Normal Nigrostriatal Innervation butDopamine Dysfunction in Mice CarryingHypomorphic Tyrosine Hydroxylase Alleles

Susanna Althini,1 Henrik Bengtsson,1 Dmitry Usoskin,1 Stine Soderstrom,1

Annika Kylberg,1 Eva Lindqvist,2 Susana Chuva de Sousa Lopes,3 Lars Olson,2

Jonas Lindeberg,1 and Ted Ebendal1*1Department of Neuroscience, Unit of Developmental Neuroscience, Uppsala University, Uppsala, Sweden2Department of Neuroscience, Karolinska Institute, Stockholm, Sweden3Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, The Netherlands

We investigated the use of the mouse tyrosine hydroxy-lase (TH) gene to drive knock-in constructs in cat-echolaminergic neurons. Two targeting constructs rep-resenting truncated forms of either of the BMP receptorsALK-2 or BMPR-II preceded by an internal ribosomeentry site (IRES) were introduced into the 3� untranslatedregion of TH. An frt-flanked neomycin-resistance (neor)cassette was placed in the 3� end of the targeting con-structs. Mice homozygous for the knock-in allelesshowed various degrees of hypokinetic behavior, de-pending mainly on whether the neor cassette was re-moved. In situ hybridization and immunohistochemistryshowed that TH mRNA and protein were variously down-regulated in these mouse strains. Reduced levels of do-pamine and noradrenalin were found in several brainareas. However, number and morphology of neurons insubstantia nigra and their projections to striatum ap-peared normal in the neor-positive TH hypomorphicmice as examined by markers for L-aromatic aminoacid decarboxylase and the dopamine transporter.Elimination of the neor cassette from the knock-inalleles partially restored TH and dopamine levels. Thepresent neor-positive TH hypomorphic mice show thatnigrostriatal innervation develops independently of THand should find use as a model for conditions of re-duced catecholamine synthesis, as seen in, for exam-ple, L-dihydroxyphenylalanine-responsive dystonia/infantile parkinsonism. © 2003 Wiley-Liss, Inc.

Key words: mouse; substantia nigra; neomycin resis-tance; internal ribosome entry site; L-dopa-responsivedystonia

To target catecholaminergic neurons genetically, wechose the tyrosine hydroxylase (TH) gene as a carrier forvarious DNA constructs. TH is the first and rate-limitingenzyme in the biosynthesis of catecholamines (Nagatsu et al.,1964) and converts tyrosine to L-dihydroxyphenylalanine(L-dopa), from which dopamine (DA), noradrenalin (NA),and adrenalin are formed in sequence. Several studies on the

expression patterns show that BMPR-II is highly expressedin dopaminergic neurons of substantia nigra (SN) and theventral tegmental area (VTA; Bengtsson et al., 1995; Carri etal., 1998; Ebendal et al., 1998; Charytoniuk et al., 2000) aswell as in sympathetic ganglia of the peripheral nervoussystem (Soderstrom et al., 1996). These and additional ob-servations in vitro suggest that BMP signaling might servefunctions in various neuronal populations (Jordan et al., 1997;Mehler et al., 1997; Bengtsson et al., 1998). We set out tostudy the role of ALK-2 and BMPR-II (type I and type IIserine/threonine kinase receptors for BMPs, respectively;Massague, 2000; ten Dijke et al., 2000; von Bubnoff andCho, 2001) in catecholaminergic neurons by attempting tointerrupt signaling via these receptors.

The embryo lethality caused by deletion of thesegenes in the mouse (Gu et al., 1999; Beppu et al., 2000)hinders evaluation of their function in the adult brain. Weconsequently used a cell-specific strategy to knock-in thedominant-negative BMP receptors (A2 and B2); we madeuse of an internal ribosome entry site (IRES; Mountford etal., 1994; Mountford and Smith, 1995; Lindeberg andEbendal, 1999) to produce bicistronic tyrosine hydroxy-lase (TH)-IRES-A2 and TH-IRES-B2 mRNAs. Theconstructs were targeted to the 3� untranslated region

Contract grant sponsor: The Swedish Science Research Council; Contractgrant sponsor: Swedish Foundation for Strategic Research; Contract grantsponsor: Fundacao para a Ciencia e Tecnologia; Contract grant number:SFRH/BD/827/2000.

The first two authors contributed equally to this work.

Stine Soderstrom’s current address is: Smerud Medical Research Group,Uppsala, Sweden.

*Correspondence to: Ted Ebendal, Department of Neuroscience, Unit ofDevelopmental Biology, Uppsala University, Box 587, SE-751 23 Uppsala,Sweden. E-mail: ted.ebendal@neuro.uu.se

Received 20 December 2002; Revised 6 February 2003; Accepted 7February 2003

Journal of Neuroscience Research 72:444–453 (2003)

© 2003 Wiley-Liss, Inc.

(3�UTR) of the TH gene (see Fig. 1). The resultingmRNA should encode two proteins: endogenous TH andthe chosen, knocked-in, dominant-negative receptor. Weexpected these modified alleles to allow TH to be pro-duced normally and with a concomitant production of A2or B2 in catecholaminergic cells. In addition to the IRES-A2/B2 elements, each construct carried a neor cassetteflanked with frt sites, to allow selection of embryonic stem(ES) cells. The frt sequences allow in vivo removal of theneor cassette using Flp deletor mice (Dymecki, 1996).

Here we characterize mice carrying these constructswith and without neor (A2�, A2–, B2�, and B2–) andmice with one TH allele carrying the A2 mutation andone allele carrying the B2 mutation, before and after neor

elimination (A2�/B2� and A2–/B2–) with respect to lo-comotion, body weight, levels of catecholamines, and THmRNA/protein expression. This paper is focused on do-paminergic neurons of the nigrostriatal system. Other sys-tems utilizing TH, such as locus coeruleus, DA neurons inthe ventral tegmental area (VTA), tuberoinfundibular DAsystem, noradrenergic nuclei of the brainstem, and adrenalchromaffin cells are also expected to be affected by theknock-in alleles. In addition, we monitored the hip-pocampus and submandibular glands, targets for noradren-ergic projections from locus coeruleus and peripheral sym-pathetic neurons, respectively.

MATERIALS AND METHODS

Isolation of the Mouse TH Gene

PCR primers (5�: 5TH- ACTGAATTCACCCTGAC-CCAAGCACT; 3�: 3TH- ACTGAATTCTTTCCTTCCTT-TATTGAGAT) were designed to cover exon 13, including the3�UTR, of the mouse TH gene (GenBank Acc. No. M69200;nucleotides 1,469–1,751). PCR using genomic DNA from129X1/SvJ (129X1) mice gave a unique fragment of the ex-pected length (280 bp). The cloned fragment was used to screena mouse 129X1 genomic �-phage library (Stratagene, La Jolla,CA). From a positive clone, the entire insert of 16 kb was cutout with Not I and subcloned in pBluescript (pBs) KS(�)(Stratagene). A 6.7-kb fragment (Pml I/Spe I) was then sub-cloned to constitute the flanking regions of the constructs de-scribed below. It contained the TH stop codon and the poly-Asignal. Between these, a unique Sph I site was identified.

Generation of Dominant-Negative ALK-2 and BMPR-II

With the polymerase chain reaction (PCR), we con-structed dominant-negative forms of ALK-2 and BMPR-II.The mutants, A2 and B2, have normal extracellular parts butlack the intracellular kinase domains. After position Gly155 forA2 and Gly182 for B2, just inside the transmembrane regions(Ebner et al., 1993; Beppu et al., 1997), an HA- tag and a c-myctag, respectively, were incorporated, followed by translationalTGA stops using the 3� primers. Forebrain cDNA was used astemplate, and the 5� primers were designed to span the startcodon of BMPR-II or ALK-2. An Nco I site was introduced atthe start ATG codon to allow accurate cloning with the IRES(see below). The 600-bp products were cloned into pMOS

(Amersham Biosciences UK Ltd., Buckinghamshire, UnitedKingdom). The dominant-negative properties of the A2 and B2on BMP signaling were confirmed in cell culture (data notshown).

Generation of IRES Knock-In Constructs

Both A2 and B2 were excised from pMOS with NcoI/BamHI, cloned into a pBS vector containing the IRES se-quence, and opened with Nco I/Bam HI to position the A2/B2constructs downstream of the IRES. The IRES-A2-frt-neor-frtand IRES-B2-frt-neor-frt were cloned into the unique Sph I sitein the 3�UTR of the TH gene described above, to give flankingregions of 3.3 kb (5�) and 3.4 kb (3�) homologous to the mouseTH gene. The constructs were linearized with Sma I beforeelectroporation into ES cells.

Generation of Genetically Modified Mice

The targeting constructs were electroporated into ES cells(GSI-1; Genome Systems Inc., St. Louis, MO), which werethen subjected to G-418 selection. After 6 days, single ES cellclones were picked for Southern blot analysis (see Lindeberg,2002); using Bgl II and an external 5� probe (1 kb) gives a 7.8-kbwild type, a 8.8-kb band for A2�, and a 8.2-kb band for B2� incells that have undergone homologous recombination (Fig. 1).One positive A2� clone (2E2) and one B2� clone (2G9) wereselected for injection into C57BL/6J (B6) blastocysts. Injectedblastocysts were transferred into one of the uterine horns ofpseudopregnant females. This resulted in one A2� and two B2�

chimeras, all transmitting the knock-in alleles. Back-crosses with129X1 and B6 (for creating congenic C57Black mice) producedstrains with homogenous genetic background and mixed hy-brids, respectively.

Genotyping

Tail biopsies were taken at weaning and lysed with pro-teinase K/sodium dodecyl sulfate (SDS), and genomic DNA wasused as template in high-stringency PCRs. To identify themodified alleles, primers SAL12–SAL3 (for A2) and HB22b–HB24 (for B2; Fig. 1A) were used. They gave amplification of600-bp fragments, corresponding to the dominant-negative re-ceptor genes. The wild-type TH allele was identified with the5TH and 3TH primers. When wild-type TH is present, a280-bp band is produced. These primers did not amplify theIRES-A2/B2 cassette introduced between them in the mutatedalleles. In the case of heterozygosity, both the 600-bp and the280-bp bands were amplified (Fig. 1C).

Elimination of the neor Cassette In Vivo

The frt-flanked neor cassette was removed by crossingA2� and B2� males with Flp deletor dams of strain 4917 fromDr. Dymecki (Dymecki, 1996). Mice carrying the A2� or theB2� allele and the Flp deletor allele were identified and back-crossed to B6. The second round of crossings resulted in A2– andB2– mice. A2– and B2– founders were further back-crossed toB6. To determine elimination of neor, a primer complementaryto neor was used (Fig. 1A). This approach gave an �800-bpband with neor still in the construct and no band after successfulremoval (not shown). Southern blots confirmed deletion of neor

in A2 mice. Genomic DNA was digested with Sph I. The

Dopamine Dysfunction in TH-Hypomorphic Mice 445

Fig. 1. Strategy for modifying the tyrosine hydroxylase locus in mice.A: Map of the mouse TH locus and the targeting constructs used toknock-in IRES-A2-neor and IRES-B2-neor between the TH stopcodon (asterisks) and poly-A signal (AAAAA). The red box indicatesA2 or B2. Blue triangles are Frt sites flanking the neomycin-resistancecassette. External probes used for Southern blots are indicated in yellowand primers for PCR by small arrows (1, 5TH; 2, 3TH; 3, SAL1b/SAL12/HB22; 4, SAL3/HB24; 5, SAL9). Large arrows indicate orien-tation of transcription. Below is shown the result of homologousrecombination before and after the elimination of the neor cassette by

FLP. B: PCR showing genotyping of the mice. TH wt band: primers1 and 2. A2– and A2� bands: primers 3 (SAL12) and 4 (SAL3). B2– andB2� bands: primers 3 (HB22) and 4 (HB24). # Indicates a band thatappears in homozygous neor-negative mice from primers 3 (SAL12/HB22) and 2. C: Southern blot showing successful removal of the neor

cassette from A2 mice in vivo. Genomic DNA was digested with SphI, separated on a 0.7% agarose gel, transferred to a Hybond N�

membrane (Amersham), and hybridized to the 5� probe. D: A ho-mozygous A2� mouse in 129X1 background and a wild-type litter-mate. Note the apparent ptosis.

446 Althini et al.

external 5� TH probe recognizes the 5� end of the Sph Ifragments produced in the different genotypes (Fig. 1A,B).

Locomotor Activity

Spontaneous movement was measured during 15-minsessions by putting single mice into 26 � 26 cm activity cham-bers (TruScan; Coulburn Instruments). The mice wereweighed, and then left undisturbed in the activity chamber.

In Situ Hybridization and Immunohistochemistry

Adult brains were rapidly dissected, frozen on dry ice, andserially sectioned (10 �m) through the midbrain level for in situhybridization. Oligonucleotide probes complementary to rat THand AADC were used. The TH probe was 5�-TGCGTGGG-CCAGGGTGTGCAGCTCATCCTGGACCCCCTCCAAGG-AGCG-3�, and the AADC probe was 5�-AGCATCAATGTGCA-GCCATACACCCTCCTGGTTGCAGATGGGACCCAC-3�.Furthermore, we used probes specific for the mRNAs produced bythe modified TH alleles [A2 (Sal14): 5�-ATATCCACCAT-ACTCCACGTCTCTGGGGTTCAGGCGCTCTT GATTG-3�; B2 (HB35): 5�-AGTTTCTGTTCACGCTGTTTCCGG-TCTCCTGTCAACA TTCTGTATCCA-3�]. These two“bicistronic mRNA”-specific probes were designed so that halftheir sequences (5�) were complementary to the truncated receptorsequence and the other half (3�) complementary to the added tag(HA and c-myc for A2 and B2, respectively). Probe labeling andhybridizations were performed as described by Bengtsson et al.(1995).

Perfusion-fixed brains of adult mice were cryosectionedfor immunohistochemistry according to standard procedures.The following antibodies were used: anti-rat TH (Pel-Freeze,Rogers, AR), anti-bovine AADC (Calbiochem, La Jolla, CA),and anti-human DAT (Chemicon International, Temecula,CA). Detection with fluorescein isothiocyanate (FITC)-labeledsecondary antibodies was evaluated by fluorescence microscopy.

Determination of Catecholamine and 5-Hydroxytryptamine Levels

Endogenous levels of NA, DA, and 5-hydroxytryptamine(5-HT) were determined in SN, striatum, hippocampus, andsubmandibular gland by high-performance liquid chromatogra-phy (HPLC) with electrochemical detection according to Luth-man et al. (1993).

Statistical Analysis

Differences between groups were tested using ANOVAand Student’s t-test (post hoc) for normally distributed data. TheMann-Whitney test was applied for nonnormally distributeddata using SigmaStat software (SPSS Inc., Chicago, IL). Signif-icance levels in the figures are given as follows: *P � 0.05,**P � 0.01, ***P � 0.001.

RESULTS

The TH-Targeted Mice Are Hypokinetic andShow Reduced Body Weight

In groups of A2� and B2� sibling crossings, a subsetof mice was smaller and less prone to move about. Thiswas obvious from 3 weeks of age, and the phenotype

coincided with homozygosity for the altered TH alleles.Spontaneous movement was recorded and the total dis-tance plotted (Fig. 2). Figure 2A shows that 129X1 mice,homozygous for A2� or B2� and mice carrying one ofeach of these alleles moved shorter distances comparedwith wild-type and heterozygous mice. The body weightwas also lower when both TH alleles carried B2� or A2�/B2� mutations (Fig. 2B). The average weight of A2�

mice was slightly lower than that of littermate controls, butthe difference was not significant. Figure 1D depicts anA2� homozygous mouse and its 129X1 wild-type litter-mate. This mutant has distinctly drooping eyelids, a featureseen in most A2� and B2� homozygous mice. Spontane-ous movement and body weight were also measured in

Fig. 2. Spontaneous locomotor activity and body weight in micecarrying altered TH alleles. A: A group of 61 129X1 mice was ana-lyzed, representing six different genotypes as follows: wild-type (wt)n � 16 (10 male, 6 female), A2� heterozygotes (het) n � 18 (12 male,6 female), B2� het n � 8 (5 male, 3 female), A2� homozygotes (hom)n � 8 (6 male, 2 female), A2�/B2� n � 5 (3 male, 2 female), B2� homn � 6 (5 male, 1 female). If both TH alleles are A2� or B2� (or theircombination), the locomotor activity is significantly reduced. B: Bodyweight in the group of 129X1 mice. Average body weight was signif-icantly reduced in mice carrying two B2� alleles or one B2� with oneA2� allele. C: Activity and body weight in a mixed genetic (129X1;B6)background. Again, the presence of B2� in both TH alleles is impairingboth locomotor activity and body size. A group of 26 mice with amixed 129X1;B6 background was studied: wt n � 7 (1 male, 6 female),B2� het n � 10 (7 male, 3 female), B2� hom n � 9 (4 male, 5 female).

Dopamine Dysfunction in TH-Hypomorphic Mice 447

two groups of B2� mice with a 129X1;B6 genetic back-ground. The hypokinesia in homozygotes is manifest alsoin this mixed genetic background (Fig. 2C). Hypokinesiaor impaired exploratory behavior in B2� homozygousmice was confirmed in an independent group of 51 malemice with mixed B6;129X1 background: wt n � 23, B2�

het n � 22, B2� hom n � 6 (not shown).

A2� and B2� Mice Are TH HypomorphsProbes complementary to TH and AADC were used

for in situ hybridization (Fig. 3). In homozygous B2�

mice still carrying the frt-neor cassette, analysis of SNshowed that the level of TH mRNA was lowered, if notabolished, compared with wild-type mice (Fig. 3A). Nochange in the expression was noted in heterozygous mice(not shown). The presence of the neuronal populationsnormally producing TH was tested by in situ hybridizationusing an AADC probe. This probe showed labeling of SNin all genotypes, with no apparent changes in levels ofAADC mRNA expression. Hence, the neurons that nor-mally are TH positive are still present in the B2� homozy-gous animals but express only very low levels of THmRNA. Data for the A2� mice were similar (not shown).

To detect TH protein, we performed immunohis-tochemistry with anti-TH antibodies (Fig. 4). No THimmunoreactivity (IR) could be detected in SN of B2�

homozygous animals. In wild-type and B2� heterozygousanimals, the antibody distinctly labeled SN and VTA, withno detectable difference in intensity, confirming the resultsof the in situ hybridization. AADC IR is similar in ho-mozygotes and wild types, indicating that the mutant alleledoes not affect neuron morphology. Controls with eitherprimary or secondary antibody were negative (not shown).TH IR was weaker in striatal nerve terminals in B2�

homozygous mice (Fig. 4K). In contrast, the expressionlevel of a marker protein for dopamine projections, DAT(membrane-bound dopamine transporter), was not alteredin these mice, confirming that dopaminergic nerve termi-nals were present in the striatum but failed to express TH.

The Knock-In Construct Results in DrasticReduction of DA Levels in Striatum

To evaluate further the functional consequences ofthe reduced TH levels, we analyzed the levels of cat-echolamines in selected areas. Mice were tested for con-tents of DA, NA, and 5-HT in SN, striatum, hippocam-pus, and submandibular gland (Fig. 5). There was a generalreduction of DA and NA levels in neor-positive homozy-gous mice. In the striatum of homozygous B2� mice, a97% loss of DA was found. This value is likely to includeDA from the caudate nucleus and putamen of the dorsalstriatum as well as from nucleus accumbens in the ventral

Fig. 3. In situ hybridization results from the TH-modified mice. Sections through midbrain areshown. A: TH mRNA expression levels in substantia nigra and ventral tegmental area wereprofoundly reduced in B2� homozygotes, whereas AADC mRNA in these neurons is unaffected.B: Expression levels of the bicistronic mRNAs containing TH-IRES-A2 or TH-IRES-B2. Probesdesigned to identify A2-HA and B2-cMyc specifically show that the amount of these mRNAs isreduced in A2� and B2� compared with neor-negative animals.

448 Althini et al.

striatum, receiving dopaminergic innervation from VTA.In addition, levels of NA were decreased in hippocampus,innervated by locus coeruleus, and in the submandibulargland, receiving noradrenergic innervation from the supe-rior cervical ganglion. It is of note that levels of 5-HT instriatum were significantly up-regulated in B2� mutantscompared with normal mice (Fig. 6).

Removal of the Neomycin Selection CassettePartially Restores TH and Catecholamine Levels

No significant alterations in spontaneous movementsamong homozygous, heterozygous, and wild-type micewere found within or between neor-negative mice [stud-

ied in 65 A2– mice (wt n � 14, het n � 30, hom n � 21)and 50 B2– mice (wt n � 11 female, 7 male, het n � 5female, 13 male, hom n � 10 female, 4 male; data notshown). Analyzing the A2– and B2– mice by in situ probesfor the corresponding mutant receptors (A2 and B2, re-spectively) showed strong expression of the knock-in al-leles (Fig. 3B), whereas SN in the wild-type mice wasnegative. Furthermore, by recognizing the bicistronicmRNAs, these probes also provide indirect evidence thatTH expression is higher in the neor-negative strains com-pared with the strains carrying the neor cassette. DAneurons in SN were TH immunopositive (Fig. 4M),whereas TH immunoreactivity was virtually absent in the

Fig. 4. Immunostaining for dopaminergic neurons in substantia nigra(A,B,E,F,I,J,M,N) and dopaminergic innervation of the striatum(C,D,G,H,K,L,O,P). TH antibodies were compared with antibodiesagainst AADC and against the dopamine transporter (DAT). TH- andAADC-positive neurons of SN appear similar in wild-type (A,B) andB2� heterozygous (E,F) mice. TH immunoreactivity is virtually un-detectable in SN of B2� homozygotes (I), whereas immunoreactivityfor AADC shows that dopaminergic neurons are still present (J). M:After elimination of the neor cassette, TH immunoreactivity is partiallyrestored in the B2– homozygotes. N: Presence of dopaminergic neu-

rons in B2– homozygotes as seen with AADC staining. Wild-type(C,D) and B2� heterozygotes (G,H) show abundant nerve fibers instriatum by both markers. In contrast, a severe reduction of THimmunoreactivity is seen in B2� homozygotes (K), whereas the pres-ence of dopaminergic nerve fibers is demonstrated in striatum of thesemice by the DAT antibody (L). After elimination of the neor cassette,TH immunoreactivity was only partially restored in striatum of the B2–

homozygotes (O), whereas DAT labelling demonstrates that the dopa-minergic nerves are still present (P).

Dopamine Dysfunction in TH-Hypomorphic Mice 449

Fig. 5. Levels of catecholamines determined in various areas of themice. A: DA is radically reduced in SN in B2� homozygous mice,whereas the A2� allele contributes less to a reduction in DA. Uponelimination of the neor cassette (neo– mice) a distinct but modest DAreduction is seen, most notably in the B2– homozygotes. B: DA instriatum was reduced to 3% of wild-type levels in the B2� homozy-gotes. Among the neo– mice, a reduction was seen only in the B2–

mice. C: NA in the hippocampal formation, indicating reduced func-tion of locus coeruleus neurons in mice carrying one or two of themodified TH alleles with neor. The most robust effects were seen whenboth TH genes carried the selection cassette. Among the mice with the

cassette eliminated by recombination, only B2– homozygotes showedsignificantly reduced NA levels. D: NA in the submandibular glandderiving from innervation by the superior cervical ganglion. The high-est levels of reduction were seen with A2� and B2� present in bothcopies of the TH gene. Wt n � 34 (12 male, 22 female), A2� het n �11 (6 male, 5 female), B2� het n � 12, A2� hom n � 6 (3 male, 3female), A2�/B2� n � 4 (2 male, 2 female), B2� hom n � 9 (4 male,5 female), A2– het n � 15 (4 male, 11 female) B2– het n � 12 (8 male,4 female), A2– hom n � 8 (8 female), B2– hom n � 5 (2 male, 3female).

450 Althini et al.

striatal nerve terminals of B2– homozygous mice (Fig.4O). The partial restoration of TH levels by eliminatingthe neor cassette was further supported by the levels of DAand NA found in the neor-negative mice (Fig. 5).

DISCUSSIONBy using a knock-in strategy, we have established

four different TH alleles showing graded TH hypomor-phism in the mouse. Two constructs were made for ho-mologous recombination, both targeting the TH locus inthe 3�UTR and carrying an frt-flanked neor selectioncassette. Our aim was to study the effects of impaired BMPsignaling in catecholaminergic neurons through the intro-duction of dominant-negative BMP receptors. To achievethis, TH alleles were constructed that allow for expressionof bicistronic mRNAs coding for both endogenous THand either of two truncated BMP receptor proteins (A2 orB2). A neor cassette flanked with frt sites allowed ES cellselection as well as removal of frt-neor in vivo, the latter bybreeding with the Flp deletor mouse. This led to thefollowing mouse strains (see Fig. 1): A2� (TH-IRES-A2-frt-neor-frt), B2� (TH-IRES-B2-frt-neor-frt), A2– (TH-IRES-A2), and B2– (TH-IRES-B2).

It is obvious from the characteristics of these mouselines that the insertion of the neor cassette is contributingstrongly to the reduced expression of TH mRNA andprotein. This, in turn, results in mild to severe reductionsof the catecholamine neurotransmitters in SN, striatum,hippocampus, and submandibular gland (Fig. 5). The mostsevere effect was found in striatum of B2� homozygotes,with only 3% dopamine, compared with wild-type con-trols. Consistently with these results, mice carrying the

neor cassette were less prone to move about and weresmaller than their littermates and mice with the neor

removed from the altered TH allele (Fig. 2). By excisingthe neor cassette, we get partial rescue of TH expressionand production of catecholamines as well as an increase inspontaneous movement.

The method used for gene alteration utilizes theendogenous TH locus to drive the expression of thetransgene, without destroying the target gene (first de-scribed by Mountford and Smith, 1995). This wasachieved by the introduction of an encephalomyocarditisvirus IRES followed by the transgene in the 3�UTR ofTH. If this alteration abolished TH expression completely,very few pups homozygous for the mutation should havebeen born, because the TH knock-out is lethal (Kobayashiet al., 1995; Zhou et al., 1995; Rios et al., 1999). Thephenotype found in the present A2� and B2� homozy-gotes, including decreased body mass and locomotor ac-tivity, is reminiscent of that found in mice described byZhou et al. (1995) with targeted disruption of the THgene. This null mutation was embryo lethal, except for asmall fraction of mice that survived to term (30 of 1,184TH–/–). These 30 mice all showed runt properties andfailed, unlike their littermates, to balance on a rotatingpencil or walk up an incline. In contrast to our knock-inmice, which live through adulthood, the TH–/– mice diedby 4 weeks of age. If catecholamine precursor compoundsare added to the drinking water of pregnant dams, THdeficient pups can be rescued in utero (Rios et al., 1999)and will survive postnatally. These rescued mice also dohave a phenotype similar to that of our homozygous A2�

and B2� mice.Given the orientation of the neor cassette (reversed

compared with that of the TH gene), the TH-IRES-A2/B2-neor mRNAs may be subjected to a knock-downeffect caused by interference of the neor transcript and theantisense neor sequence in the bicistronic mRNA (Fig. 1).A strong argument for the selection marker contributingto the TH hypomorphism is the rescuing effect on thephenotype seen after excision of the frt-neor cassette.Furthermore, another knock-in mouse provides supportfor the influence of the orientation of the neor cassette: Inthis case, the TH allele was altered to carry a TH-IRES-Cre-neor construct but with the neor cassette in the samedirection as the endogenous TH gene (Huang et al., 2002;Lindeberg, 2002; Lindeberg et al., in preparation). Thismouse is coexpressing TH and Cre recombinase andshows none of the above-reported TH hypomorphic phe-notypes.

It is clear that the reduced levels of TH mRNA inneor-positive strains cannot be explained as a result ofincreased dopaminergic cell death. The in situ hybridiza-tion shows that, whereas homozygous mice show barelydetectable labeling for TH mRNA in SN, a probe forAADC detected normal numbers and morphology of do-paminergic neurons in this area. In that AADC is anenzyme farther along the catecholamine biosyntheticpathway and is expressed in the same cells as TH, the

Fig. 6. A: 5-HT is up-regulated in the striatum of B2� homozygousmice. B: A similar alteration was not found in B2– mutants.

Dopamine Dysfunction in TH-Hypomorphic Mice 451

presence of AADC mRNA strongly suggests that thedopaminergic neurons are present but fail to express TH inthe homozygous mice (Fig. 3). It should, however, benoted that AADC is expressed also by other cells typesthroughout the body. In our midbrain sections, though, itseems clear that AADC is preferentially expressed by neu-rons in SN and VTA.

Reversions from hypomorphic to wild-type condi-tions upon excision of selection cassettes have been notedpreviously. Both Meyers et al. (1998) and Nagy et al.(1998) found that gene function was restored when theselection markers were eliminated in their FGF-8 andN-myc hypomorphs, respectively. Later, similar findingshave been made by Rucker et al. (2000) from study of aBcl-x hypomorph created by insertion of a loxP-flankedneor cassette into the promoter region of the gene. UponCre-mediated excision of the neor, Bcl-x function wasrestored. Recently, Kulessa and Hogan (2000) reported ona hypomorphic BMP4loxP-lacZ-neo allele that was pheno-typically improved by the removal of the neor cassettefrom its position in an intron. The present findings thusextend the observations of neor-mediated hypomorphiceffects to include also the insertion of the selection cassette(opposite orientation) into the 3�UTR of the gene ofinterest. The mechanisms causing the hypomorphism,however, may be of a different nature in our case com-pared with the previously reported cases.

Because it is documented that BMPs have neurotro-phic effects on catecholaminergic neurons (Jordan et al.,1997; Bengtsson et al., 1998), we examined the effects ongrowth retardation or cell survival of the dominant-negative receptors after removal of the neor cassette (i.e.,in the A2– and B2– animals). The immunomarkers TH aswell as AADC and DAT for catecholaminergic neurons inSN and nerve terminals in the striatum, respectively, gavesome indications of impaired BMP function in B2– mice.TH protein expression in SN is higher in the B2– than inthe B2� homozygotes but lower than in wild types (Fig.4). In nerve terminals projecting to striatum, TH expres-sion is down-regulated in both B2– and B2� transgenicmice (Fig. 4). The nerve terminals are still present though,in that no significant decrease in strength of the DATsignal was detected. Although mild, some effects on theSN neurons might be ascribed not solely to the hypomor-phic effect on the TH locus but also to the dominant-negative BMP receptor. This may fit with the finding that,in the B2– mice, dopamine is most markedly down-regulated in striatal neurons (Fig. 4B).

DA and NA are products in the synthesis pathway inwhich TH is the first and rate-limiting enzyme. Measuringthe concentration of these neurotransmitters in the THhypomorphic mice gives insight into the effects of ourdifferent genotypes with respect to the catecholaminergicsystems. B2� mice have only 3% of normal levels of DAin striatum. Even with this severe dopamine deficiency,the mice survive to adulthood. We find elevated levels of5-HT in striatum of the B2� homozygotes. This phenom-enon may be a way for the brain to compensate for the loss

of dopamine. Similar findings have previously been madein mice with chemical (MPTP) lesions of SN (Luthman etal., 1993). The compensatory up-regulation in our case isseen in mice with morphologically intact DA neurons,demonstrating that the altered 5-HT levels depend on thecatecholamine levels rather than being evoked by lesion-ing of dopaminergic neurons.

TH deficiency leading to altered catecholaminebiosynthesis has recently been recognized as the cause ofL-dopa-responsive dystonia and infantile parkinsonismin humans (Grattan-Smith et al., 2002). Fewer than 20cases have been described so far, and a few TH muta-tions have been linked to these cases. The severity ofthe symptoms seems to be determined by the residualTH activity caused by the different mutations. Noalterations in morphology or neuronal survival or deathin catecholaminergic systems have been linked to thesedisorders. A common feature of the TH-deficiencypatients is that they all respond to L-dopa treatment.Typically, affected infants with relatively high residualTH activity suffer from dystonia, whereas patients withlower residual activity show hypokinetic, rigid parkin-sonism. Less specific symptoms, such as swallowingdifficulties and ptosis, have also been noted.

A significant decrease in body weight was seen inthe most TH hypomorphic mice, and one cause mightbe that they eat less than their littermates because ofswallowing problems. The dopaminergic system is in-volved in the regulation of many processes, though,including the regulation of appetite. Our mice are lessprone to move about, and one possible explanation fortheir reduced size could be that they, for this reason, donot go searching for food. An interesting similaritybetween our mice and TH deficiency in man is thedrooping eyelids (ptosis, Fig. 1D). This is presumablycaused by sympathetic hypofunction in innervation oflevator muscles of the upper eyelid.

The different mouse strains described in this paperrepresent a novel spectrum of phenotypes, showing gradedTH hypomorphism and reduction of catecholamines. Wehave made efforts to gather evidence for inhibited BMPsignaling resulting from the expression of truncated recep-tors on catecholaminergic neurons, driven by the THlocus: Despite the dominant-negative properties of A2 andB2, confirmed in cell culture experiments, no clear evi-dence was obtained for neuromodulatory effects of BMPsin the nigrostriatal system using the present approach. Themice created will nevertheless provide new tools for re-search on the catecholaminergic system having relevancefor Parkinson’s disease and L-dopa-responsive dystonia.

ACKNOWLEDGMENTSHelp was obtained from the Uppsala University

Transgenic Facility. We are grateful to Liliana Minichiellofor sharing her frt-neor cassette, to Susan Dymecki forproviding the Flp deletor mice, and to Sten-Magnus Aq-uilonius for suggestions on L-dopa-responsive dystonia.

452 Althini et al.

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