Research report
4.5 kb of the rat tyrosine hydroxylase 5′ flanking sequence directs tissue specific expression during development and contains consensus sites for multiple transcription factors

https://doi.org/10.1016/S0169-328X(99)00234-XGet rights and content

Abstract

To delineate DNA sequences responsible for developmentally correct expression of the rat tyrosine hydroxylase (TH) gene, we analyzed a line of transgenic mice expressing high levels of human placental alkaline phosphatase (AP) under control of 4.5 kb of 5′ flanking DNA from the rat TH gene in embryos and adults. Several regions, such as the accessory olfactory bulb, which were not thought to synthesize TH protein or do so only transiently, were shown to express TH protein using an improved method of antigen retrieval for TH immunohistochemistry. Many of these regions had been shown to express TH-driven reporter genes in transgenic mice. In the central nervous system, AP was detected in essentially all TH-expressing cell groups throughout development and in adults. In the peripheral nervous system, transgene expression paralleled endogenous TH expression in the developing adrenal medulla and sympathetic ganglia but not in transiently TH-positive cells in dorsal root ganglia. Peripheral expression in the adult adrenal medulla was very weak and absent in sympathetic ganglia. The specificity with which the 4.5 kb region directs transgene expression in embryos is comparable to that observed with longer 5′ flanking promoter regions, implying that this region contains the control elements for appropriate expression during development. Sequence analysis of the region demonstrates a GT dinucleotide repeat, an element that resembles the neural restrictive silencer element (NRSE), which restricts transcription of neuronal genes in non-neuronal cells, and consensus sites for three families of transcription factors, Ptx1/3, Nurr1 and Gli1/2, which are required for the early differentiation of mesencephalic neurons.

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.

References (86)

  • E.J Hess et al.

    Tottering and leaner mutations perturb transient developmental expression of tyrosine hydroxylase in embryologically distinct Purkinje cells

    Neuron

    (1991)
  • M Hynes et al.

    Induction of midbrain dopaminergic neurons by Sonic hedgehog

    Neuron

    (1995)
  • M Hynes et al.

    Control of cell pattern in the neural tube by the zinc finger transcription factor and oncogene Gli-1

    Neuron

    (1997)
  • C.B Jaeger et al.

    Transient expression of tyrosine hydroxylase in some neurons of the developing inferior colliculus of the rat

    Brain Res.

    (1983)
  • G.M Jonakait et al.

    Transient expression of selected catecholaminergic traits in cranial sensory and dorsal root ganglia of the embryonic rat

    Dev. Biol.

    (1984)
  • N Kaneda et al.

    Tissue-specific and high-level expression of the human tyrosine hydroxylase gene in transgenic mice

    Neuron

    (1991)
  • D.M Katz et al.

    Developmental regulation of tyrosine hydroxylase expression in primary sensory neurons of the rat

    Dev. Biol.

    (1990)
  • K.S Kim et al.

    Both the basal and inducible transcription of the tyrosine hydroxylase gene are dependent upon a cAMP response element

    J. Biol. Chem.

    (1993)
  • K Kobayashi et al.

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

    J. Biol. Chem.

    (1995)
  • M Lazaroff et al.

    The cyclic AMP response element directs tyrosine hydroxylase expression in catecholaminergic central and peripheral nervous system cell lines from transgenic mice

    J. Biol. Chem.

    (1995)
  • J Liu et al.

    Identification of cell type-specific promoter elements associated with the rat tyrosine hydroxylase gene using transgenic founder analysis

    Brain Res. Mol. Brain Res.

    (1997)
  • L Lo et al.

    Specification of nuerotransmitter identity by Phox2 proteins in neural crest stem cells

    Neuron

    (1999)
  • N Min et al.

    5′ upstream DNA sequence of the rat tyrosine hydroxylase gene directs high-level and tissue-specific expression to catecholaminergic neurons in the central nervous system of transgenic mice

    Brain Res. Mol. Brain Res.

    (1994)
  • H Roelink et al.

    Floor plate and motor neuron induction by vhh-1, a vertebrate homolog of hedgehog expressed by the notochord

    Cell

    (1994)
  • T Sasaoka et al.

    Analysis of the human tyrosine hydroxylase promoter-chloramphenicol acetyltransferase chimeric gene expression in transgenic mice

    Brain Res. Mol. Brain Res.

    (1992)
  • J.H Son et al.

    Early ontogeny of catecholaminergic cell lineage in brain and peripheral neurons monitored by tyrosine hydroxylase-lacZ transgene

    Brain Res. Mol. Brain Res.

    (1996)
  • H.J Tae et al.

    Roles of CCAAT/enhancer-binding protein and its binding site on repression and derepression of acetyl-CoA carboxylase gene

    J. Biol. Chem.

    (1994)
  • M Takada et al.

    Tyrosine hydroxylase immunoreactivity in cerebellar Purkinje cells of the rat

    Neurosci. Lett.

    (1993)
  • G Teitelman et al.

    Proliferation and distribution of cells that transiently express a catecholaminergic phenotype during development in mice and rats

    Dev. Biol.

    (1981)
  • G Teitelman et al.

    Cell lineage analysis of pancreatic islet development: glucagon and insulin cells arise from catecholaminergic precursors present in the pancreatic duct

    Dev. Biol.

    (1987)
  • T Yamada et al.

    Control of cell pattern in the neural tube: motor neuron induction by diffusible factors from notochord and floor plate

    Cell

    (1993)
  • S.O Yoon et al.

    Tissue-specific transcription of the rat tyrosine hydroxylase gene requires synergy between an AP-1 motif and an overlapping E box-containing dyad

    Neuron

    (1992)
  • R.H Zetterstrom et al.

    Cellular expression of the immediate early transcription factors Nurr1 and NGFI-B suggests a gene regulatory role in several brain regions including the nigrostriatal dopamine system

    Brain Res. Mol. Brain Res.

    (1996)
  • C Alexandre et al.

    Transcriptional activation of hedgehog target genes in Drosophila is mediated directly by the cubitus interruptus protein, a member of the GLI family of zinc finger DNA-binding proteins

    Genes Dev.

    (1996)
  • H Baker

    Species differences in the distribution of substance P and tyrosine hydroxylase immunoreactivity in the olfactory bulb

    J. Comp. Neurol.

    (1986)
  • S.A Banerjee et al.

    5′ flanking sequences of the rat tyrosine hydroxylase gene target accurate tissue-specific, developmental, and transsynaptic expression in transgenic mice

    J. Neurosci.

    (1992)
  • F Cambi et al.

    5′ flanking DNA sequences direct cell-specific expression of rat tyrosine hydroxylase

    J. Neurochem.

    (1989)
  • A Carrier et al.

    Chicken tyrosine hydroxylase gene: isolation and functional characterization of the 5′ flanking region

    J. Neurochem.

    (1993)
  • J.M Carroll et al.

    Effects of second messenger system activation on functional expression of tyrosine hydroxylase fusion gene constructs in neuronal and nonneuronal cells

    J. Mol. Neurosci.

    (1991)
  • P Cochard et al.

    Ontogenetic appearance and disappearance of tyrosine hydroxylase and catecholamines in the rat embryo

    Proc. Natl. Acad. Sci. U.S.A.

    (1978)
  • S.R D'Mello et al.

    Isolation and nucleotide sequence of a cDNA clone encoding bovine adrenal tyrosine hydroxylase: comparative analysis of tyrosine hydroxylase gene products

    J. Neurosci. Res.

    (1988)
  • M Fauquet et al.

    The quail tyrosine hydroxylase gene promoter contains an active cyclic AMP-responsive element

    J. Neurochem.

    (1993)
  • C.A Harrington et al.

    Identification and cell type specificity of the tyrosine hydroxylase gene promoter

    Nucleic Acids Res.

    (1987)
  • Cited by (77)

    • NRSF is an essential mediator for the neuroprotection of trichostatin A in the MPTP mouse model of Parkinson's disease

      2015, Neuropharmacology
      Citation Excerpt :

      BDNF also increases the proportion of DA neurons in human embryonic mesencephalic cultures (Riaz et al., 2002; Maciaczyk et al., 2008). Previous studies show that NRSF is involved in the regulation of TH gene expression in human, and animals through binding to the NRSE site on the TH promoter (Schimmel et al., 1999; Kim et al., 2006). In the promoter-exon Ⅱ (referring to the second exon) region of human, rat and mouse BDNF gene, there is one NRSE site.

    • Epigenetic control of neurotransmitter expression in olfactory bulb interneurons

      2013, International Journal of Developmental Neuroscience
    • Epigenetic, transcriptional and posttranscriptional regulation of the tyrosine hydroxylase gene

      2011, International Journal of Developmental Neuroscience
      Citation Excerpt :

      The appearance of TH protein at an early stage of pre- and postnatal development is transitional in numerous structures and not always correlated with catecholamine synthesis (Cochard et al., 1978; Jaeger and Joh, 1983; Foster et al., 1985; Matsushita et al., 2002). During development, TH gene expression is limited to defined parts of the central and peripheral nervous system, including the adrenal chromaffin cells (Schimmel et al., 1999). Development-specific activation of the TH gene in embryogenesis coincides with the appearance of a cascade of factors in the developing brain, including factors of a homeotic nature (reviewed in Simon et al., 2003).

    • Cytokines inhibit norepinephrine transporter expression by decreasing Hand2

      2011, Molecular and Cellular Neuroscience
      Citation Excerpt :

      The Hand2 expression construct was a generous gift from Dr. Peter Cserjesi (Tulane University, New Orleans, LA). The 4.5TH-fLuc promoter construct was a generous gift from Dr. Dona Chikaraishi (Duke University Medical Center, Durham, NC), and includes the promoter region sufficient to drive tissue-specific expression of TH (Schimmel et al., 1999). The Arix/Phox2a expression construct was a generous gift from Dr. Elaine Lewis (NIH).

    View all citing articles on Scopus
    View full text