THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 35, Isaue of December 15, pp. 26494-26502,1993 Printed in U.S.A.

The 5”Flanking Region of the Rat Synapsin I Gene Directs Neuron- specific and Developmentally Regulated Reporter Gene Expression in Transgenic Mice*

(Received for publication, April 22, 1993, and in revised form, July 6,1993)

Christine HoescheSO, Angela SauerwaldSO, Rudiger W. Vehll, Bernd KripplII , and Manfred W. KilimannS** From the Slnstitut fur Physwlogische Chemie (Abteilung fur Bwchemie Supramlekularer Systeme), the llnstitut fur Anatomie, and the IlZnstitut fur Physwlogische Chemie (Abteilung fur Bwchemie des Zntermediiirstoffwechsek), Ruhr-Universitat Bochum, 0-44780 Bochum, Germany

The expression of the synapsin I gene is neuron- specific and developmentally regulated. As a step to- ward characterizing the molecular mechanisms that are responsible for its transcriptional regulation in vivo, we have generated transgenic mice that carry the chloramphenicol acetyltransferase (CAT) reporter gene under the control of -4,300 nucleotides of 5’- flanking sequence of the rat synapsin I gene. In four independent transgenic mouse lines, high level CAT expression is observed specifically in the brain and other neural tissues. Two of these lines also exhibit notable CAT expression in testis. The transgene is expressed at similar levels in many different regions of the central nervous system. Immunohistochemical staining detects the CAT marker protein in various cell populations of neuronal morphology within the brain and the spinal cord. Transgene expression is develop- mentally regulated in a way that correlates well with the expression of the endogenous synapsin I gene. Both follow a characteristic, biphasic postnatal time course with a maximum around day 20. We conclude that the DNA region investigated contains cis-regulatory ele- ments sufficient to drive the expression of a reporter gene in a spatial and temporal pattern that resembles the expression of the endogenous synapsin I gene.

Synapsin I is a peripheral protein of synaptic vesicles and is believed to be involved in the modulation of neurotrans- mitter release. It is expressed in almost all neuronal cell types but has not been found in any nonneuronal cells (for recent reviews see De Camilli et al., 1990; Valtorta et al., 1992; Greengard et aL, 1993). Therefore, the synapsin I gene is of special interest as a model system to study neuron-specific gene expression. In contrast, the expression of many other typically neuronal gene products is either restricted to certain neuronal subtypes (e.g. nerve growth factor receptor (Patil et al., 1990), peripherin (Desmarais et al., 1992), type I1 sodium channel (Kraner et al., 1992)), overlaps with nonneuronal cell

* This work was supported by the Deutsche Forschungsgemein- schaft (Ki 324/2-4) and the Fonds der Chemischen Industrie. The costa of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 These two authors contributed equally to this work. ** Heisenberg Fellow of the Deutsche Forschungsgemeinschaft. TO

whom correspondence should be addressed Institut fin Physiolo- gische Chemie, Ruhr-Universitit Bochum, D-44780 Bochum, Ger- many. Tel.: 49-234-700-7927; Fax: 49-234-709-4193.

populations such as glia or endocrine cells (e.g. neuron-spe- cific enolase (Forss-Petter et al., 1990), amyloid precursor protein (Wirak et al., 1991)), or does both (e.g. dopamine /3- hydroxylase (Mercer et al., 1991)). Mechanisms involved in the control of the synapsin I gene may be of general relevance for neuronal gene expression. It is of particular interest whether there are master regulatory molecules that coordi- nately control multiple neuronal genes, comparable to the role of the MyoD family of transcription factors in muscle devel- opment (Weintraub et al., 1991). If they exist, they could provide unifying principles for an understanding of the devel- opmental program that leads to the expression of the termi- nally differentiated neuronal phenotype. In fact, a common silencer element inhibiting transcription in nonneuronal cells has recently been characterized in the regulatory regions of the type I1 sodium channel (Kraner et al., 1992), SCGlO (Mori et al., 1992), and human synapsin I (Li et al., 1993) genes, but it does not appear to be sufficient to confer full cell type specificity to the synapsin I gene’ (Li et al., 1993). Finally, the characterization of a pan-neuronally active promoter may provide a tool to target the expression of heterologous, biolog- ically active proteins to the nervous system of transgenic animals.

We have demonstrated previously by transient expression in cell culture that -4,300 nucleotides of 5“flanking sequence from the rat synapsin I gene (4.3Syn) can efficiently drive the expression of the CAT’ reporter gene in NS20Y neuro- blastoma cells but display much weaker promoter activity in L929 fibroblastoid cells (Sauerwald et al., 1990). Preferential promoter activity in neuronal cells has subsequently been confirmed for additional neuronal and nonneuronal cell lines.’ These transient expression experiments have demonstrated that the 4.3Syn region contains cis-regulatory elements im- portant for the cell type specificity of synapsin I transcription. However, it remained unknown how much of the complete regulatory information that is required for the correct spatial and temporal expression pattern of the synapsin I gene in vivo is contained within this DNA region. TO address this question, we have introduced the 4.3Syn-CAT fusion gene into the germ line of the mouse. In four independent lines of transgenic mice, we find high marker gene expression re- stricted to the nervous system, a developmental time course in brain that is similar to that of the endogenous synapsin I

‘A. Sauenvald, C. Hoesche, and M. W. Kilimann, unpublished

The abbreviation used is: CAT, chloramphenicol acetyltransfer- data.

ase.

26494

Neuron-specific Synapsin I Gene Promoter in Transgenic Mice 26495

gene, and we can detect CAT-like immunoreactivity only in neurons.

EXPERIMENTAL PROCEDURES

Construction and DNA Analysis of Transgenic Mice-The fusion gene was excised from plasmid pBL4.3Syn-CAT (Fig. lA) with SalI and SmaI and purified by cesium chloride gradient centrifugation and Elutip-d (Schleicher & Schuell) column chromatography. It was mi- croinjected into pronuclei of fertilized eggs isolated from inbred FVB/ N mice as described by Hogan et al. (1986). Manipulated eggs were subsequently implanted into pseudopregnant foster mothers.

Transgenic founder mice were identified by dot-blot hybridization analysis of genomic DNA prepared from tail clips (Hogan et al., 1986). Filters were probed with a 301-nucleotide long CAT DNA fragment (EcoRIIStyI; probe C, Fig. 1B). Labeling of the probe with digoxy- genin-11-dUTP, hybridization and detection was performed with a chemoluminescence detection kit as recommended by the supplier (Boehringer Mannheim). Five transgenic founder mice were identi- fied, and homozygous lines were established by breeding with FVB/ N mates. Homozygosity was determined by densitometry of dot blots and confirmed by test crossings with nontransgenic mice.

Tissue Extracts and CAT Assay-Mouse tissues were homogenized in 0.25 M Tris-HC1, pH 7.8. Homogenate5 were cleared by centrifu- gation (12,000 X g, 10 min at 0 "C), supernatants heated at 65 "C for 10 min and centrifuged again. Supernatants were stored at -70 "C. Protein concentrations were determined according to Bradford (1976). CAT activity was determined according to Gorman et al. (1982) using 40 p~ acetyl-coA and 0.05 pCi of ["C]chloramphenicol (53 Cilmol, Amersham Corp.) in a 200-pl reaction volume at 37 "C. The maximal protein quantity per reaction was 120 pg; extracts from CAT-expressing tissues were diluted to be within the linear range of the reaction. Activity is given as percent conversion (total acetylated

/

. , EcoRI

0 61tnl KpnI Irogmenl

Sa E H H K R € 5 1 : . "En H K R €SI Sm

4.3 Syn ICATISVI 4.3 syn lUT lSV

A E t A 0 c - - IC" -

1 knl

FIG. 1. Transgene construction. Panel A , plan of the pBL4.3Syn-CAT expression plasmid from which the transgene was excised. The plasmid is based on pUC18 and contains, in its multiple cloning site, -4,300 nucleotides of 5'-flanking sequence of the rat synapsin I gene including its transcription start site and 105 nucleo- tides of 5"untranslated sequence (Sauerwald et al., 1990), the Esch- erichia coli CAT gene, and the SV40 splice and polyadenylation signal sequences ( S V ) from pSV2-cat (Gorman et al., 1982). Panel B, plan of the predicted transgene structure in tandem orientation and loca- tion of hybridization probes within the transgene. Approximate po- sitions of restriction sites are indicated E, EcoRI; H, HindIII; K, KpnI; R, RsaI; Sa, SalI; Sm, SmaI; St, StyI.

products versus input of ['4C]chloramphenicol). To analyze CAT expression in various tissues, hemizygous or

homozygous mice were used as indicated. Dissection of brain regions was carried out under a stereomicroscope. Spinal cord was taken from cervical vertebrae 3-7 and skeletal muscle from the thighs and hind- limbs. From the eye bulbs, the lens was removed before homogeni- zation. For developmental studies of CAT and synapsin I mRNA expression, homozygous transgenic males were bred to nontransgenic BALB/c females. Day E0.5 is defined as noon of the day that a vaginal copulation plug was present. Whole heads were used from the embryonic stages and whole brains from the postnatal developmental stages.

Immunocytochemistry-Adult mice under deep anesthesia were fixed by cardia1 perfusion with 4% paraformaldehyde, 0.05% glutar- aldehyde, 0.2% picric acid in 0.1 M phosphate buffer, pH 7.4, followed by a 30-min perfusion with 5% saccharose in the same buffer. The brains and spinal cords were dissected into pieces and, after prein- cubation in 30% saccharose, frozen at -50 "C in 8% methylcyclo- hexane, 92% isopentane. Immunocytochemistry was done on 25-pm cryosections. In brief, sections were treated with 1% sodium borohy- dride in phosphate-buffered saline followed by permeabilization with 0.3% Triton X-100, 0.005% phenylhydrazine for 30 min. Sections were incubated for 36 h with primary antibody at 4 "C (rabbit poly- clonal anti-CAT from 5Prime-3Prime Inc., Paoli, PA; diluted 10,000- 20,000-fold in phosphate-buffered saline containing 0.3% Triton X- 100 and 10% goat serum), for 24 h with secondary antibody at room temperature (biotinylated goat anti-rabbit IgG from Vector/Camon, Wiesbaden, Germany; diluted 2,000-5,000-fold) and for 6 h at room temperature with avidin-biotinylated peroxidase complex (ABC, Vec- tor/Camon; diluted 1,000-fold). Peroxidase was finally visualized with 1.4 mM 3,3'-diaminobenzidine in a solution of 10 mM imidazole in 50 mM Tris-HC1, pH 7.6, supplemented with 0.3% nickelous ammonium sulfate and 0.015% H202 for 3 min. In some experiments (e.g. Fig. 5), an anti-biotin antibody and an additional secondary antibody step were introduced before the ABC incubation. However, sensitivity was not significantly improved by this measure.

Histological structures clearly visualized under these conditions in the transgenic mice (see Figs. 3-5 and observations described in the text) did not stain in corresponding sections from nontransgenic control animals processed simultaneously under the same conditions. From strain 78, two animals were analyzed immunocytochemically and exhibited staining of the same characteristic cell populations. Staining of CAT-like immunoreactivity in line 78 was abolished by preincubation of the primary antibody with 5 pg/ml purified CAT (Sigma). Sensitivity could not be enhanced by raising the concentra- tion of the primary antibody; increasing nonspecific background staining of all cellular structures in nontransgenic as well as trans- genic animals was obtained when dilutions less than 1:10,000 were employed. This background behavior has been described by the sup- plier (Harlan et al., 1991). Even at high dilutions, the primary anti- body stained some small, circumscript cell populations very inten- sively also in nontransgenic mice. These are the nucleus supraopticus and the nucleus paraventricularis hypothalami (see Fig. 3, arrow- heads) and the posterior pituitary to which both nuclei project their neurosecretory processes (not shown). This type of nonspecific stain- ing could be antagonized by higher concentrations of purified CAT (25 pg/ml) and probably reflects the presence of an epitope in these cells related to CAT or to a contaminant of the CAT preparation. Weak to moderate staining of the cerebellar Purkinje cells was also seen in nontransgenic as well as in transgenic mice.

RNA Blot Analysis-RNA was purified from rat or mouse brains and from murine neuroblastoma cells (cultured with 10 or 0.25% fetal calf serum as described by Sauerwald et al., 1990) by the LiCl/urea method (Auffray and Rougeon, 1980), denatured by glyoxalation, resolved on an agarose gel, and blotted to nylon membranes according to conventional procedures. Blots were hybridized at high stringency (Kilimann and DeGennaro, 1985) with 32P-labeled, nick-translated cDNA probes. The rat brain and NS2OY cell RNA blots were probed with the nearly complete rat synapsin I cDNA insert from plasmid pSyn5, extending from the PstI site at codon 26 for -3,200 nucleotides downstream (Kilimann and DeGennaro, 1985; Sauerwald et al., 1990). The mouse brain RNA blot was probed with a mouse synapsin I fragment that extends from the EcoRI site at codon 244 for -2,300 nucleotides downstream.' The 18 S rRNA reference probe (with which the blots were hybridized after removal of the synapsin I probes) was a 1.95-kilonucleotide EcoRIISalI fragment of mouse genomic DNA including 1.4 kilonucleotides of 18 S sequence (plasmid pMSalC; Grummt and Gross, 1980). Autoradiograms of blots were quantified

26496 Neuron-specific Synapsin I Gene Promoter in Transgenic Mice by densitometry with a laser densitometer (Pharmacia LKB Biotech- nology Inc.), and measurements were standardized on equal quantities of 18 S rRNA per lane.

RESULTS

Production of Transgenic Mice Carrying a 4.3Syn-CAT Fu- sion Gene-The expression plasmid, pBL4.3Syn-CAT, char- acterized previously in cell culture (Sauerwald et al., 1990) contains -4,300 nucleotides of 5"flanking sequence and 105 nucleotides of 5'-untranslated sequence of the rat synapsin I gene, fused to the CAT reporter gene (4.3Syn-CAT; Fig. L4). The construct comprises the transcription start site of the endogenous rat gene (Sauerwald et al., 1990). This is also the main transcription start site recognized when pBL4.3Syn-

line no. 26 31 85 77 78 C 26 31 - 21 -

5.1 - 3.5-

2.0 -

0.9 - FIG. 2. Southern blot characterization of genomic DNA

from transgenic mice. 10 pg of genomic DNA was digested with KpnI, resolved by electrophoresis through a 0.7% agarose gel, and transferred to a nylon membrane. The blot was hybridized with a mixture of probes A and B (Fig. 1E) and developed by chemolumi- nescence. The lanes carry DNA from the five founder mice and a nontransgenic control (C) as indicated at the top. Founders 77 and 78 were apparently mosaics because their hemizygous offspring gave Southern blot hybridization signals approximately twice as intense (not shown). A t the left, the positions of DNA size standards are indicated (in kilonucleotides); in the middle, the positions of the hybridization signals caused by the endogenous synapsin I gene promoter region ( S ) and of the 6-kilonucleotide fragment derived from tandemly linked transgene units (7') are marked. The p a n e l on the rkht shows the blot of lines 26 and 31 with a shorter exposure time.

CAT is transiently expressed in NS2OY cells.' The 4.3Syn-CAT fusion gene (-6.0 kilonucleotides) was

excised from the expression plasmid with SafI and SmaI (Fig. L4) and injected into the male pronuclei of fertilized mouse eggs. Dot-blot hybridization of DNA extracted from the tails of 28 live offspring with a 301-nucleotide long EcoRI/StyI fragment from the CAT sequence (Fig. 1, A and R , probe C) showed that five animals had integrated the transgene. All five founders were successfully bred to establish homozygous lines. Founders 77 and 78 were mosaics (cf. legend to Fig. 2) but from F1 on their progeny transmitted the transgene in a Mendelian fashion and with constant copy numbers, as did the other three lines. Southern blot patterns did not change between founders and their progeny, suggesting a single in- tegration site in each line (data not shown). In line 77 the transgene is only transmitted to male offspring, suggesting integration into the Y chromosome. For animals of F1 and later generations, approximate haploid transgene copy num- bers of 25 (line 26), 20 (line 31), 5-6 (lines 77 and 78) and 2- 3 (line 85) were determined. Fig. 2 shows Southern blots of genomic DNA from the five transgenic founders and a non- transgenic control mouse digested with KpnI. The blot was hybridized with an equimolar mixture of a 550-nucleotide long Hind111 fragment upstream from the KpnI site (Fig. 1B, probe A) and a 550-nucleotide long RsaI/EcoRI fragment from nucleotide -225 of the synapsin I promoter extending into the CAT coding sequence (Fig. lB, probe B). If the transgene is integrated in the usual multicopy tandem fashion, KpnI digestion is expected to give a 6-kilonucleotide fragment with a hybridization intensity proportional to copy number, and two junction fragments differing in size from line to line with single copy intensity (cf. Fig. 1R). Fig. 2 shows that line 78 gives a pattern in agreement with such a structure, whereas lines 26, 31, 77, and 85 display absent, additional, or more intense junction fragments, suggesting that some of their transgenes have been rearranged. However, all five strains give the diagnostic 6-kilonucleotide fragment (T) , indicating that at least two copies have been integrated in tandem. In all DNA samples, the probe also labels a 2-kilonucleotide band ( S ) , resulting from the endogenous synapsin I gene of the mouse.

Neural Tissue Specificity and High Penetrance of Expression of the CAT Transgene-To investigate transgene expression,

TABLE I CAT activity in tissues of adult 4.3Syn-CAT transgenic mice

For most tissues, means f SD. ( n ) of two to four independently prepared extracts are given. Most measurements were obtained from hemizygous animals. Spinal cord, eye, and trigeminal ganglion of strains 85, 31, and 26 were analyzed from homozygous animals. These values were divided bv 2 for better comDarison. ND. not determined.

CAT activity Tissue

Line 85 Line 78 Line 31 Line 26 Line 77 Nontrann~enic control

Brain Spinal cord Eye Trigeminal ganglion Pituitary gland Adrenal gland Testis Skeletal muscle Kidney Heart Liver Lung uterus Spleen

% conoer.sbn/mg protein 4,800 f 1,700 (4) 10,900 f 2,350 (3) 9,300 f 5,100 (4) 5,900 f 230 (2) 6,800 (1) 8,ooo ( 1 )

840 f 440 (2) 190 (1 ) 210 ( 1 ) 620 k 50 (2) 920 (1) 460 ( 1 )

1,150 f 480 (2) 580 f 170 (2) 108 f 20 (2) 13 f 3.0 (2) 16 f 3.7 (2) 2.2 f 0.4 (2)

1.2 f 1.2 (3) 2.7 f 4.4 (3) 350 f 90 (3) 4.6 f 1.5 (2) 18 f 7.0 (2) 2.4 f 0.2 (2) 20 f 15 (4) 0.8 f 0.8 (3) 0.2 f 0.1 (2)

2.4 f 1.3 (2) 0.7 f 0.0 (2) 0.7 f 0.4 (2) 0.6 f 0.7 (3) 0.5 f 0.3 (2) 0.5 f 0.0 (2) 2.9 f 3.0 (3) 1.1 f 0.4 (2) 0.8 f 0.4 (2) 38 f 5.0 (3) 1.4 f 0.9 ( 3 ) 1.2 f 0.4 (2) 1.3 f 0.5 (2) 0.4 f 0.6 (2) 114 f 96 (3)

x 60 min

430 f 120 ( 3 ) 1.420 (1)

86 ( 1 ) 60 ( 1 )

8 f 1.6 (2) 0.6 f 0.6 (2) 95 f 44 (3)

0.1 f 0.1 (2) 0.0 f 0.0 (2) 0.2 f 0.3 (2) 0.6 f 0.9 ( 3 ) 0.3 f 0.3 (2) 1.0 f 1.2 (3) 0.3 f 0.1 (2)

1.7 f 2.4 (2) ND N D ND

1.7 f 2.4 (2) 2.1 f 2.6 (2) 1.8 f 2.5 (2) 1.7 f 2.3 (2) 1.7 f 2.4 (2) 1.6 f 1.9 (2) 1.9 f 2.1 (2) 3.0 f 1.7 (2)

ND 1.7 f 2.4 (2)

0.0 ( 1 ) ND N D ND N D

0.0 ( 1 ) 0.0 ( 1 ) 0.0 (1) 0.0 (1) 0.0 ( 1 ) 0.2 (1) 0.4 ( 1 ) 0.0 ( 1 ) 0.0 ( 1 )

Neuron-specific Synapsin I Gene Promoter in Transgenic Mice 26497

FIG. 3. Survey light micrographs of hemispheres of line 31 and line 78 transgenic mouse brains stained for CAT immu- noreactivity. Strongly expressing principal neurons in line 31 ani- mals are found in the CA3 region of the hippocampus (h) , and in the granular (g) and agranular ( a ) retrosplenial cortices, sharply decreas- ing at the border (arrow) to area 2 of the frontal cortex. Principal neurons of moderate immunoreactivity are found in the parietal cortex (pa; predominantly in layer 111), the piriform cortex (pi), and the nucleus of the lateral olfactory tract (not). Single heavily stained neurons, possibly interneurons, are found throughout the cortex, all areas of the hippocampus, and (seen on other sections) in the amyg- daloid nucleus. Immunopositive fibers or terminals are apparent, in addition to the above mentioned regions, in the caudatoputamen (cp), the stria terminalis ( s t ) , and the corpus callosum (cc). Arrowheads mark the supraoptic and paraventricular hypothalamic nuclei that show intense unspecific staining (see “Experimental Procedures”). In line 78 animals, intensely positive principal neurons are found espe- cially in lamina IV/V of the parietal cortex. Neurons of strong or moderate immunoreactivity are also found in the basolateral amyg- daloid nucleus (dl), the CA3 region of the hippocampus, the piriform cortex, other nuclei of the amygdaloid complex, throughout the hy- pothalamus (hy) , the anterior paraventricular thalamic nucleus (apt ) , the caudatoputamen (which is devoid of labeled cell somata in line 31), and some subnuclei of the lateral habenular complex. Heavily stained interneurons are found throughout the cortex, all areas of the hippocampus, and the other regions mentioned above. Positive fibers or terminals are apparent in the caudatoputamen, the stria terminalis, the supraoptic decussation (so), and the corpus callosum, whereas the fibers of the optic tract are completely negative. The bar represents 1 mm.

CAT activity was measured in extracts of various tissues (Table I). Line 77, apparently integrated into the Y chromo- some, displays only marginal or insignificant expression of the transgene in all tissues analyzed. Possibly, insertion into the extensive, transcriptionally silent heterochromatin re- gions of the Y chromosome is responsible for this, although other reasons for the complete loss of transgene expression cannot be excluded at present.

The other four lines all exhibit high levels of CAT activity in brain and spinal cord and lower levels in eye, trigeminal ganglion, and pituitary gland, the tissues that also express endogenous synapsin I. Expression in line 26, the line with the highest transgene copy number, is significantly lower than in the other three lines which have very similar levels of expression. In the trigeminal ganglion, neurons contribute only a small part of the tissue mass, which largely consists of myelinated nerve fibers, endoneural connective tissue, and Schwann cells. In the pituitary gland, endogenous synapsin I, expressed by hypothalamic neurons, has been found in the

neuroendocrine terminals of the posterior pituitary but not in the endocrine cells of the anterior pituitary (De Camilli et al., 1979, 1983; Goelz et al., 1981). I t is therefore likely that pituitary CAT in transgenic animals also originates from the hypothalamic neurons projecting to the posterior pituitary; as can be seen below (Figs. 3-5), CAT efficiently diffuses into the neuronal processes. CAT activity in adrenal gland is low but significant in the three highly expressing lines and may reflect eutopic expression by the sporadic sympathetic neu- rons within the adrenal medulla.

In addition to these tissues, low but significant transgene expression is found in a few ectopic tissues (line 85: muscle, kidney, and uterus; line 7 8 muscle; line 31: muscle and spleen) but is at least 2 orders of magnitude lower than in brain. Marginal CAT activities often measured in the other tissues of the transgenic lines (detection limit, -0.2%) may be a result of peripheral neurons in some tissues or diffusion of CAT into the neuronal processes and synapses innervating these tissues. The tissue pattern of ectopic expression differs from line to line. This suggests that ectopic expression is caused by position effects depending on the integration sites or by rearrangement of some of the multiple transgene copies found in lines 26, 31, or 85 (see above).

There is one notable exception to the rule that ectopic 4.3Syn-CAT expression is very low compared with the neural tissues and differs from line to line. Lines 26 and 31 express CAT in the testis, and in both lines expression in testis is relatively high, second only to brain and spinal cord, and close to or above eutopic expression in eye, trigeminal ganglion, and pituitary gland. Remarkably, in transgenic mice express- ing the 8-galactosidase gene under the control of the neuron- specific enolase promoter, high 8-galactosidase mRNA levels were found in testis of both lines that were assayed for expression in this tissue (Forss-Petter et al., 1990). Neither synapsin I nor neuron-specific enolase is physiologically ex- pressed in testis. However, more and more proteins previously thought to be typically neuronal are being found in testis too (e.g. Persson et al., 1990; Ibanez et al., 1991). I t is intriguing that in recent years several transcription factors have been identified which are expressed in both tissues, brain and testis (e.g. Suzuki et al., 1990; Stoykova et dl., 1992; Adams et al., 1992; for review see Rosenfeld, 1991). Possibly, some testicu- lar cell type(s) provide(s) nearly all of the conditions (such as transcription factors or chromatin structure) that are needed for the transcription of certain neuronal genes like those of synapsin I or neuron-specific enolase, so that positional ef- fects are then frequently sufficient to provide the last require- ments or to overcome the last constraints for the activation of their promoters.

4.3Syn Directs CAT Expression in Many Regions of the Brain-Table I1 shows that in the four transgene-expressing lines CAT activity is high in all brain regions analyzed. Relative activities between the different regions vary from strain to strain (presumably reflecting position effects) but are always within the same order of magnitude, indicating transgene expression throughout the brain.

Immunocytochemical Detection of the Transgene Product in Neurons of the Brain and the Spinal Cord-To document in more detail the histological localization of transgene expres- sion within the nervous system and particularly to address the question whether it occurs specifically in neurons, we carried out immunocytochemistry with a commercial anti- CAT antibody. In all four transgene-expressing lines, cell populations were stained; according to their morphological appearance, they were indeed neurons. Figs. 3-5 show exam- ples, and Table I11 lists a number of characteristic, CAT-

26498 Neuron-specific Synapsin I Gene Promoter in Transgenic Mice

TABLE I1 CAT activity in brain regions of adult 4.3Syn-CAT transgenic mice

Animals from lines 85,31 and 26 were homozygous. Values from homozygous animals were divided by 2 for better comparison with Table I. Activity was determined in single or in duplicate independently prepared extracts as indicated. Animals from line 78 were hemizygous.

CAT activity Area

Line 85 Line 78 Line 31 Line 26

’36 conuerswn/mg protein X 60 min Olfactory bulb 3,000/3,700 4,200 7,000 500 Medulla oblongata 7,700/3,600 5,200 13,700 2,300 Cerebellum 28,900/23,000 6,400 3,500 400 Cerebral cortex

Hindlimb area 2,400/4,300 1,400 7,600 600 Parietal area 3,600/6,200 3,900/7,200 10,700 1,400 Retrosplenial area 2,900/4,900 2,000/3,100 12,400 2,600

Amygdaloid area 4,400/2,300 4,800 5,900 1,400 Hippocampus 3,900/4,700 3,000 7,600 1,500 Thalamus 6,500/5,800 5,200 5,100 2,100 Caudatoputamen 4,600/4,100 3,300 2,600 800

TABLE I11 Occurrence of CAT-immunoreactive neurons in various regions of the

nervous system of 4.3Syn-CAT transgenic mice CAT-like immunoreactivity was characterized by visual compari-

son as high (+++), moderate (++), light (+) and undetectable (0). ND, not determined.

CAT-like immunoreactivity

Line85 Line 78 Line 31 Line26 Area

Cerebral cortex Retrosplenial area 0 ++ +++ ++ Parietal area, layer 111 0 + ++ 0 Parietal area, layers IV-V ++ +++ 0 0 Piriform area 0 ++ +++ 0

CA3 area 0 ++ +++ 0 Sporadic interneurons ++ +++ ++ 0

Paraventricular thalamic nu- + ++ + 0

Lateral habenular nucleus ++ ++ + 0 Amygdala + +++ +++ ++ Hypothalamus ++ +++ + 0 Spinal cord

Dorsal horn ++ +++ ++ 0 Ventral horn 0 + + 0

Cerebellum 0 0 0 0 Medulla oblongata ND + 0 ND Trigeminal ganglion ND 0 0 ND Olfactory bulb ND 0 ND ND

immunopositive areas of the nervous system in the four mouse lines. These cells were never stained in nontransgenic controls under the same conditions (e.g. Fig. 4, e and f ; see “Experi- mental Procedures” for additional controls).

Only certain populations of neurons are stained under these conditions. However, Tables I and I1 demonstrate that CAT expression is not restricted to these cells. CAT enzymatic activity is high also in well dissectable structures such as the cerebellum, olfactory bulb, medulla oblongata, and trigeminal ganglion, where specific staining of CAT-like immunoreactiv- ity was undetectable or very low (Table 111). Thus, it appears that the antiserum, with its limited sensitivity (see “Experi- mental Procedures”), visualizes only a subset of neurons among a much broader cell population expressing the trans- gene throughout the nervous system. Specific cell types may be immunopositive because they express particularly high CAT concentrations, but technical factors such as differences between cell types in the efficiency of CAT fixation or anti- body permeation may also contribute to selective staining. It is well known that CAT stains poorly with the available antibodies. As an alternative method, we have tried CAT

Hippocampus

cleus

enzyme histochemistry, which has been successfully employed to visualize CAT expression in muscle under the control of the myosin light chain promoter (Donoghue et al., 1991). However, we did not obtain any staining with this method, presumably because its sensitivity is even lower. Donoghue et al. were working with animals in which muscle CAT activity is 2 orders of magnitude higher than brain CAT activity in our mice.

Different levels of CAT expression in different neuronal populations could correlate with differences in endogenous synapsin I gene transcription between these cells, but they could also be artifacts of the transgene. Quantitative or qual- itative differences of transgene expression levels among the eutopic cells have been observed in other transgenic animals. For example, lacz expression under the control of the dopa- mine 8-hydroxylase promoter was found only in a subset of the dopamine 8-hydroxylase-expressing cells (Mercer et al., 1991), and CAT expression (detected with the same antibody that we have used in our experiments) in liver under the phenylalanine hydroxylase promoter was much more hetero- geneous among hepatocytes than endogenous phenylalanine hydroxylase expression (Wang et aZ., 1992).

In spite of these limitations, the following conclusions can be drawn from the immunocytochemical results. (i) All of the cells positive for CAT-like immunoreactivity which we can see are neurons, according to their morphology (Figs. 4 and 5). Glia-dominated regions, such as the corpus callosum or the internal capsule, are negative for CAT-like immunoreac- tivity except for some prominent commissural fiber tracts (Fig. 3). (ii) Although the staining patterns of the four lines of mice differ to some degree, there are common traits. Table I11 shows that certain cell populations are immunopositive in several mouse lines. The amygdala is stained in all four lines, and the retrosplenial cortex, the lateral habenula, the para- ventricular nucleus of the thalamus and the area gelatinosa of the dorsal horn of the spinal cord are stained in three of the four lines. The two most highly expressing lines, 31 and 78, have a number of additional CAT-immunopositive regions in common. (iii) Within the cerebral cortex, there is a specific difference between the pair of lines that express the transgene in the nervous system only (78 and 85) and the pair that also express it in testis (26 and 31). Lines 78 (Fig. 3) and 85 (not shown) share marked expression in the lower parietal cortex (layers IV and V), whereas CAT-immunopositive cells in lines 26 (not shown) and 31 (Fig. 3) are most prominent in the retrosplenial cortex.

A study of regional synapsin I mRNA expression in rat

Neuron-specific Sympsin I Gene Promoter in Transgenic Mice 26499

FIG. 4. Characteristic CAT-im- munopositive neuronal cell popula- tions at higher magnification. Panel a, prominent staining of the retrosple- nial cortex in line 31 animals. Immuno- positive principal neurons are restricted to the upper laminae of the granular and agranular retrosplenial cortex, whereas interneurons are additionally found in the lower layers; the border between the agranular ( top) and granular (bottom) retrosplenial cortex is sharply marked (arrow). Panel b, in the hippocampus of line 31 mice, CAT-immunopositive prin- cipal neurons are localized predomi- nantly in the CA3 region. Note the loss of immunoreactivity from CA3 to CA1, leaving the latter region largely negative. In line 78 animals, positive principal neurons are prominent in the basolateral amygdaloid nucleus (panel c) and lami- nae IV/V of the parietal cortex (panel d ) . Nontransgenic control animals are immunonegative, e.g. in the parietal cor- tex (panel e ) and hippocampus (panel f ) . The bar indicates 100 pm.

FIG. 5. CAT-immunopositive neurons and fibers in the spinal cord. In line 78 animals the survey mi- crograph (panel a ) shows CAT-immu- noreactive neurons throughout the spinal cord. Heavy staining of small in- terneurons, fibers, and terminals is ob- served in the dorsal horn, especially in lamina 2 (panel b). Moderately or lightly stained large motor neurons are seen in the ventral horn (panel c ) . The bars in- dicate 200 pm (panel a ) and 100 pm (panels b and c), respectively.

26500 Neuron-specific Synapsin I Gene Promoter in Transgenic Mice

g31 n EW 5 m 1 5 2 0 2 5 3 1

FIG. 6. Developmental time course of CAT expression in transgenic lines 26, 31, 78, and 85. CAT activity is given in percent conversion/mg of protein X 60 min. Embryonal (E), neonatal ( N ) , and postnatal ( P ) ages are indicated at the abscissa. Adult mice ( A ) were 55-71 days of age. Hemizygous offspring were analyzed, resulting from the mating of homozygous transgenic males with nontransgenic BALB/c females. CAT activity was determined in extracts of whole heads for the embryonal and neonatal stages (tri- angles) and in extracts of brains for the neonatal and postnatal stages (circles). Heads from whole litters were pooled for the embryonal samples, and brains from four to one animals, depending on age and line, were taken for the postnatal samples. Data points represent the mean of duplicate activity measurements of one extract sample. Extracts of line 85 (P15 through A ) and of line 26 (E12, E17, and N ) were prepared twice, with very similar activities (the figures give the means).

brain by in situ hybridization has been published recently (Melloni et al., 1993). In this work, particularly high levels of synapsin I mRNA were found, e.g. in the CA3 field of the hippocampus, in various regions of the cerebral cortex, and in the amygdala, i.e. in areas where we also find prominent CAT-like immunoreactivity in two or more of our mouse lines. Although other cell populations found to express high levels of synapsin I mRNA, such as the mitral and internal granular layers of the olfactory bulb and the cerebellar granule cells, were negative for CAT-like immunoreactivity in our experiments, it seems that there is at least a partial overlap of cell groups that highly express the endogenous synapsin I gene and the transgene.

Transgene Expression and Synapsin I mRNA Levels Follow Similar Developmental Time Courses-Fig. 6 shows the de- velopmental time course of CAT activity in the brains (pre- natally: heads) of the four transgene-expressing mouse lines. All four lines display similar time courses with a gradual increase in the late prenatal phase, a plateau during the early postnatal days, a steep rise between postnatal days 15 and 20, and a characteristic maximum around postnatal day 20. The three highly expressing lines show particularly similar time courses of expression.

The transcriptional time course of the transgene results from the interaction of the rat synapsin I gene promoter region with its environment (transcription factors etc.) in the murine host cells. For comparison, we have therefore deter- mined the developmental profile of the endogenous synapsin I mRNA levels in the brains of both species, rat and mouse. Fig. 7, A and B, shows that in both species, the predominant 3,400-nucleotide synapsin I mRNA bands follow very similar bimodal time courses, both with a maximum around day 20. The minor 4,500-nucleotide mRNAs go through a maximum around postnatal days 10-15, after which they decline sub-

stantially. These results are qualitatively similar to the data of Haas and DeGennaro (1988) on the postnatal expression of synapsin I mRNA in rat cerebellum, except that we can clearly detect the 4,500-nucleotide mRNA throughout post- natal development.

Thus, the developmental courses of the endogenous syn- apsin I mRNA and the transgene product are qualitatively similar, with a characteristic maximum around day 20. Quan- titatively, there is a significant difference in that the CAT expression of all four lines has a sharper developmental profile. The onset of the main postnatal induction is later and steeper, and the increase between the early postnatal days and day 20 is 6-15-fold for the reporter gene product, as compared with 3-4-fold for synapsin I mRNA.

Two reasons may account for this difference. The 4.3Syn DNA region may lack some of the regulatory elements con- tributing to developmental control, but it is also clear that posttranscriptional mechanisms modulate synapsin I mRNA levels and may account at least for part of the discrepancy. Fig. 7, A and B, shows that with increasing age the ratio of the 3,400- and 4,500-nucleotide mRNAs shifts in favor of the smaller species. The difference between the two mRNA spe- cies has not been characterized at the sequence level, but indirect evidence (Haas and DeGennaro, 1988) indicates that they differ in their 3"untranslated sequences, probably be- cause of an alternative choice of polyadenylation sites or alternative splicing. It appears that the 4,500-nucleotide mRNA is characteristic for undifferentiated neurons. It pre- dominates in NS2OY neuroblastoma cells, and if these are induced to morphological differentiation (extension of neu- rites) by serum withdrawal, a shift of relative abundance from the 4,500- to the 3,400-nucleotide species is also observed (Fig. 7C). It will be of interest to clarify how the two mRNAs differ in structure and function (e.g. stability). For example, if the 3,400-nucleotide mRNA were less stable than the 4,500- nucleotide mRNA, a sharp transcriptional time course would be blunted at the level of mRNA abundance.

DISCUSSION

The present study demonstrates that -4,300 nucleotides of 5"flanking sequence of the rat synapsin I gene drive the expression of the CAT transgene with a spatial and temporal specificity similar to that of the intact synapsin I gene. There- fore, this region apparently harbors much if not all of the cis- regulatory information required for the proper expression control of the synapsin I gene in vivo. Expression in the nervous system occurs with high penetrance and specificity, and ectopic expression in a few nonneuronal tissues is at least 2 orders of magnitude lower than in brain (with the exception of testis). In the brains of three of the four CAT-expressing mouse lines, absolute levels of CAT expression and their developmental profiles are very similar. These properties make the synapsin I promoter region an attractive tool for targeting the expression of biologically active proteins to the nervous system of transgenic animals. High levels of expres- sion in testis in two of the four transgene-expressing mouse lines may indicate a specific relationship between some as- pects of transcription control in neurons and certain testicular cells.

The CAT reporter gene product is a well established instru- ment for determining promoter activity in transgenic animals. In the present study, the sensitivity and low background of the CAT assay have allowed the quantification of transgene expression in a variety of tissues and organs over an activity range of 4-5 orders of magnitude. Immunohistochemical vis- ualization apparently highlights certain neuronal subpopula-

Neuron-specific Synapsin I Gene Promoter in Transgenic Mice 26501

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FIG. 7. Developmental regulation of endogenous synapsin I mRNAs in mouse and rat brain and regulation during differ- entiation in neuroblastoma cells. Panel A, postnatal expression of the 3.4- (circles) and 4.5-kilonucleotide (triangles) mRNAs in mouse whole brain. Animals were offspring of crosses of homozygous line 78 transgenic males and nontransgenic BALB/c females. Adult ( A ) animals were 56 days of age. A Northern blot autoradiogram is shown at the bottom (20 pg of total RNAllune), and the densitometric quantification of two independent blots (mean values) is at the top. For better visualization of the time course, the ordinute scale for the 4.5-kilonucleotide RNA is expanded 2-fold. Panel B, postnatal expres- sion of the 3.4- and 4.5-kilonucleotide mRNAs in rat forebrain. The figure is constructed as in p a n e l A. Two independent RNA prepara- tions and blots (10 pg of total RNAllune) were carried out for each time point (means f S.D.). Adult rats ( A ) were 7.5 and 8.5 months of age. Panel C, shift of relative abundance between the 3.4- and 4.5- kilonucleotide mRNAs during morphological differentiation of mouse NS2OY neuroblastoma cells by serum withdrawal. Lanes contain 20 pg of total RNA from cells seeded in separate dishes simultaneously and first cultivated in the presence of 10% fetal calf serum for 24 h. This was followed by cultivation in either 10 or 0.25% serum for the times indicated. Cells in 10% serum displayed a round morphology and continued dividing, whereas cells in 0.25% serum showed flatten- ing and decreasing growth rate after 2 days, followed by the extension of neuritic processes (not shown). Cells in 10% serum show a slight increase of the 4.5-kilonucleotide RNA in the course of time, whereas

tions, whereas biochemical analysis consistently demon- strates CAT expression in many parts of the nervous system in all four lines of mice. T o establish more rigorously whether the 4.3Syn promoter functions with exactly the same cell type specificity as the intact synapsin I gene, i.e. in practically all neurons but in no other cell type, it will be necessary to employ reporter genes that allow more sensitive histological detection. This would also permit monitoring in detail the locations and times of onset of expression during develop- ment.

As has been observed with many other transgenes, cell type specificity of the 4.3Syn-CAT construct is much more strin- gent in transgenic animals than in cell culture, where tran- sient expression in NS2OY neuroblastoma cells as compared with L929 fibroblasts (Sauerwald et al., 1990) or PC12 cells as compared with HeLa cells (Howland et al., 1991) is only 1-2 orders of magnitude higher. Similar result.. have been obtained for transient expression under the human synapsin I promoter region in various cell lines (Thiel et al., 1991). The endogenous synapsin I gene and the 4.3Syn-CAT construct are expressed a t notable levels already prenatally and in undifferentiated neuroblastoma cells. However, the activity of the 4.3Syn promoter region in transgenic mice is sharply up-regulated a t a very late developmental stage, namely, be- tween postnatal days 10 and 20. In this developmental aspect, it differs from the neuron-specific enolase and SCGlO pro- moters which, as far as they were characterized in transgenic animals, are mainly induced prenatally (Forss-Petter et al., 1990; Wuenschell et al., 1990). Although the postnatal time courses of the 4.3Syn-CAT transgene and the endogenous synapsin I gene are qualitatively similar, including a charac- teristic maximum around day 20, significant quantitative differences remain. Together with the observation of shifts between the two synapsin I mRNAs upon differentiation in cell culture and during development in vivo, this warrants an investigation of posttranscriptional components of synapsin I expression control.

In recent years, a number of transcription factors have been characterized which are predominantly or specifically ex- pressed in the nervous system or in neuronal cell lines (for recent examples see Hara et al., 1992; Tao and h i , 1992; Crosby et al., 1992; Mathis et al., 1992; Treacy et al., 1992; Brown et al., 1992; Lipkowitz et al., 1992; Akazawa et al., 1992; for reviews see Rosenfeld, 1991; He and Rosenfeld, 1991). I t will be of interest to investigate whether any of them interact with the 4.3Syn region. Further analysis of the regulatory region of the synapsin I gene may lead to the identification of binding sites for transcription factors which are expres.sed pan-neuronally, i.e. whose cell type distribution parallels di- rectly and possibly determines that of synapsin I. Alterna- tively, the cell type specificity of synapsin I expression may be caused by an interplay of several positive and negative acting transcription factors, each of which needs not neces- sarily be restricted only to neurons. In any case, the ongoing characterization of the synapsin I gene promoter region is expected to provide insight into how it orchestrates multiple transcription factors to result in i t s particular spatial and temporal pattern of transcriptional activity. Our future ex- periments will aim to define individual cis-regulatory elements in this region, to identify the transcription factors that inter- act with them, and to characterize the contribution of both to the control of transcription of the synapsin I gene in oiuo.

cells in 0.25% serum show a pronounced increase of the 3.4-kilonu- cleotide RNA. Hybridization of the blot with an 18 S rRNA probe confirmed that equal RNA quantities were applied to each lune (not shown).

26502 Neuron-specific Synapsin I Gene Promoter in Transgenic Mice

Acknowledgments-We thank Dr. Beate Lichte for the rat North- em blot data and Dr. Ingrid GNmmt (Heidelberg) for the 18 S rRNA probe.

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