Catecholaminergic neurons, which include dopaminergic,
noradrenergic, and adrenergic neurons, are located in discrete regions
in the central nervous system and have an important role in a wide
range of brain functions, such as locomotion, behavior, sleep, memory,
and learning. In the peripheral tissues, sympathetic neurons are
noradrenergic, and adrenomedullary chromaffin cells produce
noradrenaline and adrenaline as hormones. During vertebrate
development, catecholaminergic phenotypes are generated from a given
neuronal lineage. The primordial catecholaminergic neurons appear in
the intermediate zone of the neural tube at the early embryonic stage
(reviewed in (1) ), and migrating neural crest cells are
committed to the sympathoadrenal lineage that gives rise to chromaffin
cells of the adrenal gland and sympathetic neurons ((2) ;
reviewed in (3) ).
Tyrosine 3-hydroxylase (TH, (
)EC 1.14.16.2) is a monooxygenase that catalyzes the
conversion of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and
requires tetrahydropteridine as a cofactor(4) . Because it is
generally thought that the reaction of TH is the initial and
rate-limiting step of the catecholamine biosynthetic
pathway(5) , the enzyme would appear to play a central role in
the regulation of catecholamine production. TH activity is modulated by
transcriptional and post-translational mechanisms in response to
changes in the environment and to neuronal and hormonal stimuli. The
most acute regulation of TH activity occurs through post-translational
modification of the protein via phosphorylation (reviewed in (6) ). Also, various factors, including cyclic AMP, phorbol
esters, glucocorticoids, nerve growth factor, and epidermal growth
factor, enhance TH activity through transcriptional activation of the
TH gene(7, 8, 9, 10, 11) .
Alteration in expression or activity of TH is involved in the
pathogenesis of certain disorders of catecholaminergic neurons
including Parkinson's
disease(12, 13, 14) . Also, Leboyer et
al.(15) have shown that the gene for susceptibility to
manic-depressive illness is positively associated with the TH locus,
although there are conflicting results of linkage analysis that ruled
out defects in the TH gene in this
illness(16, 17, 18, 19) . On the
other hand, a null mutation (pale) in the fruit fly TH locus
is known to affect pigmentation of the cuticle and production of the
catecholamine neurotransmitter, leading to lethality at the larval
instar stage(20, 21) .
Recent advances in the
techniques of mouse genetics provide an excellent system for studying
development and function of the mammalian nervous system and for
generating animal models for several neuronal disorders. If we could
overproduce or deplete catecholamines by genetically manipulating TH
levels, it would be possible to investigate their neuronal and hormonal
functions in animal development and physiology. Previously, we produced
transgenic mice carrying multiple copies of an entire human TH gene, in
which the transgene was tissue specifically expressed with an increase
in TH activity in various catecholamine-containing
tissues(22) . An alternative approach is to disrupt the TH gene
via homologous recombination in mouse embryonic stem (ES) cells. In
this present study, we generated mice with a null mutation in the TH
locus to induce depletion of catecholamines and to investigate the
resultant phenotypic effects on the animals. Also, we examined the
influence on catecholamine metabolism of decreased TH levels in mice
heterozygous for the mutation.
EXPERIMENTAL PROCEDURES
Construction of Targeting Vector
Phage clones
containing the mouse TH gene were isolated from a DBA/2J genomic DNA
library(23) . The targeting vector, pTNH, extended from a XhoI site located 0.3 kilobase (kb) upstream of exon 1 to a SphI site located in exon 13, in which the 0.3-kb AccI-AccI fragment containing the 3`-portion of exon
7, intron 7, and the 5`-portion of exon 8 was replaced by the
phosphoglycerate kinase-neo gene cassette (24) with
the neo gene in the same transcriptional orientation as in the
mouse TH gene. A herpes simplex virus-thymidine kinase gene cassette (25) and a diphtheria toxin A-fragment gene cassette (26) driven by the MC1 promoter were used to flank the
respective 5`- and 3`-ends of the targeting vector.
ES Cell Culture and Chimera Production
E14 ES
cells (27) were cultured on feeder layers prepared from
neomycin-resistant STO cells in Dulbecco's modified Eagle's
medium supplemented with 20% fetal calf serum, 1000 units/ml leukemia
inhibitory factor, 1
nonessential amino acids, 0.1 mM
-mercaptoethanol, and 1 mM sodium pyruvate. ES cells (5
10
) were electroporated with the targeting vector
linearized at a unique SalI site and were selected in medium
containing 0.4 mg/ml G418 alone or together with 2 µM ganciclovir. Individually picked colonies were analyzed by
Southern blot hybridization with a 0.5-kb SphI-BstPI
fragment containing a 3`-external region of the targeting vector (probe
A) and with a 0.6-kb PstI-PstI fragment containing
the neo gene (probe B), both labeled with
P. ES
cells with a targeted allele were injected into C57BL/6J blastocysts,
which were implanted into the uterine horns of pseudopregnant mothers
as described in (28) . Male chimeras were mated with C57BL/6J
or MCH(ICR) females, and germline transmission was judged by the coat
color of the offspring. Mice heterozygous for the TH mutation were
identified by Southern blot hybridization of tail DNA and were used to
obtain homozygous mutant mice.
Introduction of a Human TH Transgene into the Homozygous
Mutants
hTH-1 transgenic mice carrying the entire human TH gene (22) were crossed to TH
heterozygous
mice. The offspring heterozygous for the TH mutation and hemizygous for
the human transgene were identified by Southern blot hybridization of
tail DNA, and they were further intercrossed with TH
heterozygous mice. Detection of the transgene was performed with
a 2.4-kb XhoI-XhoI fragment containing exons
4-10 and their surrounding regions of the human TH gene (29) as a probe.
RNA Analysis
Total RNA was prepared from head and
body of E12.5 mouse embryos and was subjected to reverse
transcription-polymerase chain reaction (RT-PCR) analysis as described
previously(30) . Primer A (5`-TCCCCAAGGTTCATTGGACG-3`,
nucleotides 96-115 of the mouse TH cDNA sequence from (23) ) and primer B (5`-GGTACCCTATGCATTTAGCT-3`, nucleotides
1497-1516 of the same sequence) were used for detection of TH
mRNA (see Fig. 1A). Also, the mixed primer set
containing primers C-E was used to detect differentially the mouse and
human TH mRNAs in the genetic rescue experiment (see Fig. 6B). Primer C (5`-ACTGCTGCCACGAGCTGCT-3`) is
common to the mouse and human TH cDNA sequences (nucleotides 989-1008).
Primer D (5`-TCAGGGACGCCGTGCACCTA-3`) is complementary to nucleotides
1701-1719 of the mouse TH sequence, and primer E
(5`-TAGAATACAGCATGAAGGG-3`) is complementary to nucleotides
1493-1512 of the human TH sequence.
Figure 1:
Disruption of the mouse TH locus by
gene targeting. A, structure of the targeting vector, genomic
structure of the wild-type TH allele, and predicted structure of the
mutant allele resulting from homologous recombination are shown. Exons
are represented by filled boxes and are numbered. The
sizes of diagnostic restriction fragments and locations of probes for
Southern blot analysis are shown. Probe A is a 0.5-kb SphI-BstPI fragment containing a region around the
3`-end of exon 13, and probe B, a 0.6-kb PstI-PstI fragment containing the neo gene. Arrowheads indicate primers used for RT-PCR analysis to detect
expression of TH mRNA (see Fig. 2A). B,
Southern blot analysis of ES cell DNA. Genomic DNA (8 µg) prepared
from a representative targeted clone, TH1-13, and from
untransfected E14 ES cells was digested with BstPI or EcoRI, electrophoresed, Southern blotted, and hybridized with
probe A and probe B. The sizes of the hybridizing restriction fragments
are shown. C, genotype analysis of the E12.5 embryos obtained
by intercrossing mice heterozygous for the mutated TH allele. Genomic
DNA (8 µg) prepared from the yolk sac of the E12.5 embryos was
digested with BstPI and analyzed by Southern blot
hybridization with probe A. The genotype of the wild-type
(+/+), heterozygous (+/-), and homozygous mutant
(-/-) mice is indicated on the top of each
lane.
Figure 6:
Genetic rescue of the
TH
homozygous mice. A, Southern blot
analysis of tail DNA from the offspring obtained by mating the mice
heterozygous for the TH mutation and hemizygous for the human TH
transgene to the heterozygotes. Tail DNA (8 µg) was digested with BstPI for genotyping of the mouse TH locus and analyzed by
Southern blot hybridization with probe A (upper panel). For
detection of the human TH transgene, tail DNA was digested with XhoI, and the filter was hybridized with a 2.4-kb XhoI-XhoI fragment containing the human TH gene (lower panel). The genotype of each individual F2 mouse is
indicated between the two panels. The rescued mutants correspond to
individuals shown in lanes 2 and 6. B,
detection of human TH mRNA in the rescued mutants. Total RNA was
isolated from brain and adrenal glands of wild-type mice (lanes 3 and 6), hTH-1 transgenic mice (lanes 4 and 7), and rescued mutants (lanes 5 and 8) and
subjected to RT-PCR analysis. PCR amplification was carried out with
the mixed primer set containing primers C-E as described under
``Experimental Procedures.'' Mouse TH cDNA (lane 1)
and human TH cDNA (lane 2) were added in the reaction mixture
of PCR as controls. The combination of primers C and D generates a
732-bp band corresponding to the mouse TH cDNA fragment, and the
combination of primers C and E, a 633-bp band corresponding to the
human TH cDNA fragment. C and D, recovery of TH
activity in tissues of the rescued mutants. Brain (C) and
adrenal glands (D) were dissected from adult mice and were
subjected to the TH assay. Values are mean ± S.E. of the data
obtained from three independent animals. Closed columns,
wild-type mice; hatched columns, hTH-1 transgenic mice; open columns, rescued mutant mice.
Figure 2:
Expression of transcripts and proteins
from the TH locus in the wild-type (+/+), heterozygous
(+/-), and homozygous mutant (-/-) embryos. A, detection of TH mRNA. Total RNA was isolated from head and
body regions of the E12.5 mouse embryos and subjected to RT-PCR
analysis. PCR amplification was carried out with either the TH primer
set (upper panel) or the mouse
-actin primer set (lower panel). The size of PCR products was 1.5 kb for the TH
primer set and 1.2 kb for the
-actin set. B, measurement
of TH activity. Homogenates prepared from head and body regions of the
E12.5 mouse embryos were applied to TH assay. Values represent the mean
± S.E. of the data obtained from three to six embryos. ND, not detected. Asterisks show significant
difference (p < 0.05) from the wild-type levels
(Student's t test).
Biochemical Analysis
Head and body of the E12.5
mouse embryos or neonates were homogenized in 3 volumes of 0.32 M sucrose containing 20 mM Tris-HCl buffer (pH 7.3), 1
mM EDTA, 1 mM dithiothreitol, 1 µg/ml leupeptin,
1 µg/ml pepstatin, and 0.2 mM phenylmethylsulfonyl
fluoride. Tissues removed from adult male mice (14 weeks old) were
homogenized in 9 volumes of the same buffer. Based on the measurement
of DOPA formed from tyrosine, TH activity was determined by high
performance liquid chromatography with electrochemical detection
(HPLC-ECD) as described(31) . Catecholamine levels in the
homogenates were measured with an automatic HPLC system based on a
fluorimetric method(32) .
Histological Analysis
Neonates were immersed
overnight in 4% paraformaldehyde in 0.1 M sodium phosphate
buffer (pH 7.4). Frozen sections (40 µm) were cut through a
sagittal plane with a cryostat. Immunohistochemical staining was
carried out with either anti-TH antibody (1/10,000 dilution) or
anti-L-aromatic-amino-acid decarboxylase (AADC) antibody (1/10,000
dilution) by the avidin-biotin complex procedure as detailed elsewhere (33) .
Electrocardiographic Analysis
Electrocardiograms
were obtained from newborn mice 1 h after they had been delivered by
caesarean section. Electrodes were attached to the fore and hind limbs
of the neonates, and surface electrocardiograms were monitored with an
8-channel oscilloscope using a high-gain preamplifier (Nihon Koden,
AB621C). Heart rates of the neonates were determined by calculation
based on the electrocardiographic data.
RESULTS
Generation of Mice with a Mutated TH Gene
To
introduce a mutation into the TH locus by homologous recombination in
ES cells, we used a modified procedure that combines two kinds of the
positive-negative selection strategy as described by Mansour et al.(25) and Yagi et al.(26) . The targeting
vector (Fig. 1A) included 8 kb of genomic TH sequences
consisting of the 5`-homologous region of 5.8 kb and 3`-homologous
region of 2.2 kb, in which a 0.3-kb AccI-AccI
fragment containing the 3`-portion of exon 7, intron 7, and 5`-portion
of exon 8 was replaced with a phosphoglycerate kinase-neo gene
cassette. For negative selection of random integration, the vector also
contained a herpes simplex virus-thymidine kinase gene cassette and a
diphtheria toxin A-fragment gene cassette at the respective 5`- and
3`-ends of the homologous regions. The linearized targeting vector was
introduced by electroporation into E14 ES cells, and selection was
carried out with G418 alone or together with ganciclovir. Targeted
clones were identified by Southern blot hybridization of genomic DNA
with probe A, containing the 3`-external portion of the homologous
region, and with probe B, containing the neo gene. As shown in Fig. 1B, the wild-type TH allele displayed a 4.5-kb BstPI band and a 20-kb EcoRI band when hybridized
with probe A, whereas the mutant allele gave a 6.3-kb BstPI
band and a 6.4-kb EcoRI band. Also, probe B detected only the
mutant allele in the targeted clone. The frequency of homologous
recombination was 9 out of 83 G418-resistant clones, and 13 out of 82
clones doubly selected with G418 and ganciclovir. We injected several
targeted ES clones into C57BL/6J mouse blastocysts and obtained
germline chimeras from two independent clones, designated TH1-13
and TH2-55. The offspring of the germline chimeras obtained from
the TH1-13 clone were used in this study.
TH
homozygous mice derived from the chimeras
generated with the TH2-55 clone displayed the same mutant
phenotype as described below.
Perinatal Lethality in TH
Homozygous Mice
Mice heterozygous for the TH
mutation appeared to be normal and were fertile. However, we could not
find viable homozygous mice when the TH
heterozygous mice were intercrossed and their progeny were
genotyped at 3 weeks of age (Table 1). To examine the lethality
of the TH
homozygous mutation, we genotyped
embryos at various stages of gestation and neonates obtained from the
intercross of heterozygotes (Table 1). A typical pattern of the
genotyping results is shown in Fig. 1C. Development of
TH
homozygous embryos was grossly normal up
to 12.5 days of gestation (E12.5). From E14.5 to E16.5, 55-57% of
the homozygous embryos died and became degraded, showing a varied
extent of degradation according to postmortem time, and the surviving
homozygous embryos were apparently normal. When the offspring were
delivered by caesarean section at E18.5, 75% of the homozygous mutants
were found to have been degraded and absorbed as a consequence of
death, and the remaining mutants were indistinguishable from the
wild-type and heterozygous littermates based on size, shape, and body
weight. The wild-type and heterozygous neonates as well as some of the
surviving homozygous neonates (19% of the homozygous mutants) began to
breathe and turned pink in color within 10 min. However, the remaining
homozygous neonates (6% of the homozygous mutants) failed to breathe,
responded poorly to a tapping stimulus, and died of anoxia within 10
min after birth. Subsequently, the offspring delivered by caesarean
section were cross-fostered by breeding females. The viable homozygous
neonates appeared normal within 5-6 h after birth, but they
finally died by postnatal day 1 (P1), although the symptomatic
difference was not clear. Several homozygous neonates were immediately
fixed after death and were histologically checked for abnormalities in
major organs including brain, lung, heart, liver, spleen, pancreas,
kidney, and adrenal gland. In the mutants that died at the time of
birth, the lung was atelectatic with no structural block in the
bronchi, bronchioles, and upper respiratory tract, and many cells in
the heart, liver, and kidney showed signs of degeneration possibly due
to nonspecific reactions after the death of these animals, although
there was no gross deformity in any of the organs we examined (data not
shown). This suggests that some pathological changes occurred in these
mutants at the late gestational stage and led to their inability to
breathe at birth. Moreover, in the mutants that died during nursing by
the foster mothers we could not find any particular histopathological
pictures in the examined organs that could explain the primary cause of
the neonatal death in these animals (data not shown). The animal
lethality in the TH mutants does not seem to have resulted from a
structural failure in the major organs.
Analysis of TH mRNA, Enzyme Activity, and Catecholamine
Content in the Mice
Preliminary RT-PCR and immunohistochemical
experiments on the mouse embryos showed expression of TH beginning by
E8.5. To confirm a lack of TH mRNA in the homozygous mutants, we
performed RT-PCR analysis with total RNA prepared from the E12.5
embryos. The amplification with the TH primer set detected a single
band of 1.5 kb in the head and body regions of the wild-type and
heterozygous mice, whereas the homozygous mutants lacked this band in
both regions (Fig. 2A). Subsequently, we assayed TH
activity of the embryos taken at the same gestational day (Fig. 2B). In the heterozygous mice, TH activities were
decreased to 49.5% in the head and 33.9% in the body of the values for
the wild-type mice, whereas no activity was detected in these regions
of the mutants.To determine the effects of the TH mutation on
catecholamine metabolism, we initially measured dopamine,
noradrenaline, and adrenaline levels in the E12.5 embryos (Table 2). In the heterozygous embryos, the dopamine level was
reduced to 66.7% in the head and 69.2% in the body of the values for
the wild-type embryos; and the noradrenaline level, to 89.5% in the
head and to 90.7% in the body of the wild-type values. There was only a
small amount of adrenaline detected and only in the body region of the
wild-type and heterozygous embryos. In contrast, the E12.5 homozygous
mutants showed no detectable noradrenaline and adrenaline levels, but
they had a dopamine level of 37.5% in the head and 7.7% in the body of
the wild-type values. Subsequently, we compared catecholamine levels in
the neonates delivered by caesarean section among the three kinds of
mice (Table 2). In the wild-type mice, the dopamine,
noradrenaline, and adrenaline levels detected in the neonates were much
higher than the corresponding levels in the E12.5 embryos. As compared
with the wild-type levels, there was a tendency for the levels of the
three kinds of catecholamine to decrease in the heterozygous mice, and
the difference was significant for the noradrenaline and adrenaline
levels in the head region. We observed more extensive change in the
catecholamine levels in the homozygous mutants, where the noradrenaline
and adrenaline levels were quite low in both head and body regions, and
the dopamine level was only 23.0% in the head and 41.7% in the body of
the wild-type levels.
Analysis of TH Activity and Catecholamine Content in
Tissues of the Adult Wild-type and Heterozygous Mice
To study
the effects of the mutation on the biochemical parameters in the
tissues of TH
heterozygous mice at adulthood
(14-16 weeks old), we measured TH activity and catecholamine
level in the whole brain and adrenal glands of the mice. In the
heterozygous mice, TH activities were significantly decreased to 37.7%
in the brain and 37.9% in the adrenal glands as compared with the
corresponding values of the adult wild-type mice (Fig. 3, A and B). On the other hand, the dopamine, noradrenaline,
and adrenaline levels in the brain of the heterozygous mice were
similar to the corresponding levels of the wild-type brain (Fig. 3C). Also, in the adrenal glands, noradrenaline
or adrenaline showed similar values between the wild-type and
heterozygous animals (Fig. 3D). These results indicate
that catecholamine contents in the tissues of the adult heterozygous
mice are normally maintained in spite of a significant decrease in TH
activity.
Figure 3:
TH activity and catecholamine accumulation
in tissues of adult wild-type and heterozygous mice. Tissues were
dissected from adult mice (14 weeks old), homogenized, and subjected to
TH assay and catecholamine analysis. A, TH activity in brain; B, TH activity in adrenal glands; C, catecholamine
level in brain; D, catecholamine level in adrenal glands.
Values indicate mean ± S.E. of the data obtained from six mice. Closed columns, wild-type mice; hatched columns,
heterozygous mice. Asterisks show significant difference (p < 0.05) from the wild-type value (Student's t test).
Morphology of the TH
Homozygous Mice
To determine whether a loss of TH
function would intrinsically affect development of the cells that
normally express TH, we performed an immunohistochemical analysis with
sections prepared from the newborn mice. Aromatic L-amino-acid
decarboxylase (AADC; EC 4.1.1.28), which catalyzes the second step of
the catecholamine pathway, was used as a marker of the cells that
normally produce catecholamines. As shown in Fig. 4, A and B, in the wild-type mice, TH and AADC
immunoreactivities were colocalized in the cell bodies in the midbrain
(substantia nigra or A9 nucleus and ventral tegmental area or A10
nucleus) and in the dense axon fibers originating from these nuclei. In
the midbrain of TH
homozygous mutants, the
number and distribution of the AADC-positive cells were similar to
those in the wild-type mice (Fig. 4D), although no TH
immunoreactivity was detected in the mutants (Fig. 4C).
Similar results were observed in other brain regions including
olfactory bulb, frontal cortex, thalamus, hypothalamus, and pons
medulla (data not shown). In the peripheral tissues of the wild-type
mice, TH immunoreactivity was localized in a large number of
sympathetic ganglion, trunk, and nerve plexus, as well as in the
medullary cells of the adrenal glands, showing colocalization with AADC
immunoreactivity. In the mutants, development of the AADC-positive
cells that lacked TH immunoreactivity was apparently unaffected in the
peripheral tissues as was also observed in their brain regions. The
results of immunostaining in thoracic ganglion (Fig. 4, E-H) and adrenal gland (Fig. 4, I-L) as representative tissues are shown. In addition,
similar immunohistochemical analysis with the E12.5, E14.5, and E16.5
embryos showed normal development of AADC-positive cells in the
surviving mutants (data not shown).
Figure 4:
Histological analysis of the wild-type and
homozygous mutant mice. Sections were prepared from the neonates
delivered by caesarean section at E18.5 and were stained with either
anti-TH antibody or anti-AADC antibody. Light microscopic images of the
midbrain (A-D), thoracic ganglion (E-H), and adrenal gland (I-L) of wild-type and homozygous mutant mice
are shown. Scale bar: 200 µm.
Effects on Cardiac Functions
Our observations that
there is no apparent histopathological change in the
TH
homozygous mice suggest some defects in
physiological functions of catecholamines as a specific cause of death
in the mutants. Catecholamines are known to be involved in the
regulation of cardiovascular, metabolic, and respiratory functions in
developing animals. Among these autonomic functions, we considered that
alteration of cardiovascular system in the mutants might be associated
predominantly with the animal lethality during prenatal and neonatal
stages. To examine influences on cardiac functions by the TH mutation,
we monitored electrocardiograms of newborn mice obtained from the
intercross of the TH
heterozygous parents. As
shown in Fig. 5A, the surface electrocardiograms showed
similar wave morphology among the wild-type, heterozygous, and
surviving mutant neonates, although there was a slight elevation of the
S-T segment in the mutants. When heart rate was evaluated by
calculation based on the electrocardiographic data, the value in the
mutants (210 ± 17 beats/min) was remarkably decreased as
compared with the wild-type (307 ± 9 beats/min) and heterozygous
(298 ± 8 beats/min) values, showing bradycardia in the mutant
neonates (Fig. 5B). These results suggest that
depletion of catecholamines in the mutants causes altered cardiac
functions, which may lead to the morbidity of the animals.
Figure 5:
Electrocardiographic analysis of the
newborn mice. A, surface electrocardiograms of the wild-type
(+/+), heterozygous (+/-), and homozygous mutant
(-/-) mice. Neonates delivered by caesarean section at
E18.5 were subjected to electrocardiographic recording. Typical
recording patterns are shown. P, Q, R, S, and T waves are indicated in the electrocardiogram
obtained from the wild-type neonates. B, heart rate calculated
from the electrocardiographic data. Values are mean ± S.E. of
the data. The asterisk denotes a significant difference from
the wild-type neonate value (p <
0.01).
Genetic Rescue of the Mutant Phenotype
To confirm
that the mutant phenotype observed was derived from the disruption of
the mouse TH locus without effects on other chromosomal positions in
the ES cell genome, we introduced a human TH transgene into the
TH
homozygous mice. By crossing hTH-1
transgenic mice carrying the human TH gene with the
TH
heterozygous mice, we obtained offspring
that possessed the transgene in addition to the mouse TH locus
heterozygous for the mutation, and they were further intercrossed to
the heterozygous mice. When tail DNA of F2 progeny (3 weeks old) was
analyzed by Southern blot hybridization, the mice identified as
homozygous for the TH mutation and with multiple copies of the human TH
transgene were viable (Fig. 6A). These rescued mutants
appeared normal and remained healthy for at least 5 to 6 months. To
examine the expression of human TH mRNA in these animals, we performed
RT-PCR analysis with the mixed primer set described under
``Experimental Procedures,'' which detects differentially
mouse and human TH mRNAs (Fig. 6B). When RNA samples
prepared from wild-type brain (lane 3) and adrenal glands (lane 6) were used, the amplification gave a single band of
732 bp corresponding to mouse TH mRNA. In the same tissues of
transgenic mice, the amplification detected another band of 633 bp
corresponding to human TH mRNA in addition to the 732-bp band (lanes 4 and 7). On the other hand, only a single
band of 633 bp was detected in the tissues of the rescued mutants (lanes 5 and 8). Also, assay of TH activity in the
brain and adrenal glands showed that the enzyme activity was actually
recovered in the rescued mutants and that the level in each tissue was
approximately equivalent to the difference in the activities between
transgenic and wild-type mice (Fig. 6, C and D). The catecholamine levels in the tissues of the rescued
mutants were similar to those in the wild-type mice at adulthood (data
not shown). These results indicate that the human transgene can
basically replace the function of the mouse TH gene.
DISCUSSION
The mouse TH locus of ES cells was mutated by gene targeting
with a positive-negative selection strategy. Disruption in exons 7 and
8 of the gene led to a lack of TH mRNA and enzyme activity, which was
accompanied by a significant decrease in catecholamine levels. This
resulted in animal lethality during the perinatal period. Analysis of
surface electrocardiograms of the newborn mice showed bradycardia in
the surviving mutants, suggesting alteration of cardiac functions in
these animals, which may explain the cause of the mutant death. In
addition, the lethality in the TH
homozygous
mice was negated by introducing a human TH transgene, and the same
phenotypic change in the homozygous mutants was obtained from two
separate ES clones. These facts support our contention that the mutant
phenotype can be attributable only to a mutation in the mouse TH locus
by a homologous recombination event. Our results demonstrate that TH is
required for survival of the animals during the late gestational stage
of embryonic development and after birth.
Recently, Zhou et al.(34) have reported disruption of the mouse TH locus by
gene targeting, which resulted in embryonic lethality apparently due to
cardiovascular failure, and rescue of the mutant mice in utero by DOPA administration to pregnant females. Our data reported here
support their conclusion that catecholamines are essential for mouse
fetal development and postnatal survival. However, our results include
the effects of the TH mutation on catecholamine metabolism, development
of catecholaminergic system, and cardiac functions, which were not
definitely characterized in their study. Also, there is a difference in
the results of pathohistological analysis between the two studies as
discussed below. Moreover, we used a transgenic rescue system with the
human TH gene to recover the mutant phenotype.
Defect in Catecholamine Synthesis Caused by the TH
Mutation
Analysis of catecholamines in the mouse embryos and
neonates revealed a great decrease in the levels of dopamine,
noradrenaline, and adrenaline in the TH
homozygous mutant mice, although the extent of decrease was different
among the three kinds of catecholamine. Especially, the noradrenaline
and adrenaline levels were undetectable or quite low in the mutant
mice. In the mouse embryos, a small amount of catecholamines might be
derived from maternal circulating catecholamines, although placental
transfer of catecholamines is known to be inefficient(35) .
Because we never detected expression of the TH mRNA and enzyme activity
in the mutants, changes in the catecholamine level observed would
reflect a defect in catecholamine synthesis via the TH reaction. Interestingly, the amount of dopamine that accumulated in the
homozygous mice was relatively higher in contrast to that of
noradrenaline and of adrenaline. Our observations suggest the existence
of an alternate mechanism for producing catecholamines, predominantly
dopamine, one that is not mediated by the TH reaction. Early studies
showed an enzyme activity that forms catechols from monophenols and
suggested a possible alternative pathway of catecholamine synthesis
based on the substrate specificity of several
enzymes(36, 37) . According to this hypothesis,
tyrosine is decarboxylated to tyramine by AADC activity, and the
conversion of tyramine to dopamine is catalyzed by the catechol-forming
enzyme. However, the rate of decarboxylation for tyrosine is much lower
than that for DOPA and 5-hydroxytryptamine (38) , and the
catechol-forming enzyme has not been characterized sufficiently yet.
Therefore, this possible pathway could not explain our observations. On
the other hand, Eisenhofer et al.(39) reported that
DOPA is present in plasma, where its levels are higher than those of
circulating catecholamines. Because plasma DOPA is probably transferred
from the maternal circulation to the fetuses across the placenta, it
might be decarboxylated to dopamine by AADC in the fetal tissues. AADC
is present in a wide range of non-neuronal tissues in addition to being
found in catecholaminergic and serotonergic neurons(40) . One
group of cells in such tissues is known as amino acid precursor uptake
and decarboxylation (APUD) cells, which include neuroendocrine cells in
lung and chromaffin cells in the small
intestine(41, 42) . High AADC activity is also found
in the liver and kidney. Actually, it is known that plasma DOPA is
converted to dopamine in the kidney(43) . There might be a
possible pathway for dopamine synthesis from circulating DOPA in such
non-neuronal tissues. However, this mechanism would not lead to an
efficient synthesis of noradrenaline and adrenaline, because dopamine
-hydroxylase and phenylethanolamine N-methyltransferase
are not expressed in these non-neuronal tissues except for a transient
expression of dopamine
-hydroxylase in several tissues, such as
pancreas and gut, during a midgestational stage of mammalian
development(44, 45, 46) . Also, there is a
possibility that the small amount of noradrenaline and adrenaline
detected in the TH mutants might be produced in the catecholaminergic
cells with DOPA incorporated into these cells from circulation. To
understand the alternate mechanism of dopamine synthesis in the
developing animals, we must carry out a detailed analysis of
catecholamine metabolism in these TH mutants. One possible approach is
to histologically identify the cells that have accumulated dopamine in
the TH
homozygous mice.
Adaptation of Catecholamine Accumulation during
Development
In the E12.5 embryos and adult tissues of the
TH
heterozygous mice, TH activities were less
than 50% of the corresponding values of the wild-type mice. Because TH
activity measured in our study is represented as the V
value under optimal conditions and reflects the amount of TH
protein, the results indicate that the expression level of TH protein
is affected by the gene dose. On the other hand, catecholamine levels
accumulated in the E12.5 embryos and neonates heterozygous for the TH
mutation were decreased moderately as compared with the corresponding
wild-type levels, although the extent of decrease in catecholamine
level was lower than that in TH activity. At adulthood, catecholamine
levels in the brain and adrenal glands of the heterozygous mice were
similar to the corresponding levels of the wild-type mice. These
observations suggest that there is the compensatory mechanism to
regulate catecholamine levels during animal development in spite of a
decrease in the amount of TH protein and that catecholamine
accumulation is adapted to the normal level by adulthood. There are two
possible mechanisms to explain the difference observed in changes in
the TH protein amount and catecholamine level of the heterozygotes. One
is the regulatory mechanism of in vivo TH activity. Activation
of TH is associated with phosphorylation of the pre-existing enzyme by
several protein kinases including cyclic AMP-dependent protein kinase,
calmodulin-dependent protein kinase II, and
Ca
/phospholipid-dependent protein
kinase(47, 48, 49) . In the heterozygous
mice, in vivo TH activity might be enhanced through such
post-translational modification to bring catecholamine accumulation to
the normal levels. Another mechanism is a reduction in catecholamine
turnover, which depends on the activities of monoamine oxidase and
catechol-O-methyltransferase. A decrease in the activities of
these enzymes in the heterozygotes might lead to a reduction in the
degradation rate of catecholamines.
Apparently Unaffected Development of Catecholaminergic
System in the TH Mutants
Immunohistochemical studies with
antibodies raised against catecholamine-synthesizing enzymes have shown
the appearance of dopaminergic, noradrenergic, and adrenergic
phenotypes in the brain, sympathetic ganglia, and adrenal glands during
rodent embryogenesis (50, 51, 52, 53, 54, 55) .
Our immunohistochemical analysis of the TH
homozygous mice demonstrates that development of the cells
normally showing catecholaminergic phenotypes is apparently unaffected
by the TH mutation, at least as far as the mutants can continue to
develop during the prenatal stage. A significant decrease in
catecholamines as a consequence of the TH mutation does not seem to
intrinsically influence morphogenesis of these cell types. Also, TH
immunoreactivity appears in the developing sensory ganglia,
parasympathetic ganglia, gut, and pancreas at the midgestational stage
of the rat or mouse embryos and then disappears during the subsequent
development(44, 45, 46, 56, 57, 58) .
In this study we did not observe any visible change in morphology of
these tissues in the TH
homozygous mutants
(data not shown), suggesting that the different cell types that
transiently express TH in the wild-type embryos develop normally in
spite of a loss of TH function.
Significance of Catecholamine Functions in the Developing
Embryos and Neonates
The TH
homozygous
mice showed lethality past the E12.5 stage with an increase in the
mortality rate during the progression of development. Several mutant
mice were viable at the time of birth, but they soon died, by P1.
During prenatal and neonatal stages of rodents, the central and
sympathetic neurotransmission is not fully functional due to immaturity
of these nervous systems, and the adrenal glands are thought to be
relatively more important in the catecholamine functions at that
time(59, 60) . In our analyses, adrenomedullary
chromaffin cells appeared in the developing adrenal glands of the mouse
embryos between E10.5 and E12.5, the appearance of which was followed
by a great increase of adrenomedullary catecholamines around E14.5.
This time agrees with the developmental stage at which we first found
lethality in the TH
homozygous mice. These
facts suggest the possibility that the lethality in the TH mutants is
attributable to a defect in circulating catecholamines derived from the
adrenal glands.Zhou et al.(34) proposed a failure
in the cardiovascular system as a possible cause of embryonic lethality
in their TH mutants based on the histological abnormalities including
congestion of blood and unusual pictures of cardiomyocytes, as well as
on bradycardia that was characterized by counting the heart beating of
E12.5 embryos in buffered saline. In contrast, we could not detect any
histopathological change in our mutant embryos and neonates that could
explain the primary cause of the lethality. In several mutants, we
actually observed morphological changes in the heart including dilated
atria and disorganized cardiomyocytes, but these changes seemed to be
due to nonspecific reactions occurring after death. On the other hand,
our data on cardiac functions were based on surface
electrocardiographic analysis of newborn animals and revealed a
significant decrease in heart rate in the surviving mutants, supporting
the possibility that catecholamines are required for regulating the
frequency of the spontaneous firing of the heart. Additionally, we
observed a subtle elevation of the S-T segment in the
electrocardiograms of the mutants, which might reflect modulation in
the phase of repolarization. Based on our results, it is difficult to
determine the specific cause of the lethality in the TH mutants with
the histological observations. The cause of the mutant death might well
be explained by changes in physiological parameters involved in
cardiovascular functions mediated by catecholamines.
In addition to
their action in cardiovascular functions, catecholamines are also
involved in metabolic functions through regulation of carbohydrate and
fatty acid mobilization in developing embryos (reviewed in (35) and (61) ). In newborn animals, their action also
represents the respiratory effects to prepare the lung for ventilation
after delivery, such as an increase in synthesis and release of
surfactant and a stimulation of liquid
absorption(62, 63) . In our analyses, several mutant
neonates could breathe normally at the time of birth with no apparent
histological abnormality in the lungs. Also, we checked the blood
glucose concentration in the newborns obtained from the intercross of
the heterozygotes, but the values in the surviving mutants showed no
significant difference from the wild-type values. Although our
preliminary study did not detect changes in metabolic and respiratory
functions in the TH mutants, analysis of other physiological parameters
will be required to find subtle effects of the mutation.
Pathophysiological studies of the mutant phenotype will provide a
better understanding of the catecholamine functions in the developing
embryos and neonates.
Finally, we described herein transgenic rescue
of the mutant phenotype with the human TH transgene in addition to
targeted disruption of the mouse TH locus. The availability of the
transgenic mouse technology enables us to introduce any mutations
involved in expression of the TH gene or regulation of the enzyme
activity into intact animals. Analysis of the resultant phenotypic
effects helps us to understand better the physiology of the mammalian
catecholaminergic system and to investigate the pathogenesis caused by
subtle mutations in the TH locus.