Neuronal vulnerability in transgenic mice expressing an inducible dominant-negative FGF receptor

Experimental Neurology 198 (2006) 338 – 349 www.elsevier.com/locate/yexnr

Neuronal vulnerability in transgenic mice expressing an inducible dominant-negative FGF receptor Felix P. Eckenstein ⁎, Toby McGovern, Drew Kern, Jason Deignan Department of Neurology and Department of Anatomy and Neurobiology, College of Medicine, University of Vermont, HSRF 408, VT 05405, USA Received 24 July 2005; revised 28 November 2005; accepted 1 December 2005 Available online 17 February 2006

Abstract Fibroblast Growth Factors (FGFs) and their receptors (FGFRs) are widely expressed in the mature nervous system and are thought to mediate plasticity and repair. We report the generation of transgenic mice that can be induced to express a dominant-negative FGFR (dnFGFR) in select neuronal populations. We show that a modified Thy1 promoter [Vidal, M., Morris, R., Grosveld, F., and Spanopoulou, E. 1990. Tissue-specific control elements of the Thy-1 gene. EMBO J 9 833–840] can be used to drive widespread neuronal expression of the reverse tetracycline transactivator M2 (rtTA-M2 [Urlinger, S., Baron, U., Thellmann, M., Hasan, M.T., Bujard, H., and Hillen, W., 2000. Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc. Natl. Acad. Sci. U. S. A. 97, 7963–7968]), which after stimulation with doxycycline induces co-expression of dnFGFR in mosaic subpopulations of rtTA-M2-positive forebrain neurons, but not in hindbrain and spinal cord rtTA-M2-positive neurons. Expression of dnFGFR did not cause overt neurodegeneration, but led to increased neuronal vulnerability: four days after a stab injury, cell death was marked in the hippocampus of dnFGFR-expressing animals when compared to controls. The nuclear morphology of dying CA1 pyramidal cells suggested an apoptotic mechanism of cell death. These observations demonstrate the importance of endogenous FGFs in the maintenance of the nervous system. © 2005 Elsevier Inc. All rights reserved.

Introduction Fibroblast Growth Factors (FGFs) are important regulators of neuronal development with potent broad-spectrum neurotrophic and mitogenic activities (Eckenstein, 1994; Reuss and von Bohlen und Halbach, 2003). Many of the over 20 members of the FGF family are expressed in the developing and adult nervous system (Ornitz and Itoh, 2001). Some FGFs contain signal peptide sequences and are released through the secretory pathway. Others, such as FGF1 and FGF2, lack signal peptides, are present in extremely high levels in select neuronal populations (FGF1 (Elde et al., 1991; Stock et al., 1992) or astrocytes (FGF2; Chadi et al., 1994; Kuzis et al., 1995; Woodward et al., 1992) and appear to depend on alternate mechanisms for release (Prudovsky et al., 2003). Four genes encoding high affinity transmembrane tyrosine kinase FGF receptors (FGFRs) are currently known (Dionne et al., 1991; Itoh and Ornitz, 2004; Johnson and Williams, 1993). Differential splicing of FGFR RNAs creates receptors with altered selectivity ⁎ Corresponding author. Fax: +802 656 8704. E-mail address: [email protected] (F.P. Eckenstein). 0014-4886/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.12.020

for FGFs, although many FGFs can activate more than one type of FGFR (Klint and Claesson-Welsh, 1999; Ornitz and Itoh, 2001). Many neuronal populations preferentially express FGFR1, while glia primarily express FGFR2 and FGFR3 (Asai et al., 1993; Reuss and von Bohlen und Halbach, 2003; Yazaki et al., 1994). Gene knockouts of FGFs or FGFRs can result in embryonic lethality or significant abnormalities in the developing CNS. For example, FGF8 knockouts and FGFR1 knockouts die at an early stage of development (Amaya et al., 1991; Deng et al., 1997), and knockout of these genes specifically targeted to the midbrain–hindbrain border or olfactory bulb strongly affects the development of these structures (Chi et al., 2003; Hebert et al., 2003; Meyers et al., 1998; Trokovic et al., 2003). In addition, expression of dominant-negative FGFRs (dnFGFR) targeted to cerebral cortex (Shin et al., 2004) or GnRH neurons results in the generation of fewer neurons in these tissues. On the other hand, FGF1 knockouts do not exhibit a marked phenotype (Miller et al., 2000), and FGF2 knockouts show a relatively subtle loss of cortical neurons (Ortega et al., 1998). The absence of a severe phenotype in FGF1 and FGF2 knockouts may be due to compensation by other members of the FGF family since FGF1

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and FGF2 likely function to promote plasticity and repair in the adult CNS (Fagan et al., 1997; Kiprianova et al., 2004; Yoshimura et al., 2001). Thus, current genetic approaches are often highly successful in deciphering FGF function during development, but analogous approaches to address the roles of FGF signaling in the adult nervous system require the development of methods that allow targeted interference with FGFs without affecting FGF function during development. We report here the generation and characterization of transgenic mice designed to attenuate FGF signaling selectively in mature neurons through inducible expression of dnFGFRs. We show that a modified Thy1 promoter (Vidal et al., 1990) can be used to drive widespread neuronal expression of the reverse tetracycline transactivator M2 (rtTA-M2; Urlinger et al., 2000) which, after stimulation with doxycycline, induces the expression of dnFGFR and EGFP in mosaic subpopulations of rtTA-M2positive forebrain neurons, but not in hindbrain and spinal cord rtTA-M2-positive neurons. Induction of dnFGFR in forebrain neurons does not lead to overt neurodegeneration but increases the vulnerability to injury of CA1 hippocampal neurons. Material and methods Generation of inducible FGFR1 truncations DNA fragments coding for truncations of FGFR1 were generated by polymerase chain reaction (PCR) using adult mouse brain cDNA as a template. Thirty cycles of amplification with high fidelity KOD polymerase (Novagen) using the following primers resulted in amplicons of the expected size: Forward primer: CAA CCT CTA ACC GCA GAA CTG G Reverse primers: CTC TTC CAG GGC TTC CAG AAC G: truncation at AA 462 CTT CTT GGT GCC GCT CTT CAT C: truncation at AA 503 GTC CTT ATC CAG CCC GAT GG: truncation at AA 591 CCC AAT CAT TTT CAT CAT CTC CAT C: truncation at AA 629 AAG ATA CTC CAT GCC CCG AGC C: truncation at AA 704 CTA GTG TGT CCA ACA GGG GAT TTG: full length

These amplicons were then cloned into the MluI site of pBiEGFP followed by verifying the nucleotide sequence of the insert. Determining the effect of FGFR1 truncations on FGF activity The ability of the different FGFR1 truncations to block FGF-induced neurite outgrowth was determined in tTAexpressing PC12 cells (from Clontech), using a subclone that responds vigorously to NGF and FGF (a gift from Dr. Rae Nishi). Cells in 24-well plates were grown in RPMI containing 10% fetal calf and 5% horse serum until 50% confluent followed by co-transfection using Lipofectamine (Invitrogen) with 0.25 μg full-length FGFR1 (in pBi-EGFP) and 1.75 μg of varying ratios of truncated FGFR1 (in pBi-

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EGFP) and empty pBi-EGFP. One day after transfection, the culture medium was changed to RPMI containing 1% N2 supplement (Invitrogen) and 100 ng/ml of either FGF2, FGF5, FGF9 or NGF. Neurite outgrowth was analyzed 3 days after transfection by measuring the length of the longest neurite in over 50 strongly green fluorescing cells per condition, using a Diagnostic Instruments digital camera fitted to an inverted microscope and Spot 3.5 software. Standard PCR methods were used to add a FLAG epitope to the FGFR1 construct that blocked FGF-induced neurite outgrowth most efficiently (truncation at AA 629). The ability of epitope-tagged truncation to block neurite outgrowth was confirmed, and Western blot analysis was used to determine the effect of this construct on FGF-induced ERK phosphorylation. PC12 cells were transfected as described above, transferred to serum-free medium 1 day after transfection, followed 1 day later with exposure to 100 ng/ml FGF2 for 5 min. Cells were immediately harvested in SDS sample buffer, and 10 μg protein extract per lane was loaded on a 12% polyacrylamide gel and, after electrophoresis, transferred onto a nitrocellulose membrane. The membrane was sequentially incubated in Odyssey blocking buffer, phospho-ERK-specific antibodies (produced in rabbit, 1:100 dilution, Cell signaling) and IR800 anti-rabbit antibodies (1:500 dilution, Rockland), with washes in PBS between incubation steps. The bound IR800 signal was visualized using an Odyssey Infrared Imaging System (Licor Biosciences). Generation of the Thy1-rtTA-M2 plasmid The rtTA-M2 coding sequence was excised from the pUHrT-62-1 plasmid (a gift from Dr. H. Bujard) using EcoRI and BamHI and blunt cloned into the XhoI site of the modified Thy1 promoter plasmid pTs-2 (a gift from Dr. J. Sanes). The nucleotide sequence of the resulting Thy1-rtTAM2 plasmid was confirmed. The Thy1-rtTA-M2 plasmid and pBi-EGFP were co-transfected into N2A cells which were treated with doxycycline (Dox, 0–1 μg/ml) 2 days later. Induction of EGFP fluorescence was monitored 2 days later, with little fluorescence seen at 0 μg/ml Dox and maximum fluorescence at 50 ng/ml. Generation of transgenic mice The Thy1-rtTA coding sequence was excised by digestion with EcoRI and PvuI, and the coding sequence of pBi-EGFP/ dnFGFR was excised using AseI and SfoI. Both fragments were gel-purified, and the transgenic mice were generated by injecting the fragments into F2 B6C3H fertile eggs at the University of Vermont Transgenic Mouse Facility. Genotyping DNA was extracted from a small piece of tail, and genotype was determined by PCR using the REDExtract-N-Amp kit (SIGMA) according to the manufacturer's instructions. Primers specific for dnFGFR and M2-rtTA were used, with

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both primer sets present in the same reaction. Primer sequences are: rtTA-M2: GGA CAA GAG CAA AGT CAT AAA CGG GGC ATA GAA TCG GTG GTA GGT G EGFP: TCT TTG CTC AGG GCG GAC TG GCA CCA TCT TCT TCA AGG ACG AC

Analysis of transgene expression Initially, dnFGFR founder mice were bred to NSE-tTA (B6. Cg-Tg(Eno2tTA)5030Nes/J) and CMV-tTA mice (Tg (tTAhCMV)3Bjd/J from Jackson Laboratories). Breeding pairs and nursing dams were maintained on Dox containing feed (0.6 g/kg, from Biosource) to prevent transgene expression. Offspring received feed without Dox after weaning (21 days postnatal) to induce transgene expression and harvested 2 weeks later. Transgene expression was analyzed in 15 CMVtTA/dnFGFR and 23 NSE-tTA/dnFGFR double transgenic mice. Thy1-M2 transgenic mice were bred to dnFGFR transgenic mice. Weaned offspring were maintained on Dox feed (6 g/kg) to induce transgene expression for 7 days prior to harvest. Some litters were never exposed to Dox to determine whether transgenes were detectable in the absence of Dox. Transgene expression was analyzed in a total of over 90 Thy1-M2/ dnFGFR double transgenic mice (see Table 1 for breakdown of numbers for individual lines). Needle stab injury was performed according to a protocol approved by IACUC. Mice were deeply anesthetized with isoflurane, mounted in a stereotaxic headholder and a 2 mm diameter hole was drilled in the skull over the right hemisphere with the center located 3 mm posterior to the bregma and 2.5 mm lateral to the midline. A 22-gauge needle was then lowered 3.5 mm deep into the brain using a micromanipulator and allowed to remain in place for 5 min, after which the mice were removed from the headholder and allowed to recover. Animals were harvested 4 days after the needle stab injury. Animals were euthanized, and tissue was harvested according to protocols

Fig. 1. Panel A shows five different PCR products corresponding to different sequential truncations of FGFR1, as seen in an ethidium-bromide-stained agarose gel after electrophoresis. Lane 1: amplicon leading to truncation at amino acid 462, lanes 2 through 5 at amino acids 503, 591, 629 and 704 respectively. These PCR products (dnFGFR) were subsequently cloned into the bi-directional Tetracycline Response Element (TRE) containing plasmid pBi, as shown in panel B (pAsv = SV40 polyadenylation signal, EGFP = enhanced green fluorescent protein, pAglob = beta-globin polyadenylation signal). In some subsequent experiments, expression of dnFGFR and EGFP was induced by the reverse tetracycline transactivator M2 (rtTA-M2), which was expressed under control of a modified Thy1 promoter as shown in the panel (1a, 1b, 2 and 4 correspond to the exons of the Thy1 gene).

approved by IACUC. Dissected tissues were immersion-fixed while gently shaking for 24 h at 4°C in freshly made Zamboni's fixative, washed with PBS and sunk in 15%, then 30% sucrose in PBS, all at 4°C. Fifty-μm-thick serial coronal frozen sections were cut on a freezing microtome and collected in PBS containing 0.05% sodium azide. This protocol preserved the native fluorescence of EGFP. Immunohistochemistry Sections were stained for rtTA-M2 (rat antiserum, see below, diluted 1:500 in PBS containing 10% horse serum, 0.5% Triton X-100, 0.05% sodium azide), FLAG epitope (goat antibodies, diluted 1:2000, Bethyl laboratories), MAP2 (chicken antibodies, 1:2,000, EnCor Biotechnology), CD45 (rat monoclonal, 1:200, Chemicon International) and GFAP (rabbit antiserum,

Table 1 The number of double transgenic mice produced by breeding different TRE and (r)tTA lines, binned according to the intensity of doxycycline-induced neuronal EGFP expression TRE line ×

(r)tTA line

EGFP ++++

EGFP +++

EGFP ++

EGFP +

EGFP −

dnFGFR20 dnFGFR20 dnFGFR20 H dnFGFR20 dnFGFR20 dnFGFR21

Thy1-M2 1 Thy1-M2 17 Thy1-M2 17 H Thy1-M2 19 NSE-tTA Thy1-M2 17

1 3 12 0 0 0

0 6 4 1 0 0

0 18 0 1 0 0

7 5 4 2 13 0

4 0 0 2 14 8

EGFP ++++: large number of intensely fluorescent neurons. EGFP +++: large number of neurons with significant fluorescence. EGFP ++: some neurons with significant fluorescence. EGFP +: few neurons with significant fluorescence. EGFP −: no fluorescence observed. Note that dnFGFR20 H and Thy1-M2 H denote F1 and F2 offspring of a single breeding pair derived from the dnFGFR20 and Thy1-M2 lines, respectively.

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1:500, EnCor Biotechnology). Floating sections were incubated overnight at room temperature while gently shaking, washed three times with PBS, incubated in appropriate CY3 labeled secondary antibodies (1:500 from Jackson ImmunoResearch) for 2 h, then for 20 min in PBS containing 0.5 μg/ml of Hoechst 34580, washed twice with PBS and coverslipped using Citifluor mounting medium. Sections were imaged using a Nikon Eclipse C1 confocal microscope. rtTA-M2 antigen was produced by cloning the rtTA-M2 coding sequence from the pUHrT-62-1 plasmid into pET101/DTOPO plasmid (Invitrogen), which provides a carboxy-terminal 6-His tag. Recombinant antigen was produced from this plasmid and purified using nickel-affinity column chromatography as suggested by the manufacturer. Three Long–Evans rats were immunized with 200 μg of this antigen each four times in monthly intervals. All rats exhibited high antibody titers against rtTA-M2 2 weeks after the last immunization. Titers and specificity of the antisera were tested by serial dilution immunofluorescence staining of sections from Thy1-M2 transgenic and control mice.

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Results The ability of different FGFR1 truncations to block FGF action was determined by generating a series of FGFR1 truncations by PCR, with each truncation terminating shortly before a tyrosine residue (Fig. 1A) that is known to be phosphorylated after stimulation with FGF (Klint and ClaessonWelsh, 1999). The truncated FGFR1 fragments were cloned into a vector (pBi-EGFP, Fig. 1) containing a bi-directional TETResponse Element (TRE) which allows tetracycline-inducible expression (Baron et al., 1995) of the truncated FGFR1 and coexpression of EGFP in cells that express tetracycline transactivator proteins (tTA). PC12 cells that constitutively express tTA were co-transfected with pBi containing full-length FGFR1 and varying concentrations of the pBi-FGFR1 truncation plasmids. One particular construct (truncation after amino acid 629) strongly inhibited neurite outgrowth induced by stimulation with FGF2, FGF5 and FGF9 (Fig. 2). A FLAG epitope tag was added to the carboxy-terminus of this dominant-negative FGFR (dnFGFR) construct, which did not affect the ability of the

Fig. 2. Transfection of dnFGFR, truncated after amino acid 629, blocks FGF signaling. Stimulation with FGF2 (100 ng/ml) of PC12 expressing FGFR1 causes process outgrowth (A), which is inhibited when the cells co-express dnFGFR (B, scale bar = +35 μm). For this experiment, tTA expressing PC12 cells were transfected with equal amounts of full-length FGFR1 (cloned into pBi) and pBi (A) or equal amounts of full-length FGFR1 and dnFGFR (both cloned into pBi, B). dnFGFR can block process growth stimulated by 100 ng/ml of FGF2, FGF5 or FGF9 (C). The inhibition of process growth is dependent on the ratio of dnFGFR to full-length FGFR1 plasmids co-transfected, and equimolar co-transfections (1:1 ratio) result in over 50% inhibition of growth. Inhibition of ERK phosphorylation after stimulation with dnFGFR is shown in (D), using a Western blot assay employing phospho-ERK-specific antibodies. tTA expressing PC12 cells were transfected with either full-length FGFR1 (lanes 3, 4 and 5) and co-transfected with dnFGFR (lane 5) or pBi (lanes 3 and 4) followed by stimulation with 100 ng/ml of FGF2 for 5 min. Note that transfection with full-length FGFR1 significantly enhances ERK phosphorylation after stimulation, while co-transfection of full-length FGFR1 and dnFGFR (1:4 ratio) reduces ERK phosphorylation to the level observed in control cultures. This reduced level of ERK phosphorylation is likely due the stimulation of non-transfected cells (about 20% of all cells were transfected in these assays).

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Fig. 3. Doxycycline-induced EGFP expression in Thy1-M2/dnFGFR double transgenic mice is variable. (A) (scale bar = 1 mm) shows confocal micrographs of 50-μm-thick coronal sections taken from two double transgenic littermates that had equal access to DOX. Sytox-orange was used to counter-stain DNA. The upper two sections are from animal 2, the lower two sections are from animal 6 of this litter, with genotypes determined by PCR (B). Animal 2 shows strong expression of EGFP in many neurons in cerebral cortex, hippocampus, striatum and amygdala, while animal 6 showed EGFP expression in only very few neurons in the amygdala (not visible in this micrograph).

construct to block stimulation of neurite outgrowth by FGF. In addition, this dnFGFR construct blocked ERK phosphorylation after FGF stimulation of transfected PC12 cells (Fig. 2D) and did not affect the ability of transfected cells to respond to nerve growth factor.

Three transgenic mouse lines with germ-line transmission of dnFGFR were obtained (dnFGFR8, dnFGFR20, dnFGFR21). These lines were bred with commercially available lines expressing the tetracycline transactivator (tTA), which allows induction of target gene expression in the absence of tetracycline, while expression is blocked in the presence of tetracycline (Mansuy and Bujard, 2000). These lines expressed tTA either under control of the promoter for neuron-specific enolase (NSE; Chen et al., 1998) or cytomegalovirus virus (CMV; Kistner et al., 1996). Breeding pairs were maintained on a diet containing the stable tetracycline analog doxycycline (DOX, 0.6 g/kg) to block expression of dnFGFR and to assure normal development of double transgenic (tTA/dnFGFR) offspring. At weaning, offspring were put on a DOX-free diet to allow dnFGFR and EGFP expression, and tissue was harvested 14 days later. Some NSE-tTA/dnFGFR breeding pairs did not receive DOX feed, allowing for expression of dnFGFR at the time when the NSE promoter becomes active. The expected number of double transgenic offspring was obtained in such litters, which were harvested shortly after weaning. Double transgenic offspring were screened for EGFP expression in the brain and peripheral organs. No EGFPpositive cells were ever observed in offspring of dnFGFR8, while double transgenic first generation (F1) offspring of dnFGFR20 and 21 showed weak and variable EGFP expression in some blood cells (when crossed CMV-tTA) and variable but strong EGFP expression in axons in the olfactory bulb (when crossed with NSE-tTA). In a few NSE-tTA/dnFGFR21, double transgenic mice EGFP-positive neurons were also observed in the forebrain. EGFP expression was reduced or absent in later generations (F2 and F3) of double transgenic mice. Overall, the variability in EGFP expression observed in both the CMV-tTA/ dnFGFR or NSE-tTA/dnFGFR mice precluded further analysis of these mice. Recently, rtTA-M2, an improved version of a reverse tTA, has been described (Urlinger et al., 2000). Reverse transactivators allow induction of expression of target genes after

Fig. 4. Antisera to rtTA-M2 were produced in rat. One of these antisera shows specific detection of recombinant rtTA-M2 in a Western blot assay (A), where the antiserum detects a single band of the expected molecular weight in a lane loaded with 10 μg of protein prepared from rtTA-M2 expressing E. coli. The same antiserum strongly stains neurons in Thy1-M2 transgenic mice (B, red immunofluorescence) but shows no detectable staining in non-transgenic littermates (C, scale bar = 25 μm). Coronal sections through the dentate gyrus are shown. Hoechst dye (blue color) was used as a DNA counterstain.

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stimulation with DOX, which has the advantage that breeding pairs do not need to be maintained on a DOX diet and allows rapid induction of target genes after stimulation with DOX. rtTA-M2 was chosen since it exhibits only extremely low levels of leaky expression in the absence of DOX, and induction can be achieved with low levels of DOX. In addition, codon usage of the sequence coding for rtTA-M2 has been optimized for expression in eukaryotic cells. A construct driving rtTA-M2 expression under control of a neuron-specific modified Thy1 promoter (Fig. 1; Feng et al., 2000; Vidal et al., 1990) was used to produce transgenic mice. Six lines with germ-line transmission were obtained (Thy1-M2 lines) and crossed with both dnFGFR20 and dnFGFR21 mice. EGFP expression was analyzed shortly after weaning. The presence of DOX (6g/kg) in the feed of either the nursing dam or the weaned pups was found to lead to a significant expression of EGFP in forebrain neurons of some double transgenic offspring, while other double transgenics showed little induction. Such variability was present even among littermates (Fig. 3). To test whether the observed variability in transgene induction was due to variable expression of the transactivator protein, we produced an antibody to rtTA-M2. Rat antisera to recombinant rtTA-M2 recognized the transactivator on Western

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blots and strongly stained many neuronal populations in Thy1M2 mice. Specificity of the antisera was established by the lack of staining in non-transgenic mice (Fig. 4). The distribution and levels of rtTA-M2 immunoreactivity showed little variability between individuals in three of the Thy1-M2 lines (lines 1, 17 and 19), with line Thy1-M2 17 exhibiting the most robust and widespread expression of the transactivator. In all lines, expression of rtTA-M2 immunoreactivity was confined to cells with neuronal morphology, with strong staining of the nucleus and more faint staining of the cytoplasm. Treatment of animals with DOX did not alter the cellular or subcellular distribution of the transactivator. The distribution of the transactivator was characterized in more detail in six transgenic mice from the Thy1-M2 17 line. Transactivator expression was reproducible and reliably observed in many neurons in cerebral cortex (all layers), hippocampus (all areas), caudate, thalamus, hypothalamus, midbrain, brainstem and spinal cord, including many motor neurons and mesencephalic sensory neurons. Induction of EGFP expression was analyzed by breeding the three Thy1-M2 lines with two dnFGFR lines, providing DOX feed to offspring at weaning, harvesting and analyzing EGFP fluorescence 4 days later. A total of 72 double transgenic (Thy1M2/dnFGFR) mice were analyzed. Significant variability of

Fig. 5. The distribution of EGFP (green fluorescence) and rtTA-M2 expression (red immunofluorescence) are compared in six areas in the CNS of DOX-treated Thy1M2/dnFGFR double transgenic mice. All panels show confocal micrographs of Hoechst dye (blue fluorescence) counterstained coronal sections. EGFP expression is high in many neurons of the forebrain (A: dentate gyrus, B: CA1 hippocampal pyramidal cells, C: layer 6 of cerebral cortex, D: striatum) but is absent in trigeminal sensory neurons (E) or motor neurons in the facial nucleus (F, scale bar = 45 μm), although the latter two neuronal populations express levels of rtTA-M2 immunoreactivity that is indistinguishable of that seen in EGFP-positive neurons in the forebrain. Note that EGFP-negative/rtTA-M2-positive neurons are also present in forebrain areas that contain many EGFP-positive/rtTA-M2-positive neurons and that these two populations appear to intermingle in a random pattern. In addition, all areas contain at least a few neurons that do not express either marker, for example, the rim of blue fluorescing granule cells in panel A.

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Table 2 The percentage of total neurons expressing transgenes in different CNS areas of four DOX-treated double transgenic Thy1-M2/dnFGFR mice Area

% rtTA-M2

% EGFP

% FLAG

Hippocampus CA1 CA3 Dentate gyrus

70–90 70–90 60–80

65–85 5–45 50–70

65–85 35–70 50–70

Cerebral cortex Layer 6 Layer 5 Layer 2/3 Caudate Facial motor Trigeminal sensory

50–80 50–80 30–50 50–70 90–100 90–100

25–40 0–5 15–25 10–35 0 0

20–35 20–35 15–25 10–35 0 0

Numbers given are estimates of percentages derived from counting three fields of view, rounded to the closest 5th percentile. The range of percentages given indicates the values for the lowest and highest scoring single animals. Note that almost no neurons expressing EGFP but not FLAG immunoreactivity were observed in any of the areas analyzed. However, FLAG- but not EGFPexpressing neurons were found in cortical layer 5 and hippocampal area CA3.

EGFP expression was again observed in all possible crosses of the different lines, with EGFP levels ranging from very high to barely detectable. Overall, double transgenic animals generated by crossing line rtTA-M2 17 with dnFGFR 20 showed the most

robust expression (Table 1). Breeding of these lines into defined genetic backgrounds for three generations (C57Bl6 and FVB/N) resulted in double transgenic mice that still expressed rtTA-M2 but could no longer be induced to produce detectable levels of EGFP. However, continued brother–sister mating of single transgenic animals (Thy1-M2 17 × dnFGFR 20) from litters that contained high level EGFP expressing double transgenic littermates allowed us to obtain a single brother–sister pair (Thy1-M2 17 male and dnFGFR20 female) that generated offspring with a high likelihood to express high levels of EGFP in many forebrain neuronal populations (Table 1). All further studies were conducted with mice descending from this one brother–sister breeding pair. Strong rtTA-M2 expression, but no EGFP fluorescence or FLAG staining (for dnFGFR), was observed in the CNS of three double transgenic (Thy1-M2 17/dnFGFR 20 positive genotype) mice that never received DOX. The distribution and extent of overlap in expression of rtTAM2 and EGFP were investigated in four double transgenic mice that showed strong induction of EGFP after receiving DOX. EGFP was only observed in neurons that also expressed rtTAM2 immunoreactivity. In general, the highest overall percentage of EGFP expressing neurons was observed in hippocampal CA1 pyramidal cells, with almost no EGFP expression observed in rtTA-M2-positive neurons in brainstem and spinal cord. Overall,

Fig. 6. The distribution of EGFP (green fluorescence) and dnFGFR (red immunofluorescent stain for FLAG epitope) is compared in four areas of the CNS of Thy1M2/dnFGFR double transgenic mice. All panels show confocal micrographs of Hoechst dye (blue fluorescence) counterstained coronal sections. Since both transgenes are driven by the same TRE, it is expected that the two proteins are co-expressed. This is largely the case in hippocampal granule cells (A), hippocampal pyramidal cells in CA1 (B) and neurons in the striatum (D, scale bar = 30 μm), although the ratio of the intensity of the signal for the two antigens can vary somewhat. However, large pyramidal neurons in layer 5 of cerebral cortex preferentially stain for dnFGFR (C). Note that EGFP fluorescence is present throughout the cell body, dendrites and axons of stained cells, while dnFGFR immunoreactivity is strongest in intracellular organelles and clusters along dendrites.

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the ranking of the percentage of co-expression of EGFP in rtTAM2-positive neurons was: hippocampus: CA1 N hippocampus: dentate gyrus N caudate N cerebral cortex: layer 6 N cerebral

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cortex: layer 2/3 N cerebral cortex layer 5 N midbrain N hindbrain N spinal cord (Fig. 5). EGFP expression in area CA3 of hippocampus was somewhat variable between individuals.

Fig. 7. Needle stab injury leads to significant neurodegeneration in the hippocampus of DOX-treated Thy1-M2/dnFGFR double transgenic mice, but not in control littermates. Panel A (scale bar = 100 μm) demonstrates the loss of EGFP-expressing CA1 pyramidal neurons in a coronal section taken about 400 μm rostral to the injury. The following panels show higher power confocal micrographs of sections taken at the edge of the area of cell death at roughly equivalent distances from the injury (∼400 μm) in a Thy1-M2/dnFGFR double transgenic mouse (B, D, F) and a Thy1-M2 single transgenic control littermate (C, E, G, scale bar = 40 μm). These coronal sections were stained in red immunofluorescence for dendrites (anti-MAP2, B and C), leukocytes (CD45, D and E) and astrocytes (GFAP, F and G), and DNA was counterstained with Hoechst dye (blue fluorescence). Note the almost complete degeneration of CA1 pyramidal cells in the double transgenic animal (B), while a very high percentage of neurons survive in the control animal (C). In addition, many highly fragmented and condensed nuclei are seen in the pyramidal cell layer of the double transgenic animal, while only few such nuclei can be observed in the control animal. The majority of these condensed nuclei are not contained in cells expressing the pan-leukocyte antigen CD45 nor the astrocyte-specific antigen GFAP, indicating that they represent the remnants of nuclei of dead pyramidal cells. The number of cells staining for CD45 or GFAP and the intensity of staining for the two antigens are roughly similar in the two animals, demonstrating that the neurodegeneration seen in the injured Thy1-M2/dnFGFR hippocampus does not lead to strong infiltration and activation of lymphocytes and microglia or activation of astrocytes.

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Estimates of the percentage of rtTA-M2-positive and EGFPpositive neurons are given in Table 2. The distribution of the dnFGFR protein (identified by immunofluorescent localization of the FLAG epitope) and colocalization with EGFP were investigated in the same four double transgenic mice. We expected to observe complete overlap in the expression pattern of the two transgenes since a bi-directional promoter is used to drive their expression. Indeed, in hippocampal area CA1 and the dentate gyrus, FLAG immunoreactivity and EGFP fluorescence were almost always co-expressed, and the extent of overlap of the two markers was also very high in caudate and cerebral cortical layers 6 and 2/3. However, many FLAGpositive EGFP-negative neurons were observed in cortical layer 5 of all individuals and in area CA3 of hippocampus of one individual analyzed (Fig. 6). Immunofluorescence localization of EGFP confirmed the absence of EGFP expression in these layer 5 neurons (not shown). Numerical estimates of the percentage of FLAG positive neurons are given in Table 2. The subcellular distribution of the two transgenes was different in double positive neurons. EGFP was distributed evenly throughout the cytoplasm and nucleus and filled dendrites, the entire axon and terminals. FLAG immunoreactivity was strongest in intracellular organelles and puncta in neurites, consistent with localization of dnFGFR to membranebound compartments. Cell membrane staining was less intense and gradually faded farther from the cell body, although very weak staining could be seen in some axons. The nuclear morphology of EGFP- and FLAG-positive neurons appeared undistinguishable from that of neighboring non-labeled neurons, demonstrating that expression of dnFGFR in postnatal neurons does not lead to overt neurodegeneration. In addition, immunofluorescent staining for CD45 and BM8 (a leukocyte marker that detects macrophages and microglia) and GFAP (a marker for astrocytes) showed no evidence for an activation of glial injury responses in the CNS of the four double transgenic mice induced to express high levels of dnFGFR, when compared to control littermates (not shown). The effect of dnFGFR expression on the response to injury was studied by placing a needle stab wound (22 gauge needle, 3.5 mm deep) through cerebral cortex and hippocampus of an additional four double transgenic mice and six control littermates (three Thy1-M2 and three dnFGFR single transgenic mice), which all had received DOX feed. Mice were allowed to survive for 4 days after injury. Neuronal survival in cerebral cortex appeared indistinguishable between the control and dnFGFR-expressing groups. The four double transgenic mice expressed high levels of EGFP fluorescence and FLAG immunoreactivity and showed a significant loss of CA1 pyramidal cells in a circular halo of about 700 to 1000 μm distance surrounding the core of the stab wound (Fig. 7). In this halo, almost all EGFP fluorescence and MAP2 immunoreactivity were lost, and most nuclei exhibited a highly pyknotic morphology. This strongly suggests that most injured CA1 pyramidal cells in dnFGFR-expressing animals undergo cell death. In contrast, no marked cell death or condensed nuclei were seen at a distance further than 200 μm from the core of the lesion in control littermates. Overall, the mean size of CA1

exhibiting loss of over 95% of neuronal cell bodies was 1.65 mm2 (±0.037 mm2, STD) in the dnFGFR-expressing mice and 0.09 mm2 in the control mice (±0.02 mm2 STD). Since about 20% of CA1 pyramidal neurons in these animals did not express EGFP or FLAG immunoreactivity, the almost complete lack of MAP2 staining indicates that more than just dnFGFR-expressing neurons are lost in the area of cell death. dnFGFR-negative neurons may be lost through a “bystander effect” due to the massive loss of dnFGFR-positive neurons. CD45 and BM8 immunoreactivity in this transition zone and in other areas of equivalent distance from the lesion showed only a moderate level of staining in both dnFGFR-expressing and control animals, suggesting that the death of the pyramidal cells in the double transgenic animals did not induce a strong inflammatory response. Analysis of GFAP immunoreactivity demonstrated a similar moderate activation of astrocytes in both groups of mice (Fig. 7). These results indicate that endogenous FGFs function to minimize the extent of damage and cell death after hippocampal injury. Discussion The main findings of this study are that transgenic mice engineered to inducibly express a truncated FGFR1 that inhibits FGF signaling can overcome the limitations caused by the early embryonic lethality of FGFR gene knockout mice. Using this system, we were able to show that blocking FGFR signaling in hippocampal neurons of mice results in enhanced vulnerability of these neurons to injury. In addition, we analyzed the possible reasons that can cause variability in the inducible expression of transgenes using the TET system. These reasons likely include epigenetic silencing of transgene inducibility, genetic background of the mice used and possibly also doxycycline availability. The truncated FGFR1 used in the present study can block signaling for several FGFs, similar to what has been reported previously for such truncations (Amaya et al., 1991). The bioassay employed here established that this dnFGFR1 can significantly reduce signaling by several FGFs through FGFR1. Similar FGFR1 truncations have been shown to block FGF signaling by FGFR2 and FGFR3 (Ueno et al., 1992) and to affect neuronal development (Shin et al., 2004; Trokovic et al., 2003; Tsai et al., 2005). In addition, neurons express predominantly FGFR1 (Belluardo et al., 1997; Reuss and von Bohlen und Halbach, 2003), thus expression of the dnFGFR1 used here will block most if not all FGF signaling in neurons. We employed the TET system to induce dnFGFR expression and co-expression of EGFP since this system had been used previously to regulate transgene expression in the CNS (Mansuy and Bujard, 2000), and the specific variant rtTA used (rtTA-M2) to drive transgene expression had been previously reported to be highly sensitive to DOX in vitro while allowing only very low levels of background expression (Urlinger et al., 2000). By repeatedly breeding single transgenic littermates (dnFGFR or rtTA-M2) of high-expressing double transgenic (dnFGFR/rtTA-M2) mice, we were able to obtain a line of mice that showed reproducible high level of dnFGFR

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and EGFP expression in most hippocampal and many cerebral cortical neurons after stimulation with DOX. In general, EGFP and dnFGFR were co-expressed, with the exception of cortical layer 5 pyramidal cells, which preferentially expressed dnFGFR and not EGFP. This may be due to the differences in the poly-A tail of the transgenes (beta-globin for dnFGFR and SV40 for EGFP). Induction of dnFGFR and EGFP expression for up to 1 week did not affect the overall morphology of transgene expressing neurons, suggesting that blocking FGFR signaling for several days in adult neurons does not lead to overt neurodegeneration. In addition, markers of glial activation, which are expected to provide an indirect measurement of degeneration, were also not induced in these animals. It is unclear at present whether blocking of FGFR signaling for days may cause more subtle changes in morphology or whether more prolonged blocking may cause neurodegeneration. However, blocking of FGFR signaling was found to significantly increase the vulnerability of hippocampal, but not cortical neurons to injury. Dead hippocampal CA1 pyramidal cells were found at a significant distance from a stab injury in dnFGFR-expressing, but not in control animals. Four days after injury, many pyknotic and condensed nuclei remained in affected areas. This is reminiscent of previous studies that demonstrated that hippocampal pyramidal cells are particularly sensitive to ischemic and excitotoxic injury, which often causes a delayed apoptotic cell death (Back et al., 2004; Böttiger et al., 1998; Ouyang et al., 1999; Snider et al., 1999). Identification of DNA fragmentation and caspase activation in injured dnFGFR-expressing neurons will be needed to ascertain whether these cells indeed underwent apoptosis. This observation strongly indicates that endogenous FGFs are important in limiting the damage caused by injury of the adult hippocampus. We propose that endogenous FGFs are released from astrocytes (FGF2; Woodward et al., 1992), basal forebrain afferents (FGF1; Elde et al., 1991; Stock et al., 1992) and hippocampal neurons (FGF5; Haub et al., 1990) after the stab injury and counteract excitotoxic or ischemic damage by activating antiapoptotic signals in stressed hippocampal neurons. FGFs may have a similar function in cortical neurons, but the stab injury used here may not have provided a sufficiently strong insult to reveal that function. This proposed model is in agreement with findings of previous studies demonstrating that application of exogenous FGFs can rescue neurons after ischemic or excitotoxic insults in vitro (Alzheimer and Werner, 2002; Kirschner et al., 1995; Mattson et al., 1995) and in vivo (Cuevas et al., 1998; Koketsu et al., 1994; Nakata et al., 1993). An obstacle for the present study was that not all rtTA-M2positive neurons could be induced to express dnFGFR or EGFP. In hippocampus and cerebral cortex, for example, EGFPnegative/rtTA-M2-positive neurons were surrounded by EGFPpositive/rtTA-M2-positive neurons. In this instance, it is highly likely that both kinds of neurons are exposed to identical concentrations of DOX, suggesting that transgene inducibility has been silenced in EGFP-negative/rtTA-M2-positive hippocampal neurons. On the other hand, the general rostro-caudal gradient of dnFGFR/EGFP induction observed (with no such

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gradient in rtTA-M2 expression) may not be solely due to silencing of inducibility. For example, neither dnFGFR nor EGFP expression could be induced in rtTA-M2-positive motor neurons. The complete lack of inducibility through a larger area of tissue could also be due to a lack of DOX availability. Obtaining transgenic mice that expressed dnFGFR and EGFP in a reproducible manner was hampered initially by a great variability between individual mice. It is of importance for future studies to determine the possible reasons for variability of transgene induction using the tetracycline system. In general, variability or lack of transgene induction might be caused by transgene silencing (possibly due to DNA methylation and/or chromatin condensation; Chevalier-Mariette et al., 2003; Chiurazzi and Neri, 2003), limitations in DOX availability, differences in the chromosomal location of transgene integration or differences in the genetic background of transgenic animals. These mechanisms may affect either one or both of the transgene components of the tetracycline system (here: TREdriven EGFP/dnFGFR and rtTA-M2). Most of the Thy1-M2 line of transgenic mice generated here expressed rtTA-M2 reproducibly, thus this transgene is not the source of variability in EGFP or dnFGFR induction. We recently generated transgenic mice where the TREdriven transgenes are flanked by the HS4 insulator (RecillasTarga et al., 2002; Yusufzai and Felsenfeld, 2004), which can prevent transgene silencing through multiple mechanisms. Early generations of these mice show little variability in transgene induction between individuals, and transgene induction can be observed in the spinal cord (unpublished observations, J.D. and F.E.). These observations suggest that silencing of TRE represents a likely mechanism responsible contributing to the variability of transgene induction in the TET system. Several other groups have observed similar variability and silencing when employing the TET system in different cell types or tissues (Chang et al., 2000; Chevassut and Lim, 2003; Pankiewicz et al., 2005; Robertson et al., 2002), thus use of HS4 insulators may prove of widespread use. In contrast to the rtTA-M2 transgenic mice generated here, commercially available lines of tTA transgenic mice showed variable expression of tTA immunoreactivity, thus this transgene has the ability to contribute to overall variability of induction. The difference in the variability of expression between rtTA-M2 and tTA likely is due to that the nucleotide sequence coding for rtTA-M2 has been optimized for expression in eukaryotic cells, while the sequence coding for tTA has not and is prone to silencing (Valencik and McDonald, 2001). Differences in the genetic background of individual mice can also affect transgene expression (Opsahl et al., 2002). We attempted to reduce variability of transgene induction between individuals by breeding both the rtTA-M2 and the dnFGFR transgene into the C57B6 or the FVB/N background, but double transgenic mice, while expressing rtTA-M2, could not be induced to express EGFP or dnFGFR. This indicates that the genetic background can exert a significant control over transgene induction in the TET system, but the current data cannot be used to identify one of the known inbred mouse strains as well suited for this use.

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