Research report4.5 kb of the rat tyrosine hydroxylase 5′ flanking sequence directs tissue specific expression during development and contains consensus sites for multiple transcription factors
Introduction
Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in catecholamine synthesis, hydroxylating tyrosine to dihydroxyphenylalanine (dopa), which is sequentially converted to dopamine, norepinephrine and epinephrine. Thus TH is critical for acquisition of a catecholaminergic phenotype. In adults, TH is expressed in two major groups in the peripheral nervous system (PNS) and in 18 discrete regions of the central nervous system (CNS) that are otherwise extremely diverse.
TH is present at several sites in the embryo that do not maintain TH expression in the adult such as the spinal cord, the anterior prosencephalon, cranial sensory and dorsal root ganglia 17, 27, 31, 55, 73. Hence, the embryonic pattern of TH expression is somewhat different from that seen in the adult. TH can first be detected by RT-PCR at embryonic day (E) 8.5 in the mouse [75]. A day later TH can be seen immunohistochemically in neuroblasts shortly after their birth in the periphery. In some groups, such as those in the ventral lateral spinal cord, TH is present but catecholamines are not, because other enzymes in the pathway are missing [17]. Expression in the CNS is first detected in the ventral midbrain and lags that of the PNS by at least a day and is less robust until later in development. The importance of TH during mid-gestation has been unappreciated until recently when several groups showed that 60–70% of TH knockout mice die between E11.5 and E14.5, with greater than 90% lethality by birth 36, 56, 86. Since functional catecholaminergic innervation of targets is not established until after birth, the role of TH in the developing embryo is distinct from its contribution to synaptic transmission. Based upon the critical role of TH in the embryo, identifying sites of TH expression in the embryo and determining which DNA sequences direct expression are of significant interest.
The spatial and temporal regulation of TH transcription is regulated by a complex interaction of protein transcription factors acting at sites within the 5′ region of the rat TH gene to activate the basal transcriptional machinery. These promoter and enhancer elements have been intensively studied in cultured cells 37, 58, 71. Early work showed that 272 bases of 5′ flanking DNA of the rat TH gene supported maximal transcription of a linked reporter gene in TH-expressing cultured cells and was much less effective in TH-negative cell lines 7, 9. Mutational analysis in numerous TH-expressing cells lines, including rat PC12 lines (PC12 18, 33PC8b [82]), human neuroblastomas lines (SK-N-BE 33, 34) and in transgenically-derived mouse CNS and PNS lines (Catha, Path2 [40], CAD [41]), has shown that two enhancer sites mediated basal transcription: the activator protein 1 (AP-1) site at −205 bp and the cAMP response element (CRE) at −45 bp, for exception, see [79]. Interestingly, various cell lines relied on these two elements to varying extents. In addition, a proximal promoter element which resided between the TH TATA box and the start of transcription was required in all cell lines, even those that do not express the endogenous TH gene [53]. Consistent with the primacy of these elements, sequence analyses revealed that the AP-1, CRE and the proximal promoter element are conserved in sequence and position in the 5′ flank of all mammalian TH genes examined to date 13, 49, 51, 53. The importance of the AP-1 and CRE sites in vivo was recently demonstrated by Trocme et al. [77]who showed that mutation of either the AP-1 or CRE site abolished expression in adult transgenic mice, but not in the embryo.
Based on the cell culture experiments, transgenic mice bearing 272 bp of 5′ flank were generated but, contrary to expectation, they did not express a linked reporter in any tissue [5]. Similar results were obtained when 150 bases of the rat promoter or 200 bases of the human TH promoter were used in transgenic studies 43, 48, 60. To achieve tissue-specific expression, longer 5′ flanking sequences of the rat gene were required. The results of these studies indicated that lengthening the flanking sequence to 4.5 kb or greater produced a pattern of CNS reporter expression closely mirroring the endogenous TH gene in adult mice, with minimal ectopic expression 6, 43, 48, 77. Peripheral expression, however, varied with different reporters driven by the same 5′ sequence [77]. Interestingly, common areas of ectopic expression were found with all constructs tested, suggesting that even the longest 5′ flanking sequence (9.0 kb) may not be sufficient to repress ectopic expression. In contrast to the studies using the rat gene, those using the human TH gene demonstrated that 5 kb of 5′ flanking DNA did not recapitulate endogenous TH expression [60]but other intronic or 3′ flanking regulatory elements were required [29].
We have analyzed a line of transgenic mice utilizing a human placental alkaline phosphatase (AP) reporter gene, under the control of 4510 bp of rat TH 5′ flanking sequence through embryonic development and in adults. Due to the high level of expression associated with this line and sensitive immunohistochemistry for TH, we demonstrate that 4.5 kb drives correct expression of AP to almost all CNS TH-positive areas including several traditionally thought to be transient. Thus, 4.5 kb is the shortest construct yet examined that directs proper CNS expression through development and in the adult.
Section snippets
Animals
Animals were housed and maintained in accordance with the National Institutes of Health guide for the care and use of laboratory animals. Efforts were made to minimize animal suffering and use the minimum number of animals.
The transgenic mice were generated as previously described [19]. The mouse strain used to generate the founder mice was C57Bl/6 X SJL/J F2. This strain was then backcrossed onto C57Bl/6 and animals homozygous for the transgene were obtained and used for the experiments
Localization of TH-AP expression in adult brain
4.5 kb of the rat TH 5′ flank drove expression of AP to the same areas as endogenous TH immunoreactivity (IR) (Fig. 1A, B, C, D). In general, the AP reaction product was more intense in fibers and terminals compared to cell bodies, probably because the transgene encodes a membrane bound form of AP, whereas staining for TH IR was localized to both fibers and cell bodies.
TH-AP expression in well characterized adult mouse brain TH containing cell groups
Examination of frontal brain sections of adult TH-AP transgenic mice showed AP expression in all of the well characterized
5 kb of rat TH 5′ flank drives proper expression of AP
In the CNS, the AP transgene is expressed in all the regions that express TH in the adult and during development, suggesting that the necessary elements to direct proper tissue specific and developmentally correct TH gene expression reside within 4.5 kb of the start of transcription. In the PNS, AP is weakly co-expressed with TH IR during development, but not in the adult. AP was not observed in adult sympathetic ganglia and was very weak in the adrenal medulla, both regions of robust TH
Acknowledgements
This study was supported by NS 22675 (DMC) and NS 20181 (SR-T). The authors thank Marybeth Groelle for her expert technical assistance and Dr. Rod Bronson for helping us to understand the anatomy of the embryos.
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