remaining
at near normal levels (Fig. 4, B and C). High
levels of nucleoside kinases and AdoHcy hydrolase are found in rodent
livers(24, 25) , suggesting that either of these
metabolic pathways may be involved in the hepatocellular damage seen in
ADA deficient fetuses. Recent studies have shown that AdoHcy hydrolase
activity is inhibited in tissues of ADA-deficient fetuses ()and tissues of newborn ADA-deficient pups(9) ,
suggesting that disturbances in transmethylation reactions may be
involved in the phenotypes seen. Based on the overall metabolic
findings presented here, we hypothesize that deoxyadenosine
cytotoxicity likely provides the metabolic basis for the liver damage
and subsequent perinatal lethality of ADA deficient fetuses.
Naturally occurring null alleles of the Ada gene have been
observed in the human population and are associated with immune
deficiency(17) . In contrast to what we observed in mice,
ADA-deficient human fetuses survive prenatal development and are not
known to suffer significant hepatocellular damage, although mild liver
findings have been noted(17) . These differences may suggest
that there is a greater need for ADA during mouse prenatal development.
Genetic restoration of ADA in the placentas of ada/ada
fetuses allows them
to survive prenatal development, providing the opportunity to
investigate whether postnatal mice develop phenotypes similar to those
seen in humans. Preliminary observations suggests that rescued ADA
deficient animals exhibit lymphopenia and immune deficiency.
ADA is expressed at high levels at three different places and times during the murine life cycle: first in the trophoblasts of the placenta during prenatal development(6, 7) , next in the gastrointestinal epithelium of the adult(16) , and then in the deciduum of the pregnant uterus(6) . The lack of ADA in the first of these places, the placenta, results in a phenotype that precludes our ability to investigate its importance in adult tissues. Placental expression of an ADA minigene on an ADA-deficient background allowed for survival through the prenatal phenotypic bottleneck and provided adult mice that can now be used to assess the role of ADA in adult tissues such as the gastrointestinal tract, the deciduum, and the immune system.