Targeted disruption of the tyrosine hydroxylase locus results in severe catecholamine depletion and perinatal lethality in mice.

Tyrosine 3-hydroxylase (TH, EC 1.14.16.2) catalyzes the first and rate-limiting step of the catecholamine biosynthetic pathway in the nervous and endocrine systems. The TH locus was disrupted in mouse embryonic stem cells by homologous recombination. Mice heterozygous for the TH mutation were apparently normal. In these mice, TH activity in the embryos and adult tissues was less than 50% of the wild-type values, but the catecholamine level was decreased only moderately in the developing animals and was maintained normally at adulthood, suggesting the presence of a regulatory mechanism for ensuring the proper catecholamine level during animal development. In contrast, the homozygous mutant mice died at a late stage of embryonic development or shortly after birth. Both TH mRNA and enzyme activity were lacking in the homozygous mutants, which thus explained the severe depletion of catecholamines. These changes, however, did not affect gross morphological development of the cells that normally express high catecholamine levels. Analysis of electrocardiograms of surviving newborn mutants showed bradycardia, suggesting an alteration of cardiac functions in the homozygous mice that may lead to the lethality of this mutation. In addition, transfer of a human TH transgene into the homozygous mice corrected the mutant phenotype, showing recovery of TH activity by expression of the human enzyme. These results indicate that TH is essential for survival of the animals during the late gestational development and after birth.

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 inter-mediate zone of the neural tube at the early embryonic stage (reviewed in Ref. 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 (Ref. 2; reviewed in Ref. 3).
Tyrosine 3-hydroxylase (TH, 1 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 Ref. 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 -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 recombina-tion 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 3Ј-portion of exon 7, intron 7, and the 5Ј-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 Afragment gene cassette (26) driven by the MC1 promoter were used to flank the respective 5Ј-and 3Ј-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 ϫ nonessential amino acids, 0.1 mM ␤-mercaptoethanol, and 1 mM sodium pyruvate. ES cells (5 ϫ 10 7 ) 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 M ganciclovir. Individually picked colonies were analyzed by Southern blot hybridization with a 0.5-kb SphI-BstPI fragment containing a 3Ј-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 32 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 Ref. 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 ϩ/Ϫ 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 ϩ/Ϫ 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 (5Ј-TCCCCAAGGTTCATTGGACG-3Ј, nucleotides 96 -115 of the mouse TH cDNA sequence from Ref. 23) and primer B (5Ј-GGTAC-CCTATGCATTTAGCT-3Ј, 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 (5Ј-ACTGCTGCCACGAGCTGCT-3Ј) is common to the mouse and human TH cDNA sequences (nucleotides 989-1008). Primer D (5Ј-TCAGGGACGCCGTGCACCTA-3Ј) is complementary to nucleotides 1701-1719 of the mouse TH sequence, and primer E (5Ј-TAGAATACAGCATGAAGGG-3Ј) is complementary to nucleotides 1493-1512 of the human TH sequence.
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 g/ml leupeptin, 1 g/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).
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 5Ј-homologous region of 5.8 kb and 3Ј-homologous region of 2.2 kb, in which a 0.3-kb AccI-AccI fragment containing the 3Ј-portion of exon 7, intron 7, and 5Ј-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 5Ј-and 3Ј-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 3Ј-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 G418resistant 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 Ϫ/Ϫ homozygous mice derived from the chimeras generated with the TH2-55 clone displayed the same mutant phenotype as described below.
Perinatal Lethality in TH Ϫ/Ϫ 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 ϩ/Ϫ heterozygous mice were intercrossed and their progeny were genotyped at 3 weeks of age (Table I). To examine the lethality of the TH Ϫ/Ϫ homozygous mutation, we genotyped embryos at various stages of gestation and neonates obtained from the intercross of heterozygotes (Table I). A typical pattern of the genotyping results is shown in Fig. 1C. Development of TH Ϫ/Ϫ 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 immunohisto-FIG. 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 3Ј-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 g) 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 g) 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 (ϩ/ϩ), heterozygous (ϩ/Ϫ), and homozygous mutant (Ϫ/Ϫ) mice is indicated on the top of each lane. chemical 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  II). 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 II). 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 ϩ/Ϫ 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 het-erozygous 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.
Morphology of the TH Ϫ/Ϫ 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 Ϫ/Ϫ 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).
Effects on Cardiac Functions-Our observations that there is no apparent histopathological change in the TH Ϫ/Ϫ 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  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 Ϯ S.E. of the data obtained from three to six embryos. ND, not detected. Asterisks show significant difference (p Ͻ 0.05) from the wild-type levels (Student's t test). 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 ϩ/Ϫ 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 Ϯ 17 beats/min) was remarkably decreased as compared with the wild-type (307 Ϯ 9 beats/min) and heterozygous (298 Ϯ 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.
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 Ϫ/Ϫ homozygous mice. By crossing hTH-1 transgenic mice carrying the human TH gene with the TH ϩ/Ϫ 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 identi-  fied 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 Ϫ/Ϫ 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 conten-tion 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 preg- FIG. 5. Electrocardiographic analysis of the newborn mice. A, surface electrocardiograms of the wild-type (ϩ/ϩ), heterozygous (ϩ/Ϫ), and homozygous mutant (Ϫ/Ϫ) 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 Ϯ S.E. of the data. The asterisk denotes a significant difference from the wild-type neonate value (p Ͻ 0.01).
FIG. 6. Genetic rescue of the TH Ϫ/Ϫ 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 g) 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 Ϯ 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. nant 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 Ϫ/Ϫ 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 -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 Ϫ/Ϫ homozygous mice.
Adaptation of Catecholamine Accumulation during Development-In the E12.5 embryos and adult tissues of the TH ϩ/Ϫ 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 max 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 2ϩ / 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 -55). Our immunohistochemical analysis of the TH Ϫ/Ϫ 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 -46, 56 -58). In this study we did not observe any visible change in morphology of these tissues in the TH Ϫ/Ϫ 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 Ϫ/Ϫ 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 Ϫ/Ϫ 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 Refs. 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 result-ant 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.