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Inducible Gene Manipulations in Serotonergic Neurons

An impairment of the serotonergic (5-HT) system has been implicated in the etiology of many neuropsychiatric disorders. Despite the considerable genetic evidence, the exact molecular and pathophysiological mechanisms underlying this dysfunction remain largely unknown. To address the lack of instruments for the molecular dissection of gene function in serotonergic neurons we have developed a new mouse transgenic tool that allows inducible Cre-mediated recombination of genes selectively in 5-HT neurons of all raphe nuclei. In this transgenic mouse line, the tamoxifen-inducible CreERT2 recombinase is expressed under the regulatory control of the mouse tryptophan hydroxylase 2 (Tph2) gene locus (177 kb). Tamoxifen treatment efficiently induced recombination selectively in serotonergic neurons with minimal background activity in vehicle-treated mice. These genetic manipulations can be initiated at any desired time during embryonic development, neonatal stage or adulthood. To illustrate the versatility of this new tool, we show that Brainbow-1.0LTPH2-CreERT2 mice display highly efficient recombination in serotonergic neurons with individual 5-HT neurons labeling with multiple distinct fluorescent colors. This labeling is well suited for visualization and tracing of serotonergic neurons and their network architecture. Finally, the applicability of TPH2-CreERT2 for loxP-flanked candidate gene manipulation is evidenced by our successful knockout induction of the ubiquitously expressed glucocorticoid-receptor exclusively in 5-HT neurons of adult mice. The TPH2-CreERT2 line will allow detailed analysis of gene function in both developing and adult serotonergic neurons.

offered by gene knockout technologies in mice. Unfortunately, germline knockouts can not address the specifi c serotonergic function of widely expressed genes without affecting the gene in other tissues as well. Moreover, germline gene inactivation may result in a lethal phenotype or induction of compensatory, homeostatic mechanisms or pleiotropy during development. Therefore, delineation of the particular function of the serotonergic system in relationship to other neuronal systems as well as the exact developmental and adult role of genes in 5-HT neurons remains elusive.
To overcome these obstacles, we have developed a CreERT2/ loxP-recombination system that allows temporal control of conditional gene manipulation specifi cally in serotonergic neurons. Temporal control of recombination is a prerequisite for distinguishing the developmental role of a gene from its present function during adulthood. For that reason, we took advantage of a fusion protein consisting of Cre recombinase and a mutated ligand-binding domain (LBD) of the human estrogen receptor (ER) that was developed to achieve tamoxifen dependent Cre activity (Feil et al., 1997;Indra et al., 1999).
We have chosen regulatory sequences of the tryptophan hydroxylase 2 (Tph2) gene which is exclusively expressed in serotonergic neurons during development and adulthood (Cote et al., 2003(Cote et al., , 2007 to accurately direct Cre expression to 5-HT neurons of the brain. Transgenic TPH2-CreERT2 mice were generated by DNA microinjection of a modifi ed DNA construct containing the Tph2 open reading frame together with large upstream and downstream
Furthermore, 5-HT plays a key role during development, being an essential neurotransmitter for proper neuronal division, differentiation, migration and synaptogenesis (Gaspar et al., 2003). Even transient alterations in serotonin homeostasis during development cause permanent changes to adult behavior (Ansorge et al., 2004;Gross and Hen, 2004).
In order to dissect the molecular basis of the multiplex serotonergic involvement in a plethora of physiological and pathophysiological brain functions, researchers have turned to the specifi city fl anking regions (177 kb). Breeding to three different mouse lines with loxP-fl anked alleles shows reliable and effi cient serotonergic neuron specifi c induction of recombination following tamoxifen treatment during development and adulthood and so represents a new and powerful tool for conditional gene manipulation in the serotonergic system.

GENERATION OF TPH2-CreERT2 TRANSGENIC MICE
A 196-kb PAC (RP24-243J21, RZPD, Deutsches Ressourcenzentrum für Genomforschung GmbH) that contains the full-length mouse Tph2 gene (107 kb) with 51 kb upstream and 19 kb downstream DNA sequences was selected for recombineering in EL250 bacteria (Lee et al., 2001). First, the kanamycin resistance gene of the pPAC4-backbone was replaced with a chloramphenicol resistance. Next, a cassette encoding a fusion protein (CreERT2) consisting of a Cre-recombinase (Cre) and a mutated LBD of the human ER (ERT2) as well as a kanamycin resistance gene fl anked by two FRT sites was integrated into the ATG-start codon of the TPH2-gene. A 23-bp sequence downstream of the ATG-translation start of Tph2 was intentionally deleted since it contained additional in-frame ATG-start sites in Exon 1 ( Figure S1 in Supplementary Material). The FRT-fl anked kanamycin resistance cassette was then deleted by arabinose-induced expression of Flp recombinase. The CreERT2modifi ed genomic Tph2 sequence was separated from the PAC backbone by NotI digestion and subsequent preparative pulse-fi eld electrophoresis. The purifi ed, linearized DNA was microinjected into the pronucleus of C57BL/6N mouse oocytes. Transgenic offspring (founders) were identifi ed by PCR genotyping of tail DNA. TPH2-CreERT2 transgenic mice were always crossed with C57BL/6N mice periodically purchased from Charles River Laboratories.

IN VIVO INDUCTION OF Cre-MEDIATED RECOMBINATION WITH TAMOXIFEN
Tamoxifen (Sigma) was dissolved in a corn oil/ethanol (10:1) mixture at a fi nal concentration of 10 mg/ml. Adult R26R TPH2-CreERT2 and GRfl ox/GRfl ox TPH2-CreERT2 mice (8-12 weeks) were injected intraperitoneally twice daily for fi ve consecutive days (protocol 1) with 1 mg of tamoxifen. Adult Brainbow1.0L TPH2-CreERT2 mice (8-12 weeks) were injected intraperitoneally twice daily for one to fi ve consecutive days. Control animals were injected with 100 µl of a corn oil/ethanol vehicle mixture (vehicle). Experimental animals for immunohistochemistry (IHC) or X-gal staining were sacrifi ced 5-14 days after the last injection. For recombination analysis during embryogenesis, pregnant R26R TPH2-CreERT2 mothers were injected for four consecutive days between E15 and E18 with 1 mg tamoxifen twice daily. Offspring were delivered by C-section at E20 and raised by foster mothers. For recombination analysis in postnatal pups, lactating transgenic mothers were injected intraperitoneally for fi ve consecutive days with either 1 mg tamoxifen twice daily (protocol 1), with 2 mg tamoxifen twice daily (protocol 2), with 3 mg tamoxifen twice daily (protocol 3) or one single injection of 1 mg tamoxifen daily (protocol 4). R26R TPH2-CreERT2 litters from embryonal and postnatal tamoxifen injections were analyzed at P60 for β-gal expression as described below. All experimental procedures were approved by the Animal Welfare Committee (Regierungspräsidium Karlsruhe) and carried out in accordance with the local Animal Welfare Act and the European Communities Council Directive of 24 November 1986 (86/609/EEC).

STATISTICAL METHODS
Coronal slices of two to four adult R26R TPH2-CreERT2 mice of each founder line were processed with dual-label fl uorescent IHC detecting βgal and TPH2. Image stacks of all slices that showed TPH2 staining were acquired using a confocal laser-scanning microscope. The ratio of βgal+/TPH2+ neurons to all TPH2+ neurons was calculated separately for caudal, median and dorsal raphe nuclei. Confi dence-bounds (CI) for recombination effi cacy and background recombination in adult mice were calculated using the Clopper-Pearson method based on signifi cance level 95.0% (Clopper and Pearson, 1934). For adult R26R TPH2-CreERT2 mice which had been injected during E15-18, P2-6 and P12-16 (n = 3 each group) the ratio of βgal+/TPH2+ neurons to all TPH2+ neurons per group was expressed as the mean and as the range of expression.

GENERATION OF TPH2-CreERT2 TRANSGENIC MICE
We used the CreERT2/loxP-system which provides inducible, Cre-mediated recombination upon tamoxifen administration to generate transgenic mice which allow conditional manipulation of individual genes in serotonergic neurons at any desired time. Targeted spatial control in serotonergic neurons was achieved by using elements of the Tph2 locus as a promoter to control Cre expression. Since precise information about the regulatory elements which govern timely and 5-HT specifi c TPH2 expression is not available, we used a large genomic fragment of the Tph2 locus to drive CreERT2, thereby enhancing the probability to include most of the important regulatory components of Tph2. To this end, a P1-derived artifi cial chromosome (pPAC4) with a 177 kb genomic insert containing the entire Tph2 gene with large additional fl anking sequences (see Section "Materials and Methods") was used to generate the TPH2-CreERT2 construct. The coding sequence of the CreERT2 fusion protein was inserted in place of the ATG-start of the Tph2 gene (Figure 1) by recombineering in bacteria (Lee et al., 2001). The modifi ed genomic fragment was released from the PAC backbone and fi nally introduced into the mouse germline of C57BL/6N by DNA microinjection. Five TPH2-CreERT2 founders were obtained (1.21, 1.23, 1.27, 1.35 and 1.38).

EFFICIENT AND SPECIFIC RECOMBINATION IN SEROTONERGIC NEURONS OF ADULT R26R Tph2CreERT2 MICE
TPH2-CreERT2 founder were crossed to ROSA26 Cre reporter mice (R26R) (Soriano, 1999) to obtain R26R Tph2CreERT2 mice. Temporal and spatial control of tamoxifen-induced CreERT2-mediated recombination in 5-HT neurons was determined and the effi cacy of serotonergic recombination in the caudal, dorsal and median raphe (MR) nuclei was calculated. Adult R26R Tph2CreERT2 mice were injected twice daily with either tamoxifen (1 mg) or vehicle for fi ve consecutive days. Animals were sacrifi ced for analysis 5 days after the last tamoxifen injection. X-Gal staining of tamoxifen-treated R26R Tph2CreERT2 mice from all lines revealed recombination in the brain stem and midbrain in regions shown to contain serotonergic somata (Figures 2A-I) while extraserotonergic brain regions showed no staining. Vehicle treated R26R Tph2CreERT2 mice showed minimal background recombination among all lines (Figures 2J-L). TPH2-CreERT2 lines 1.21, 1.35 and 1.38 showed the highest recombination effi ciency and lowest background recombination.
Next, we performed dual-label fluorescent IHC with anti-β-galactosidase (βgal) and anti-TPH2-antibodies to investigate selectivity and effi ciency of recombination in serotonergic neurons. While β-gal antibodies confi rm Cre-mediated recombination in the Rosa26 locus, TPH2 antibodies detect TPH2 which is the rate-limiting enzyme in brain serotonin synthesis and solely found in serotonergic raphe nuclei. As suggested by X-gal staining, recombination occurred exclusively in serotonergic neurons of all raphe nuclei with comparable effi ciency (Figures 3A-I). Vehicle treated mice showed only minimal background recombination (Figures 3J-L).
Lines 1.21 and 1.38 showed the highest recombination efficacy in serotonergic neurons when tamoxifen was injected (91 and 90%, respectively) ( Table S1 in Supplementary Material) while background recombination in vehicle treated mice was below 2% ( Table S2 in Supplementary Material). All further experiments were carried out with line 1.38.

RECOMBINATION IN SEROTONERGIC NEURONS OF R26R TPH2CreERT2 MICE DURING EMBRYOGENESIS AND POSTNATAL DEVELOPMENT
To investigate whether tamoxifen inducible recombination is also feasible during embryogenesis we injected pregnant mothers intraperitoneally with 1 mg tamoxifen twice daily for four consecutive days between E15 and E18. Offspring were delivered by C-section   Table S3 in Supplementary Material) but recombination rates were below those achieved during adulthood. Next, we determined whether recombination during early and later postnatal development is feasible. Lactating mothers were injected intraperitoneally for fi ve consecutive days starting at P2 and at P12, respectively. Different tamoxifen protocols were used: 1 mg tamoxifen twice daily (protocol 1), 2 mg tamoxifen twice daily (protocol 2), 3 mg tamoxifen twice daily (protocol 3) and 1 mg tamoxifen once daily (protocol 4).
Intraperitoneal injections were well tolerated by lactating mothers during the fi ve consecutive days of tamoxifen treatment but mothers tended to abandon their offspring thereafter, in particular those treated with 2 mg × 2 mg tamoxifen during early postnatal development (P2-6). Recombination during early and later postnatal development could be successfully induced in serotonergic neurons (Figures 4D-I; Figures S2D-I,M-R in Supplementary Material) but recombination effi ciency varied signifi cantly with protocols. Tamoxifen injections twice daily (2 mg × 1 mg) led to superior recombination effi cacy (68%) compared to single daily injections (1 mg × 1 mg) whereas doubling the amount of tamoxifen (2 mg × 2 mg) during early postnatal development (P2-6) did not result in better recombination effi cacy than with 2 mg × 1 mg tamoxifen per day. In contrast, further increasing the amount of tamoxifen (2 mg × 3 mg) in lactating mothers with older offspring (P12-16) led to recombination effi cacies (72%) comparable to those achieved during early postnatal development with 2 mg × 1 mg tamoxifen ( Table S3 in Supplementary Material).
In conclusion, signifi cant recombination effi cacies could be attained in R26R TPH2-CreERT2 offspring from tamoxifen injected pregnant or lactating mothers but recombination rates did not reach the level of adult recombination (90%). We also noted that insubstantial recombination occurred during embryogenesis and early postnatal development in regions which do not contain 5-HT neurons in adult mice. A few β-gal-positive/TPH2-negative cells were detected in the caudal and MR nuclei, the hypothalamus and deep layers of the cortex.

INDUCIBLE RECOMBINATION IN BRAINBOW-1.0L TPH2-CreERT2 MICE RESULTS IN COMBINATORIAL EXPRESSION OF FLUORESCENT PROTEINS WITH MULTIPLE, DISTINCT COLORS IN 5-HT NEURONS
The recently described Brainbow-1.0L transgenic mouse line (Livet et al., 2007) allows individual fl uorescence labeling of living neurons. In this line, Cre-mediated recombination of loxP-fl anked fl uorescence transgenes leads to a switch from RFP (red fl uorescent protein) expression to a stochastically determined combinatorial expression of CFP and YFP. Double transgenic Brainbow-1.0L TPH2-CreERT2 mice were induced with 1 mg tamoxifen twice daily for one to fi ve consecutive days and recombination was analyzed by fl uorescence emission. Confocal laser-scanning microscopy of RFP, CFP and YFP fl uorescence detected the combinatorial expression of CFP and YFP leading to mosaic fl uorescent labeling of serotonergic neurons (Figure 5; Figure S3 in Supplementary Material). The length of tamoxifen treatment correlated with the proportion of recombined 5-HT neurons. Three days of tamoxifen treatment were suffi cient to achieve maximal serotonergic recombination whereas in animals given 1-2 days of tamoxifen treatment the proportion of recombined 5-HT neurons was signifi cantly reduced.

EFFICIENT KNOCKOUT OF THE GLUCOCORTICOID-RECEPTOR GENE IN SEROTONERGIC NEURONS OF ADULT GRfl ox/GRfl ox TPH2-CreERT2 MICE
Both R26R and Brainbow mice are reporter mice which express fl uorescent proteins or βgal following Cre-mediated recombination. To demonstrate that loxP fl anked target genes can also be effi ciently deleted in the adult serotonergic system we generated TPH2-CreERT2 mice that are homozygous for the loxP-fl anked glucocorticoid-receptor (GR) gene (GRfl ox/GRfl ox TPH2-CreERT2 ) (Tronche et al., 1999). The GR protein is ubiquitously expressed throughout the brain. In particular, 5-HT neurons show abundant GR-antibody staining. Adult mice (2 months old) were treated with tamoxifen (1 mg twice daily for 5 days) and sacrifi ced 14 days later. Double-immunostaining against GR and TPH revealed that 80-90% of 5-HT neurons had lost their GR-immunoreactivity whereas GR expression was unchanged in all other brain regions examined (Figure 6; Figure S4 in Supplementary Material).

DISCUSSION
In this study, we describe a novel transgenic tool for conditional somatic manipulation of loxP-fl anked target genes in serotonergic neurons. Using the Tph2 genomic locus as a regulatory element to direct CreERT2 expression specifi cally to serotonergic neurons we show effi cient, tamoxifen-inducible recombination with minimal background activity in R26R TPH2-CreERT2 mice during development and adulthood.
Traditionally, transgenic mouse lines are generated via pronuclear injection of plasmid-derived DNA resulting in random integration of the transgene into the genome. The level and pattern of plasmid-derived transgene expression is determined both by the promotor and its integration site in the genome. Such transgenic mice show copy-number independent transgene expression in regions or cell types not associated with the original specifi city of the promoter (Bronson et al., 1996;Wallace et al., 2000;Chandler et al., 2007).
Because of the large insert size of BAC/PAC clones, they can often accommodate an entire gene of interest, including long-range cis-acting regulatory elements required for correct temporal and tissue-specifi c expression (Chandler et al., 2007). This is especially desirable for experiments where precise transgene expression is critical, such as for promotors driving Cre-recombinase (Lee et al., 2001). These large constructs allow copy-number dependent expression of the transgene resembling the expression of the endogenous gene independent of the integration site (Schedl et al., 1993;Casanova et al., 2001;Giraldo and Montoliu, 2001;Gong et al., 2003).  sections at E20. While behavioral effects of tamoxifen treatment have not been identifi ed in adult mice (Vogt et al., 2008) there is no comparable data on mice available that received tamoxifen treatment during development. Taken together, Pet1-Cre mice are preferable if embryonal serotonergic gene deletion starting at E12.5 (Scott et al., 2005) is desired as they do not require tamoxifen and convey high recombination rates. Inducible TPH2-CreERT2 mice are advantageous if recombination is to start later than E12.5. As such, TPH2-CreERT2 mice are currently the only alternative for postnatal and adult induction of serotonergic recombination. Nonetheless, further research needs to be conducted to improve tamoxifen application during development and to evaluate the biochemical, molecular and behavioral impact this treatment exerts on developing offspring.
Serotonergic recombination in Brainbow-1.0L TPH2-CreERT2 mice (Livet et al., 2007) creates a stochastic choice of Thy1-promotor controlled expression between two different loxP-fl anked fl uorescence genes as a result of tandem integration of Brainbow-1.0L copies. To date, it was unclear whether the Thy1-promotor is active in 5-HT neurons. Here we show that, upon tamoxifen induction, Brainbow-1.0L TPH2-CreERT2 mice display highly effi cient recombination in 5-HT neurons. Virtually all serotonergic neurons can be labeled with combinatorial CFP and YFP expression. An advantage of the TPH2-CreERT2 system in conjunction with the Brainbow mice is the possibility of titrating the proportion of 5-HT neurons exhibiting recombination by reducing the duration of tamoxifen treatment. This should facilitate anterograde tracing experiments of single, distinctly color-labeled serotonergic cell projections by three-dimensional reconstruction from serial sections which will help to defi ne the connectivity of individual 5-HT neurons or its cell clusters (B1-9) with other brain regions and within the raphe nuclei.
Tissue-specifi c recombination effi cacies derived from artificial Cre reporter lines should be interpreted carefully regarding their value to predict recombination frequencies for loxP-fl anked target genes (Weber et al., 2001). On this account, we substantiated our results obtained with R26R Cre reporter and Brainbow mice by inducing a selective knockout of the endogenous GR gene exclusively in 5-HT neurons of the adult mouse. With the GRfl ox/GRfl ox TPH2-CreERT2 mice it will be possible to decipher the reciprocal interactions between the HPA axis and 5-HT neurons, systems that have been implicated together in the etiology of several pathological conditions like escalated aggression and violence or anxiety and depression (Haller et al., 2005;Miczek et al., 2007;Lanfumey et al., 2008).
The neurotransmitter serotonin plays a diverse and important role in both the developing and adult brain (Gaspar et al., 2003). With our TPH2-CreERT2 line we provide a tool for highly effi cient and inducible recombination specifi cally in the serotonergic system. This system will allow the study of the roles of many genes in the regulation of serotonin systems and hence its role in brain development and function. Moreover, the possibility of manipulating gene expression selectively in mature serotonergic neurons allows direct molecular and behavioral effects of candidate gene deletion to be distinguished from compensatory, homeostatic mechanisms during development. In the pathology of complex neuropsychiatric diseases, serotonergic genes, in conjunction with stressors, may act at In this study we were able to demonstrate that tissue-specifi c regulatory elements of the mouse Tph2 locus, present on a 177 kb genomic DNA fragment and comprising the entire Tph2 gene plus large fl anking regions, reliably direct Cre expression selectively to serotonergic neurons in all fi ve analyzed transgenic TPH2-CreERT2 lines.
To date only two mouse lines published have been shown to target loxP-fl anked gene knockouts to the serotonergic system. A knockin of the Cre recombinase into the Sert gene has been used to achieve recombination in serotonergic neurons (Zhuang et al., 2005). However, this strategy is compromised by the fact that it results in a hemizygous Sert knockout. Furthermore, SERT is expressed in extraserotonergic neurons during development (Gaspar et al., 2003). Therefore, using the endogenous Sert locus to control Cre expression will lead to developmental recombination in neurons outside the serotonergic system. Finally, the Sert-Cre mouse line does not contain regulatory elements that allow temporal control of Cre-mediated recombination.
Recently, a Pet1-Cre transgenic mouse line was published (Scott et al., 2005) in which cell-type specifi c serotonergic gene inactivation of loxP-fl anked target genes will be irrevocably initiated at E12.5 during embryogenesis. Despite its excellent characteristics to create tissue-specifi c embryonal knockouts in the serotonergic system, Pet1-Cre lacks the possibility of temporal control. As Pet-1 is a key player in a transcriptional mechanism that controls embryonal 5-HT neuron differentiation (Hendricks et al., 2003) it would be of interest whether its regulatory elements are suffi cient to achieve inducible CreERT2-mediated gene inactivation in the entire serotonergic system of the postnatal and adult brain as well.
TPH2 is the rate-limiting enzyme of 5-HT synthesis in the serotonergic raphe nuclei (Walther et al., 2003). In the brain, it is strongly and exclusively expressed in serotonergic neurons starting during embryogenesis around E11 and continuing throughout adult life (Cote et al., 2003(Cote et al., , 2007. Thus, regulatory elements of the Tph2 locus should ensure strong serotonergic Cre expression in TPH2-CreERT2 mice at any time from embryogenesis to adulthood. To test this, we induced recombination in R26R TPH2-CreERT2 mice during embryogenesis, postnatal development and adulthood. In adult mice, tamoxifen injections twice daily for fi ve consecutive days lead to highly effi cient induction of recombination specifically in serotonergic neurons. During embryogenesis and postnatal development intraperitoneal injections to the mothers led to effi cient serotonergic recombination (66-72%) in their offspring but rates were inferior to adult recombination (90%). This fi nding is most likely a result of the tamoxifen application route rather than of attenuated activity of Tph2 regulatory sequences driving CreERT2 during development as expression of TPH2 commences already at E11. Moreover, we used the identical Tph2 regulatory sequences to direct the expression of other genes to serotonergic neurons during development and found strong expression already at E15 (unpublished observation). In our opinion, these results demonstrate that ip application to the mother does not lead to suffi cient tamoxifen concentrations in their offspring to achieve high recombination rates. Furthermore, tamoxifen is an active substance which inhibits ERs and stimulates progesterone receptors. In pregnant mice, tamoxifen treatment inhibits parturition (Boyle et al., 2008;Erdmann et al., 2008) which necessitated scheduled cesarean any time from development to adult life to predispose an individual to psychiatric disorders (Caspi et al., 2002(Caspi et al., , 2003Champoux et al., 2002). The action of perturbed serotonergic genes may even produce opposite phenotypes depending on time of action (Gross and Hen, 2004). This TPH2-CreERT2 mouse model may help to delineate time windows in which gene inactivation is crucial for the pathogenesis of neuropsychiatric disorders dependent on serotonergic abnormalities. Furthermore, varying the onset of gene manipulation during different periods of life and combining genetic manipulation with pharmacological or behavioral interventions will help to unravel gene-environment interactions that are crucial for the development or maintenance of pathological phenotypes.