Catecholamine synthesis is mediated by tyrosinase in the absence of tyrosine hydroxylase

Abstract

Catecholamine neurotransmitters are synthesized by hydroxylation of tyrosine to L-dihydroxyphenylalanine (L-Dopa) by tyrosine hydroxylase (TH). The elimination of TH in both pigmented and albino mice described here, like pigmented TH-null mice reported previously (Kobayashi et al., 1995; Zhou et al., 1995), demonstrates the unequivocal requirement for catecholamines during embryonic development. Although the lack of TH is fatal, TH-null embryos can be rescued by administration of catecholamine precursors to pregnant dams. Once born, TH-null pups can survive without further treatment until weaning. Given the relatively rapid half-life of catecholamines, we expected to find none in postnatal TH-null pups. Despite the fact that the TH-null pups lack TH and have not been supplemented with catecholamine precursers, catecholamines are readily detected in our pigmented line of TH-null mice by glyoxylic acid-induced histofluorescence at postnatal day 7 (P7) and P15 and quantitatively at P15 in sympathetically innervated peripheral organs, in sympathetic ganglia, in adrenal glands, and in brains. Between 2 and 22% of wild-type catecholamine concentrations are found in these tissues in mutant pigmented mice. To ascertain the source of the catecholamine, we examined postnatal TH-null albino mice that lack tyrosinase, another enzyme that converts tyrosine to L-Dopa but does so during melanin synthesis. In contrast to the pigmented TH-null mice, catecholamine histofluorescence is undetectable in postnatal albino mutants, and the catecholamine content of TH-null pups lacking tyrosinase is 18% or less than that of TH-null mice with tyrosinase. Thus, these extraordinary circumstances reveal that tyrosinase serves as an alternative pathway to supply catecholamines.

Fig. 1.

a, Disruption of the TH gene. Sequence between exons 6 and 8 of the mouse TH gene was replaced by aTK-Neo cassette without a polyadenylation signal.Vertical rectangles represent exons, and theshort shaded horizontal bar represents the location of the 5′ DNA probe. b, Southern blot analysis of an E12.5 litter obtained from a heterozygous cross. DNA extracted from E12.5 fetuses was digested with BamHI andHindIII and hybridized to the 5′ probe. The 13 and 10 kb bands represent the wild-type and targeted alleles, respectively.c, Western blot containing protein extracted from heads of E14.5 wild-type (lane 2), heterozygous (lane 3), and TH-null (lane 4) fetuses. Protein samples from J1 embryonic stem cells were a negative control (lane 1). Reduced content of TH protein was observed in the heterozygous fetuses compared with wild types. A low level of mutant TH was detected in extracts from the TH-null fetus.

Fig. 2.

Transverse section through the thoracic region containing the paravertebral sympathetic ganglia. Transverse sections of fetal sympathetic ganglia (arrows) treated with glyoxylic acid revealed catecholamine fluorescence in the wild-type fetus (left), whereas histofluorescence was undetectable in the mutant (right). A, Aorta.

Fig. 3.

Catecholamine histofluorescence in peripheral tissues of rescued pigmented and albino TH-deficient mice. Adrenal medullae from P6 mice (a, b,c). Adrenal chromaffin cells of pigmented wild-type mice (a) possess intense catecholamine fluorescence. Adrenal chromaffin cells of pigmented TH-null mice (b) contain catecholamines, but the fluorescence is less intense than that of wild-type mice. In contrast, adrenal chromaffin cells of albino TH-null mice (c) are almost devoid of catecholamine histofluorescence. The medullary region with sparse granular fluorescence is distinguishable from the surrounding cortex by the lipid inclusions in the cortical cells. Most sympathetic SCG neurons in pigmented P6 wild-type mice (d) contain bright granular fluorescence. Principal sympathetic neurons of pigmented P6 TH-nulls (e) lacked catecholamine fluorescence; however, fluorescence was seen in SIF cells (arrow). No catecholamine fluorescence could be detected in SCG from TH-nulls that were also tyrosinase-deficient (f). Brightly fluorescent sympathetic fibers were present in the ventricular smooth muscle wall in pigmented P6 wild-type mice (g). A reduced number of brightly fluorescent fibers (arrow) were present in the ventricles of pigmented P6 TH-null mice (h). No catecholamine fluorescent fibers could be detected in the ventricles of albino TH-null mice (i). Brightly fluorescent sympathetic fibers were associated with piloerectors in the hairy skin of pigmented P6 wild-type mice (j). Fibers exhibiting intermediate or weak fluorescence were present in pigmented P6 TH-null mice (k). No catecholamine histofluorescent fibers were detected in the hairy skin of albino TH-null mice (l).

Fig. 4.

Catecholamine histofluorescence in the striata and ventral midbrains of rescued pigmented TH-null P6 pups. Coronal sections of the rostral striatum from heterozygous P6 pups (A) revealed clusters of brightly fluorescent nigral fibers, the dopamine islands (arrows). In contrast, glyoxylic acid treatment of coronal sections of rostral striatum from a pigmented TH-null littermate (B) revealed very few clusters of fluorescent fibers. Those detected were weakly fluorescent (arrow). In heterozygous pups, brightly fluorescent cells in ventral midbrain (C) were abundant in the dopamine-containing substantia nigra, pars compacta. In contrast, the substantia nigra of pigmented TH-null pups (D) contained few catecholamine fluorescent cells. Although such cells were rare, they were brightly fluorescent (arrow). c, Cortex.

Fig. 5.

Glyoxylic acid-induced catecholamine fluorescence in the striata and midbrains of albino (tyrosinase-deficient) wild-type and TH-null P6 pups. Striata from wild-type albino mice (A) contain clustered brightly fluorescent fibers, representing dopamine islands (arrows). No catecholamine histofluorescence was seen in the striata of albino TH-deficient mice (B). In the wild-type albino midbrain (C), many fluorescent dopamine cells can be observed. In contrast, no catecholamine histofluorescence could be detected in the midbrains of TH-null mice also deficient in tyrosinase (D). c, Cortex.

Fig. 6.

Catecholamines were extracted from tissues harvested from TH-null and wild-type pups that were 14- and 15-d-old. Both the albino and pigmented mice were treated until birth withl-Dopa supplied to pregnant dams. Values represent the mean ± SEM of six or seven samples, except skin of the albino TH-null, which is the mean of three samples. Values are calculated as percent of the appropriate (albino or pigmented) wild-type value. *p < 0.005 indicates a significant difference from values for pigmented mice by Student’s t test.