Tetracycline Inducible Gene Manipulation in Serotonergic Neurons

The serotonergic (5-HT) neuronal system has important and diverse physiological functions throughout development and adulthood. Its dysregulation during development or later in adulthood has been implicated in many neuropsychiatric disorders. Transgenic animal models designed to study the contribution of serotonergic susceptibility genes to a pathological phenotype should ideally allow to study candidate gene overexpression or gene knockout selectively in serotonergic neurons at any desired time during life. For this purpose, conditional expression systems such as the tet-system are preferable. Here, we generated a transactivator (tTA) mouse line (TPH2-tTA) that allows temporal and spatial control of tetracycline (Ptet) controlled transgene expression as well as gene deletion in 5-HT neurons. The tTA cDNA was inserted into a 196 kb PAC containing a genomic mouse Tph2 fragment (177 kb) by homologous recombination in E. coli. For functional analysis of Ptet-controlled transgene expression, TPH2-tTA mice were crossed to a Ptet-regulated lacZ reporter line (Ptet-nLacZ). In adult double-transgenic TPH2-tTA/Ptet-nLacZ mice, TPH2-tTA founder line L62-20 showed strong serotonergic β-galactosidase expression which could be completely suppressed with doxycycline (Dox). Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system. For functional analysis of Ptet-controlled, Cre-mediated gene deletion, TPH2-tTA mice (L62-20) were crossed to double transgenic Ptet-Cre/R26R reporter mice to generate TPH2-tTA/Ptet-Cre/R26R mice. Without Dox, 5-HT specific recombination started at E12.5. With permanent Dox administration, Ptet-controlled Cre-mediated recombination was absent. Dox withdrawal either postnatally or during adulthood induced efficient recombination in serotonergic neurons of all raphe nuclei, respectively. In the enteric nervous system, recombination could not be detected. We generated a transgenic mouse tTA line (TPH2-tTA) which allows both inducible and reversible transgene expression and inducible Cre-mediated gene deletion selectively in 5-HT neurons throughout life. This will allow precise delineation of serotonergic gene functions during development and adulthood

Efficiency of Ptet-controlled βgal expression in TPH2-tTA/Ptet-nLacZ offspring of eight independent TPH2-tTA founder lines.

<p>TPH2-tTA/Ptet-nLacZ nice which had received Dox throughout development until P60 were sacrificed at P90 after 30 days of Ptet-controlled gene activation without Dox and analyzed with dual-label IHC. Efficiencies for caudal (CR), median (MR) and dorsal (DR) raphe nuclei were separately and jointly (total) calculated. Confidence-bounds (CI) were calculated using the Clopper-Pearson method based on significance level 95.0%.</p

Functional characterization of Ptet-controlled βgal expression in TPH2-tTA/Ptet-nLacZ mice.

<p>TPH2-tTA mice were mated with Ptet-nLacZ mice to generate double-transgenic TPH2-tTA/Ptet-nLacZ mice. In Ptet-nLacZ reporter mice, a bidirectional promoter Ptetbi which contains seven tet operator sequences flanked by two minimal promoters <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038193#pone.0038193-Baron1" target="_blank">[77]</a> allows bidirectional Ptet-controlled transcription of nuclear localized lacZ and luciferase cDNA. Only βgal reporter expression was analyzed in TPH2-tTA/Ptet-nLacZ mice. In the presence of Dox (+Dox), βgal expression is not initiated since tTA binding to Ptet is prevented by Dox. In the absence of Dox (-Dox), tTA binds to Ptet which activates lacZ reporter gene transcription.</p

Functional characterization of Ptet-controlled, Cre-mediated recombination in TPH2-tTA/Ptet-Cre/R26R mice.

<p>TPH2-tTA mice were mated with Ptet-Cre/R26R mice to generate triple-transgenic TPH2-tTA/Ptet-Cre/R26R mice. In Ptet-Cre mice, a bidirectional promoter Ptetbi allows co-regulated Ptet-controlled transcription of Cre and luciferase cDNA. Luciferase activity can be used to indirectly quantify tissue-specific Cre expression but was not needed to assess Cre-mediated recombination. In the presence of Dox (+Dox), Ptet-controlled Cre expression does not occur. In the absence of Dox (−Dox), tTA activates Ptet-controlled Cre expression. Cre then deletes a loxP-flanked stop cassette in the Rosa26 locus, thereby initiating βgal expression which indicates successful Cre-mediated recombination.</p

Non radioactive <b><i>in-situ</i></b><b> hybridization of TPH2-tTA founder lines.</b>

<p>A tTA-antisense probe was used to detect tTA-mRNA in transgenic TPH2-tTA mice. Each TPH2-tTA line was screened by non-radioactive ISH. (A-C) Exemplary tTA <i>in situ</i> hybridization of founder line 62-20 showed abundant tTA-mRNA in brain stem and midbrain areas where raphe nuclei with serotonergic neurons are located. CR, caudal raphe nuclei; DR, dorsal raphe nuclei; MR, median raphe nuclei.</p

Embryonic serotonergic recombination in TPH2-tTA/Ptet-Cre/R26R mice.

<p>In the absence of Dox, Ptet-controlled, Cre-mediated recombination was analyzed in the embryonic brain. Double-fluorescence IHC with βgal- and 5-HT neuron-specific TPH2 antibodies was done in brains from embryos at E12.5–E15.5. Already at E12.5, extensive βgal-staining was observed in developing 5-HT neurons (A,C,E). Later during development at E15.5, X-Gal staining allows easy visualization of the strong and ubiquitous βgal activity in caudal (B), dorsal (D) and median (D,F) raphe nuclei.</p

Protocols for inducible and reversible reporter gene expression in TPH2-tTA/Ptet-nLacZ mice.

<p>Dox was administered with 5 µg/ml from initiation of matings until birth. At P0, Dox concentration was changed to 50 µg/ml. Suppression of Ptet-controlled gene expression in adult mice was also done with 50 µg/ml Dox. In protocol 1, mice were maintained under chronic Dox administration throughout life until sacrifice at P90. In protocol 2, transgenic mice were maintained in the absence of Dox until P60 when Dox administration was initiated until sacrifice at P90. In protocol 3, Dox was given from conception until P60 when Dox was withdrawn until sacrifice at P90. Protocol 4 is based on protocol 3. Here, Ptet-controlled βgal expression was re-suppressed from P90 until sacrifice at P120. Protocol 5 is based on protocol 4, but removal of Dox at P120 until sacrifice at P150 reactivated Ptet-controlled βgal expression. +Dox, administration of Doxycycline; θDox, no administration of Dox.</p

Inducible and reversible 5-HT neuron-specific transgene expression in TPH2-tTA/Ptet-nLacZ mice.

<p>Inducibility and reversibility of Ptet-controlled transgene expression is a hallmark of the tet-system. To test these potential merits in TPH2-tTA mice, double-transgenic TPH2-tTA/Ptet-nLacZ mice were generated. Adult mice (>P90) were sacrificed and analyzed with dual-label fluorescence IHC using βgal and 5-HT neuron-specific TPH2 antibodies. (A,F,K) With permanent Dox administration (+Dox), Ptet-controlled βgal expression could be fully suppressed in TPH2-tTA/Ptet-nLacZ mice. Furthermore, Ptet-controlled βgal expression was reversible with Dox administration, regardless whether prior βgal expression occurred during development (−/+Dox; B,G,L) or during adulthood (+/−/+Dox; D,I,N). Vice versa, after chronic Dox administration during embryonic and postnatal development, Ptet-controlled βgal expression could be fully induced (C,H,M) and re-induced (E,J,O) via Dox withdrawal. Scale bars: 100 µm.</p

5-HT neuron-specific transgene expression in TPH2-tTA/Ptet-nLacZ mice.

<p>Exemplary results from TPH2-tTA line L62-20 mated with Ptet-nLacZ mice to generate double-transgenic TPH2-tTA/Ptet-nLacZ mice which never received Dox. Adult mice (P90) were sacrificed and analyzed with X-Gal staining and IHC. (A,C,E) Without Dox administration, X-Gal staining of coronal sections of brain stem and midbrain showed intense βgal activity in the raphe nuclei where 5-HT neurons reside. (B,D,F) Dual-label fluorescence IHC with βgal and 5-HT neuron-specific TPH2 antibodies. Co-labelling indicates 5-HT neuron-specific βgal transgene expression. IHC confirmed that βgal expression occurred tissue-specifically in the majority of all 5-HT neurons (B,D,F). Grey arrowheads show scarce βgal-negative TPH2+5-HT neurons. Scale bars: 100 µm.</p

Inducible, 5-HT neuron-specific recombination in TPH2-tTA/Ptet-Cre/R26R mice.

<p>Adult mice (P90) were sacrificed and analyzed with X-Gal staining and IHC. (A–L) X-Gal staining of coronal sections of the brain stem and midbrain. (M–X) Dual-label fluorescence IHC with βgal and 5-HT neuron-specific TPH2 antibodies. Co-labelling indicates 5-HT neuron-specific recombination. Without Dox administration (−Dox), X-Gal staining showed intense βgal activity in all areas of the raphe nuclei where 5-HT neurons are located (A,E,I). Dual-label IHC confirmed that βgal expression occurs tissue-specifically in virtually all 5-HT neurons (M,Q,U). Of note, extraserotonergic βgal expression was found in the median raphe nuclei of untreated mice (U, arrows). (B,F,J,N,R,V) With permanent Dox administration (+Dox), Cre-induced recombination and consequently βgal expression did only occur in a minor fraction of 5-HT neurons of TPH2-tTA/Ptet-Cre/R26R mice (V, arrow). To assess inducibility of Cre-mediated recombination, Dox was either administered during embryonic development until P0 (+Dox –P0; C,G,K,O,S,W) or during embryonic and postnatal development until P60 (+Dox –P60; D,H,L,P,T,X), when Dox was withdrawn until sacrifice at P90. X-Gal staining showed equally strong βgal activity in the raphe nuclei of TPH2-tTA/Ptet-Cre/R26R mice which were induced at P0 (C,G,K) or at P60 (D,H,L). As shown with IHC, recombination occurred tissue-specifically in the majority of 5-HT neurons, irrespective of the time point of induction at P0 (O,S,W) or P60 (P,T,X). The efficiency of tissue-specific recombination was indistinguishable from mice which never received Dox (M,Q,U) which proves that induction of Cre-mediated recombination after Dox suppression is equally efficient. Of note, when Cre recombination was suppressed with Dox until P0, extraserotonergic recombination in the median raphe nuclei could not be detected anymore (W,X). Grey arrowheads indicate scarce non-recombined, βgal-negative TPH2+5-HT neurons. Scale bars: 100 µm.</p