It is known that transgenes are randomly integrated in the host genome

3. RESULTS

3.1 Generation of Pet1/Cre transgenic mouse line

One of the most limiting problems in the generation of transgenic mouse models is the effect of the integration site of the transgene within the host genome on the expression of the transgene itself, known as positional effect.

It is known that transgenes are randomly integrated in the host genome;

therefore the same transgene can be differentially expressed in distinct mouse lines, depending on the region of the genome in which it is integrated. Positional effect may result even in the silencing of the transgene, or, in the opposite scenario, in ectopic expression of the transgene outside the expected expression domains. The use of bacterial artificial chromosome (BAC) technology for transgenesis has shown that integration site effects resulting in unwanted expression patterns are largely eliminated.

Therefore, in our lab a bacterial artificial chromosome (BAC) containing a 210 kb genomic fragment corresponding to the entire Pet1 gene locus has been used to drive the expression of the Cre recombinase in the Pet1/Cre transgenic mouse line (Fig. 3.1 a-c). This approach reduces positional effects, and likely guarantees the presence of all Pet1 enhancers and regulatory elements that can be located even many kilobases away from the promoter region.

In order to generate the Pet1/Cre transgene, an appropriate BAC clone containing the mouse genomic locus of Pet1 gene was first identified via in silico analysis on NCBI data bank (www.ncbi.nlm.nih.gov). Among the available clones, we chose rp23_165_D11 BAC clone, as it contains 170 kb upstream and 40 kb downstream Pet1 gene, likely ensuring a faithful transcriptional control of the Cre recombinase in Pet1/Cre transgene.

The generation of Pet1/Cre transgene was based on a homologous recombination approach in E.coli, strain DY380. DY380 cells harbour a defective λ-prophage carrying three λ-recombination proteins, exo (α), bet

(β) and gam (γ), collectively known as the Red proteins, under the tight control of the λ temperature-sensitive repressor (Lee et al., 2001).

Incubation of DY380 cells at 42°C results in the inactivation of the temperature-sensitive λ repressor, and hence the production of the exo (α), bet (β) and gam (γ) proteins, enabling recombination. In order to allow the

homologous recombination event to occur, the

Pet1(LA)Cre/pSVKanaFRT/Pet1(RA) construct was prepared as described in Materials and Methods.

Upon recombination in E. coli the recombined Pet1/Cre BAC was linearized and microinjected into the male pronucleus of fertilized eggs obtained from C57BL/6 females. The injected zygotes were subsequently transferred into the uterus of pseudopregnant recipient females, and the offspring was screened by PCR and Southern Blot assay to identify founders harbouring the Pet1/Cre transgene integrated into the host genome.

Three distinct Pet1/Cre transgenic mouse founders, namely founder female 9 (FF9), founder female 3 (FF3) and founder male 3 (FM3), were identified and intercrossed to wild-type animals in order to asses for germline transmission and to subsequently establish the three independent transgenic lines.

In order to validate the newly generated mouse line, I first characterized the expression of the Cre recombinase in the three distinct founder lines, by assessing whether the expression of Cre recombinase mirrors that of the endogenous Pet1 gene.

In order to characterize Cre expression, I used the Pet1/Cretransgenic line in combination with two different Cre-dependent conditional reporter transgenic mouse line: the ROSA26R reporter line (Soriano, 1999) in which the expression of the β-galattosidase reporter gene is inhibited by a stop cassette flanked by two loxP sites (floxed), and the ROSA26RYFP (Srinivas et al., 2001), which differs from the former because it expresses the enhanced yellow fluorescent protein (eYFP) reporter gene in place of β- galattosidase as reporter gene. In both reporter lines the Cre recombinase,

a

b c

under the transcriptional control of the Pet1 gene, recognizes loxP sites and promotes a conservative somatic recombination event that results in the excision of the stop cassette and the subsequent expression of the reporter gene in those cells in which the recombination event occurred and in their progeny. By mating the Pet1/Cre mouse line with either ROSA26R or ROSA26RYFP reporter lines I analysed double transgenic embryos (namely Pet1/Cre-ROSA26R or Pet1/Cre-ROSA26RYFP) starting from 9.5 dpc to postnatal stages for the presence of the reporter gene expression. Either a chromogenic reaction for the detection of β-galattosidase activity or immunohistochemistry (IHC) experiments to reveal the presence of YFP were performed.

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Figure 3.1 Pet1/Cre transgene drives Cre recombinase activity in the serotonergic system. Diagram showing the genomic region present within the BAC_rp23_165D11 (a), the targeting vector Pet1/Cre for the homologous recombination in DY380 bacteria (b) and the resulting recombined BAC_rp23_165D11 (Pet1/Cre transgene) (c). (d-j) X-gal staining of E12.5 Pet1/Cre- ROSA26R mouse embryos (d,f-g) and P1 Pet1/Cre-ROSA26R mice (e,h-j). At E12.5 X-gal staining highlights that Cre recombination have specifically occurred with a temporal progression in the rostral serotonergic nuclei (intense staining) (d,f) and in the caudal raphe nuclei (faint staining) (d,g). In P1 double transgenic animals Pet1/Cre is expressed in all B1-B9 serotonergic nuclei along the rostro-caudal extent of the hindbrain (h-j).

In the mouse hindbrain Pet1 expression starts to be detectable between 10.5 dpc - 11 dpc in the rostral serotonergic domain, and about one day later appears in the caudal domain (Pfaar et al., 2002; Pattyn et al.,2003). X-gal staining revealed that Cre recombinase was already expressed in E12.5 Pet1/Cre-ROSA26R embryos and showed a specific pattern along the rostro-caudal extent of the hindbrain, with the exception of the r4-derived territory, reflecting the endogenous expression of Pet1 (Fig.

3.1 d). Importantly, the three founder lines showed a specific Cre expression in a domain where serotonergic neurons develop, with only FM3 line exhibiting scarse staining in extraserotonergic brain regions. Moreover, the analysis of β-galattosidase expression at 12,5 dpc on coronal sections highlighted stronger expression intensity in the two lateral wings defining the rostral domain, as compared to the caudal raphe group (Fig.3.1 f,g), thus reflecting the temporal order in the generation of raphe serotonergic neurons. As development proceeds, the rostral and caudal hindbrain domains that will give rise to the B1-B3 caudal nuclei and the B4-B9 rostral raphe nuclei exhibit profound morphogenetic changes (Dahlström and Fuxe, 1964). At P1, Pet1/Cre-ROSA26R β-galattosidase-expressing neurons have migrated from their original position in the ventral region of the hindbrain to their final location forming the B1-B9 raphe nuclei, and also further increased in number (Fig. 3.1 e) Detailed analysis on coronal sections of P1

Pet1/Cre-ROSA26R mice showed that X-gal staining clearly identified all rostral raphe nuclei such as B8 median nucleus, B7 and B6 dorsal nuclei, and the B1-B3 caudal group (Fig.3.1 h-j). Thus, in the Pet1/Cre transgenic mouse line, Cre recombinase expression mirrors Pet1 spatio-temporal expression both in caudal raphe nuclei and in rostral raphe nuclei, in all the Pet1/Cre lines analyzed. As line FF9 showed the highest similarity to the Pet1 expression pattern without recombination in extraserotonergic domains, all further analyses were carried out using Pet1/Cre FF9 line, unless otherwise specified.

3.2 Pet1/Cre mouse line reveals the presence of a non-serotonergic Pet1-positive neuronal population in the raphe nuclei

In rodents, Pet1 is known as a precise and selective marker of serotonergic neurons, from early embryonic development up to adulthood (Hendricks et al., 1999; Hendricks et al., 2003). Therefore, in order to assess whether in our Pet1/Cre transgenic mouse line Cre expression is driven in all neurons producing 5-HT I performed combined immunohistochemistry experiments on P1 Pet1/Cre-ROSA26RYFP mouse brains using specific antibodies against YFP and 5-HT (Fig. 3.2).

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Figure 3.2 Pet1/Cre transgenic mouse line promotes somatic recombination in a non-serotonergic cell population within the raphe nuclei. (a-l) Coronal sections of P1 Pet1/Cre-ROSA26RYFP from different rostro-caudal levels of the hindbrain as indicated by the schematic representations on the left. Square boxes highlight the region of the raphe nuclei presented in the panels. Double immunohistochemistry for YFP and 5-HT shows the distribution of YFP-positive and double YFP /5-HT-positive cells in caudal B1-B3 (a-c), dorsal B6 (d-f), dorsal B7 (g-i) and median B8 (j-l) raphe nuclei. Merge images highlight that a higher level of discrepancy between YFP-positive and double YFP/5-HT-positive cell population is seen in B8 nucleus (l).

Surprisingly, the analysis of P1 Pet1/Cre-ROSA26RYFP coronal sections along the rostro-caudal extent of the hindbrain highlighted a discrepancy in 5-HT immunoreactivity among cells of the raphe nuclei.

Indeed, several YFP-positive cells resulted to be devoid of serotonin (Fig.

3.2). In particular, the ratio of YFP-positive cells on YFP/5-HT double positive seemed to be higher in the rostral nuclei, specifically in median (B8)

(Fig. 3.2 j-l), and B7 (Fig. 3.2 g-i) dorsal raphe nuclei, as compared to the B1-B3 caudal raphe neurons (Fig. 3.2 a-c). We observed that the discrepancy between cells in which YFP colocalizes with 5-HT and cells expressing YFP only was maintained up to postnatal stages.

Figure 3.3 Discrepancy between YFP-positive and double YFP/5-HT-positive cell population within the raphe nuclei persists at later stages of postanal development. (a-l) Coronal sections of P30 Pet1/Cre-ROSA26RYFP from different levels along the rostro-caudal axis of the hindbrain as indicated by the schematic representations on the left. Boxes highlight the region of the raphe nuclei presented in the panels. Double immunohistochemistry for YFP and 5-HT shows the distribution of YFP-positive and double YFP/5-HT-positive cells in caudal B1-B3 (a-c), dorsal B6 (d- f), dorsal B7 (g-i) and median B8 (j-l) raphe nuclei. Merge images highlight that a non- serotonergic cell population expressing YFP in neurons persists up to this stage (l).

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Analysis of coronal sections through the hindbrain of P30 Pet1/Cre- ROSA26RYFP mice immunostained for both YFP and 5-HT immunoreactivity confirmed that the ratio of YFP-positive/5-HT-negative on YFP-positive/5-HT-positive neurons was higher in the median raphe nuclei as compared to the caudal neuron groups (Fig. 3.3). The persistence of a YFP-positive/5-HT-negative cell population even at late postnatal stages led us to ask whether this unexpected lack of complete co-localization between YFP and 5-HT was due to either an ectopic expression of the Cre recombinase or to a specific, novel expression domain of Pet1 gene in a non-serotonergic cell subtype. To answer this question, I performed double in situ hybridization (ISH) experiments on P14, P30 and P60 (young adults) wild-type mice, using specific riboprobes for both Pet1 and Tph2, with the latter being expressed selectively in all mature serotonegic neurons in the hindbrain, as it encodes for the rate-limiting enzyme for the biosynthesis of brain serotonin (Fig. 3.4). In particular, I used NBT/BCIP and HNPP/Fast Red as alkaline-phosphatase (AP) substrates to detect Pet1 and Tph2 expression, respectively. The former reacts with AP to produce a dark blue precipitate, while the latter results in a red fluorescence also visible with diascopic illumination when overstained (Fig. 3.4). Cells expressing both Pet1 and Tph2 genes thus showed a combination of blue and red staining (see Materials and Methods for the complete description of the procedure).

Results showed that in B6 caudal dorsal and B7 dorsal serotonergic nuclei few cells presented Pet1 but not Tph2 expression, consistently with what observed in the immunohistochemistry experiments above described (Fig.

3.4 d-i’). In all the three stages analyzed Pet1-positive/Tph2-negative cells were still present and more frequently observed in median B8 nucleus, where the number of cells expressing only Pet1 resulted to be markedly increased respect to the amount of cells expressing both Pet1 and Tph2, particularly in the ventral region (Fig.3.4 g-i’).

Therefore, in Pet1/Cre transgenic mouse line, the expression of YFP in non-5-HT cells reflects the presence of a rostral population of cells expressing Pet1, which do not express Tph2.

Fig. 3.4 Pet1 gene is expressed in a non-serotonergic cell population in adult mice. (a-i’) Double ISH performed on coronal sections obtained from P14 (a,d,g), P30 (b,e,h) and P60 (g,h,i) wild-type animals, at the level of dorsal B6 (a-c), dorsal B7 (d-f) and median B8 (g-i) raphe nuclei. In each picture, Pet1 expression is highlighted by a dark blue staining, while Tph2 gene is visualized by a red precipitate (inset in a). In each picture the boxed areas indicate the region corresponding to the higher magnification on the right. In all the raphe nuclei analysed two distinct populations of neurons expressing either only Pet1 (arrows) or both Pet1 and Tph2 (arrowheads) are present. Median B8 raphe nucleus resulted the region in which neurons expressing Pet1 alone are more numerous (g’, h’, i’).

Since it is known that both dopaminergic and rostral serotonergic neurons originate closely to the midbrain-hindbrain boundary and common factors are important for their specification, such as Shh and Fgf8 (Hynes and Rosenthal, 1999), I first wanted to exclude that these rostral YFP- positive/5-HT-negative cells could be dopaminergic neurons. I performed immunohistochemistry experiments on P1 Pet1/Cre-ROSA26RYFP mice

using antibodies against YFP, which can be considered as a specific marker of Pet1 expression, and tyrosine hydroxylase (TH), the rate-limiting enzyme for the biosynthesis of dopamine (Fig. 3.5). Results showed that, consistently with what previously reported on the ontogenesis of dopaminergic and serotonergic systems (Hynes and Rosenthal, 1999;

Goridis and Rohrer, 2002), YFP-positive, either positive or not for serotonin, and TH-positive neurons are present in adjacent, non-overlapping regions, so that TH-positive neurons of the VTA did not express YFP and YFP- expressing cells in the hindbrain did not show immunoreactivity for TH (Fig.

3.5). Neurons expressing YFP are intermingled with TH-expressing cells only in the region of the rostral linear nucleus, but none of them resulted to express TH (Fig. 3.5 c,f). The use of other neuron subtype-specific markers, which is in progress, will help to define the molecular characteristics of these Pet1 non-serotonergic neurons.

Figure 3.5 Pet1 gene is expressed in a non-dopaminergic cell population. (a-c) Images of coronal sections of P1 Pet1/Cre-ROSA26RYFP mouse at the level schematically indicated in the black boxed area stained with anti-TH (a,d) and anti-YFP (b,e) antibodies. The white dotted box indicates the area showed at higher magnification below. Merge images (c,f) show that none of the YFP-positive cells are also TH-immunoreacitve, excluding that YFP- expressing cells are dopaminergic neurons.

3.3 Pet1/Cre transgenic mouse line highlights Pet1 expression in developing pancreas and kidney

Thanks to the combination of the properties of Cre/loxP system and mouse reporter lines it is possible to highlight cells that express Pet1 either transiently or at sub-detectable levels, that conversely would be difficult to be detected with canonical immunohistochemistry- or in situ hybridization- based methods. As the combination of our Pet1/Cre transgenic mouse line with inducible reporter lines allowed us to identify a pool of non-serotonergic, Pet1-expressing cells in the raphe nuclei not reported so far, I decided to better analyze Cre expression also in a broader array of tissues during embryonic development. To test the possibility that Pet1 is expressed also in other tissues than the hindbrain, I performed X-gal staining on Pet1/Cre- ROSA26Rmouse embryos starting from 11.5 dpc (Fig. 3.6). Results showed in all the three founder lines Cre-mediated recombination in extraserotonergic domains, with X-gal staining identifying two distinct stereotyped structures: a grape-like structure beneath the growing liver, and a parasagittal tubular structure on each side of the caudal abdominal region, ending with a small branch at the level of the hindlimbs (Fig. 3.6 a-c).

Furthermore, X-gal staining in these non-neural tissues resulted to be intense already at 11.5 dpc, while Pet1/Cre expression in the hindbrain was barely detectable at this stage, and only about one day later the rostral and caudal raphe groups could be clearly identified in whole mount preparations (Fig. 3.6 d-f). The precocious and intense X-gal staining in the caudal embryo made it reasonable to think that Cre-mediated recombination event in these cells have begun even earlier than in the raphe nuclei. At 13.5 dpc, the morphological analysis on sagittal sections of Pet1/Cre-ROSA26R mouse embryos showed that Pet1-mediated recombination had occurred in the developing pancreas and kidneys (Fig.3.6 g-l).

Fig 3.6 Pet1 drives Cre-mediated recombination in extraserotonegic domains. (a-l) X-gal staining performed on Pet1/Cre-ROSA26R whole mount 11.5 dpc embryos (a-c), flat mount preparations of 12.5 dpc hindbrains (d-f) and on sagittal sections at the level of the pancreas (g-i) and kidney (j-l) of 13.5 dpc embryos obtained from the three

founder mouse lines. At 11.5 dpc X-gal staining highlights a faint staining in the hindbrain (arrowheads), which at 12.5 becomes more evident in the rostral raphe group than in the caudal nuclei, with no staining in r4, mirroring Pet1 gene expression. Cre- mediated somatic recombination is occurred in two unexpected non-serotonergic domains in all the three founders line, namely the pancreas (g-i) and the kidney (j-l).

The presence of X-gal staining outside the canonical Pet1 domains raised the question about its possible origin as Cre recombinase expression in the pancreatic and nephric primordium could reflect an ectopic expression of Pet1/Cre transgene, or a specific Pet1 expression previously not detected. To test between these possibilities, I performed in situ hybridization experiments on E15.5 Pet1/Cre-ROSA26RYFP embryos, using specific riboprobes for Pet1 and YFP, to compare the expression of our transgene and that of the endogenous Pet1. I used also molecular markers such as Lmx1b and Nkx2.2, which are both expressed in serotonergic neurons, as well as in the kidney and pancreas, respectively (Fig. 3.7).

Figure 3.7 Pet1 drives Cre-mediated recombination in developing pancreas and kidney. ISH performed on coronal sections of Pet1/Cre-ROSA26RYFP 15.5 dpc embryos, at the level of the dorsal raphe nucleus (a-e), pancreas (f-j) and kidney (k-o).

At this stage, Pet1 is expressed not only in serotonergic neurons of the raphe (a), but also in the pancreas (f), and in few cells of the developing kidney (k). In these tissues, Pet1 expression correlates with the expression of the reporter gene (b,g,l), and with that

of specific markers for pancreas, such as Nkx2.2 (h), or that of both serotonergic neurons and kidney, such as Lmx1b (c,m), which in the kidney is expressed in podocytes. In the hindbrain, Tph2 is expressed in serotonergic neurons (e), while Tph1 is expressed in the pineal gland. Conversely, neither Tph2 nor Tph1 are expressed in the kidney and in the pancreas at this stage (i,j,n,o).

Results showed that Pet1 was strongly expressed in the pancreas, and that the YFP reporter well correlated with Nkx2.2, which is expressed in the pancreatic islet cells (Fig. 3.7 f-h). In the kidney, Pet1 was expressed at very low levels and only in a subset of cells, as compared to YFP expression, indicating that in this region Pet1 may have a faint expression that resulted sufficient to genetically label the progeny of the recombined cells (Fig. 3.7 k-l). Interestingly, Lmx1b, which in the kidney is expressed exclusively in podocytes, showed an expression pattern that was complementary to that of Pet1, suggesting that in the kidney Pet1 is expressed in distinct cell populations than podocytes (Fig. 3.7 k-m).

As in the hindbrain Pet1 gene expression leads to serotonergic neuron differentiation, I asked whether also in the pancreas and in the kidney Pet1 is needed to promote a serotonergic fate. Neither Tph1 nor Tph2, the known enzymes for the synthesis of 5-HT, resulted to be expressed in the kidneys and in the pancreas of 15.5 dpc Pet1/Cre- ROSA26RYFP embryos (Fig. 3.7 i,j,no). Furthermore, at the same stage, I could not observe any 5-HT immunoreactivity (not shown), thus indicating that, at least in E15.5 pancreas and kidney, serotonin is neither synthesized nor uptaken in the extraserotonergic Pet1-positive cells.

To better visualize the presence of Pet1 expression in the kidney, I performed RT-PCR and radioactive ISH on coronal sections of 10.5 dpc, 11.5 dpc and 12.5 dpc Pet1/Cre-R26RYFP embryos, using specific ³⁵S- labeled riboprobes against YFP and Pet1 (Fig. 3.8). Both the experiments showed sub-threshold levels of Pet1 expression within the hindbrain at 10.5 dpc, consistently with the reported timing of appearance of Pet1 gene expression, which starts between 10.5 dpc and 11 dpc in this region (Fig.

3.8 a, h,i). Analysis of autoradiograms showed a clearly detectable Pet1 expression in the developing kidney, which correlated with YFP expression in double transgenic mice at 10.5 dpc (Fig. 3.8 b,c). In 11.5 dpc embryos radioactive ISH showed Pet1 expression within the nephric ducts, where again it parallels with YFP expression (Fig. 3.8 d, e). In the hindbrain, both Pet1 and YFP expression are detectable in the raphe nuclei at 11.5 dpc (Fig.

3.8 j,k). At 12.5 dpc, Pet1 was strongly expressed in the hindbrain (Fig. 3.8 a,l,m), and it was readily visible by means of non-radioactive ISH, while in the kidney Pet1 expression was only detectable in wild-type animals by means of radioactive ISH, even though it resulted to be less intense than in the hindbrain (Fig. 3.8 a,f,g).

Fig 3.8 Pet1 is expressed during kidney development. Both RT-PCR and radioactive ISH confirmed Pet1 expression in the kidneys. (a) RT-PCR showing Pet1 expression at both 10.5 dpc and 12.5 dpc within the hindbrain, kidney, limbs and gut.

(b-m): radioactive ISH performed on coronal section of 10.5 dpc, 11.5 dpc and 12.5 dpc Pet1/Cre-R26RYFP (b-e, h-m) and wild-type (f,g) embryos. Results show presence of Pet1 expression within the nephric duct (yellow arrows) already present at 9.5 dpc (b), thus mirroring YFP reporter expression (c). Pet1 expression within the hindbrain is not yet present at 10.5 dpc (h,i), is detected at 11.5 dpc (yellow arrowheads) (j,k) and is clearly detectable with non-radiactive ISH at 12.5 dpc (l,m). In the kidney, Pet1 expression is in accordance with eGFP expression starting from

Hb, hindbrain Kd, kidney L, limbs G, gut

10.5dpc in the nephric duct (right panel). YFP reporter expression is not detected in 12.5 dpc wild-type embryos, confirming that Pet1 expression in the nephric duct is specific and not due to a stickiness of the tissue.

Thus, Cre activity in the Pet1/Cre mouse line faithfully recapitulates Pet1 expression, and can be used as an ideal tool to study the development of Pet1-expressing tissues. In this regard, as Pet1 expression in the kidney has not been described before, I used Pet1/Cre transgenic mouse line as a tool to follow in detail the development of the kidney cell population in which Pet1 was expressed (Fig 3.9).

Mammalian kidney organogenesis proceeds through three successive steps, giving rise to specific structures termed pronephros, mesonephros, both of which are transient structures, and metanephros, which will be the definite and functional kidney (reviewed in Lechner and Dressler, 1997). The first step of kidney development takes place between 8 and 10 dpc with the formation of the pronephric duct (also known as the nephric or Wolffian duct). At 10.5 dpc, at the caudal end of the nephric duct, which is about at the level of the hindlimbs (27–28^th somite), an outgrowth of the nephric duct called the ureteric bud (UB) invades the metanephric mesenchyme and is induced to branch; in turn, the branching ureteric bud tips induce the surrounding metanephric mesenchyme to undergo to a mesenchymal-to- epithelial transition. The UB forms a T-shaped bifurcation at 11.5 dpc, and then undergoes ∼11 cycles of branching and elongation to generate the metanephric collecting duct system (Fig. 3.9 a) (Cebrian et al., 2004).

Epithelial cells originated from metanephric mesenchyme give rise to nephron progenitors that proliferate, differentiate into glomerular and tubular epithelial cells, and fuse with the UB-derived collecting duct. This system of branching and differentiation is reiterated until nephrogenesis is completed, in mice shortly after birth (reviewed in Bouchard, 2004; Dressler, 2006).

Fig 3.9 Cre recombinase activity is present in the precursors of ureteric bud- derivatives. (a) Schematic representation of kidney development stages. Ureteric bud forms at 11.0, and starts invaginating within metanephric mesenchyme. At 11.5 dpc UB starts branching and by 13.5 dpc it has undergone many rounds of branching, forming the characteristic tree-like structure. Postnatally, UB-derivatives form the collecting duct, which elongate to form the medulla and the papilla. (b-g) X-gal staining performed on whole mount 9.5 dpc (b,b’), 10.5 dpc (c,c’), 11.5 dpc (d,d’), 13.5 dpc Pet1/Cre-ROSA26R mouse embryos and on coronal sections of a P10 Pet1/Cre-ROSA26R kidney (f,g). Staining performed at different stages of development recapitulates the main phases of UB-derivatives development, as highlighted in the magnifications (b’,c’,d’,e’). Cre-mediated recombination occurs already at 9.5 dpc in some scattered cells in the caudal nephric duct (b,b’), which increase at 10.5 dpc (c,c’) and later during development X-gal staining highlights the typical branching of ureteric bud (d-e’). At P10, X-gal staining highlights that Cre recombinase is expressed in the collecting duct system (f) and ureter (g). ND, nephric duct; NC, nephrogenic cord; NT, neural tube; LPM, lateral plate mesoderm; S, somites (Adapted from: Costantini and Kopan, Developmental Cell, 2010).

In order to highlight Pet1 expression during metanephric development I performed X-gal staining on Pet1/Cre-ROSA26R mouse embryos at different developmental stages known to be relevant in kidney development,

from 9.5 dpc to adulthood. Results showed that β-galactosidase expression was already present at 9.5 dpc, and it was detectable only at higher magnification as a group of scattered cells along the developing nepric duct (Fig. 3.9 b,b’). At 10.5 dpc X-gal staining is present with higher density in the cells at the caudal end of the nephric duct (Fig. 3.9 c,c’), which, one day later, will invade the surrounding metanephric mesechyme to form the ureteric bud (Fig. 3.9 d,d’), consistently with the timing of metanephric development. At 13.5 dpc Pet1/Cre expression highlighted that UB underwent already through different rounds of branching forming the typical tree-like structure (Fig. 3.9 e,e’). As expected, in P10 mice X-gal staining marked the epithelium of collecting duct (Fig. 3.9 f,g). Importantly, no X-gal staining was observed within the metanephric mesenchyme, thus suggesting that in Pet1/Cre transgenic mouse line Cre-mediated recombination occurred specifically within the ureteric bud derivatives (Fig.

3.9 f,g).

In order to confirm that Pet1 expression is localized within the ureteric bud derivatives, I used both ISH and IHC in order to compare YFP expression in Pet1/Cre-ROSA26RYFP kidneys to cadherin16 and calbindin- D28K expression, two well-known specific markers of ureteric buds (Davies, 1994; Thomson et al., 1995; Thomson et al., 1998) (Fig. 3.10). ISH performed on coronal sections of an E13.5 Pet1/Cre-R26RYFP embryo showed that YFP and cadherin16, which at this stage of development is strongly expressed in the branching ureteric bud, are expressed in overlapping domains (Fig. 3.10 a,b). Additionally, immunohistochemistry performed using anti-calbindin-D-28K and anti-YFP on whole-mount urogenital system dissected from E13.5 Pet1/Cre-R26RYFP embryos showed that cells expressing YFP are also immunoreactive for calbindin-D- 28K, which stains both tips and stalks of ureteric bud derivatives (3.10 c,f’).

Fig 3.10 Cre recombinase is co-expressed with the UB-derivatives markers calbindin-D28k and cadherin16 during early kidney development. (a,b) Parallel coronal sections of a 13.5 dpc Pet1/Cre-R26RYFP embryo, at the level shown in the schematic representation on the left, showing that YFP mRNA is expressed within the same domain of cadherin16, which is a specific marker for UB-derivatives during development. (c-f) Immunohistochemistry performed on whole-mount urogenital system dissected from a 13.5dpc Pet/1Cre-R26RYFP embryo (c,c’) using antibodies specific for YFP (d,d’) and calbindin-D28k, another specific marker for UB. Images show that ureteric bud derivatives strongly express YFP. Higher magnification images of the boxed areas highlight co-localization of YFP and calbindin, even though some calbindin- positive cells do not express YFP, indicating that Pet1 is expressed in a subset of UB- derivatives.

Since Pet1 has an important role in hindbrain serotonergic neuron development, where its expression precedes and specifically promotes that of Tph2, the key enzyme of 5-HT synthesis in the brain, I asked whether Pet1 has the same role in the kidney development. I performed IHC on coronal vibratome sections obtained from 13.5 dpc Pet1/Cre-R26RYFP embryos. Results showed that in double transgenic mice 5-HT is not present in the kidney, while at this developmental stage there are already many cells strongly positive for 5-HT in the hindbrain (not shown). We can thus

presume that Pet1 function in the kidney, if it has one, likely does not involve serotonergic fate.

Therefore, I demonstrated that Pet1/Cre mouse line drives Cre activity in Pet1-specific expression domain, such as seorotonergic neurons, confirming previous reports. Interestingly, Pet1/Cre mouse line allowed the identification of a Pet1-positive/5-HT-negative cell population both in the hindbrain and in two other distinct and so far ignored Pet1 expression domains, namely the developing pancreas and kidney.

3.4 Effect of serotonin depletion on serotonergic circuitry formation A Tph2::eGFP(FRT-neo-FRT) knock-in mutant allele has been generated in our lab, in which the Tph2 gene is replaced by the coding region of the vital reporter gene eGFP (Migliarini et al., 2008; Migliarini et al., 2009; Migliarini et al., submitted) (Fig. 3.11 a). In the recombinant allele thus generated Tph2 enzyme activity is disrupted and, at the same time, the presence of eGFP allows monitoring of Tph2 expressing-cells. In order to compare eGFP levels between heterozygous and mutant animals, the removable selection marker cassette PGK-neo has been cloned in the opposite transcriptional orientation than the eGFP cDNA, as previous reports have demonstrated that this particular arrangement repressed the reporter gene (Pasqualetti et al., 2002). Removal of the PGK-neo cassette by means of a Flp-mediated recombination resulted in the generation of the Tph2::eGFP^(FRT) allele, in which eGFP expression was activated (Fig. 3.11 b,c) and its presence was readily observed in all 5-HT positive neurons (Fig. 3.11 d-f). Therefore, as in Tph2::eGFP(FRT-neo-FRT) allele eGFP was not activated, while in Tph2::eGFP^(FRT) allele eGFP was strongly expressed, Tph2::eGFP^(FRT) +/- and Tph2::eGFP(FRT)(FRT-neo-FRT) -/- mice showed comparable eGFP expression levels.

In Tph2 mutants Tph2 activity was disrupted and, as a consequence, brain 5-HT was not synthesized any more. Strikingly, 60% of Tph2 -/- mice died within the first 3 weeks after birth, with apparently no defects in suckling

ability. Moreover, mutants showed a reduced growth rate starting from the very first days after birth, regaining body weight only after weaning.

Figure 3.11 Generation of the Tph2::eGFP mouse line expressing eGFP in

serotonergic neurons. (a) Diagram showing the targeting strategy for knock-in replacement of Tph2 and representative Southern blot analysis confirming homologous recombination in ES cells (BglI and StuI digests) and genotyping of mice upon flp- mediated recombination (HindIII digest). (b,c) Ventral view of adult brain rhombencefalic region of Tph2::eGFP(FRT-neo-FRT) +/- and Tph2::eGFP(FRT) +/- mice which showed live eGFP fluorescence of the boxed area in the inset (b) upon flp-mediated recombination. (d-f) Representative confocal images of adult Tph2::eGFP^{(FRT) +/-} dorsal raphe nuclei showing the presence of eGFP in all serotonin positive neurons. (g,h) Sagittal section of a 12.5 dpc Tph2::eGFP(FRT) +/- mouse immunostained for eGFP showing that eGFP expression highlights serotonergic system (g), and higher magnification of serotonergic fibers projecting rostrally (h). B: BglI, H: HindIII, S: StuI, RPa: raphe pallidus nucleus, RMg: raphe magnus nucleus, LPGi: lateral paragigantocellular nucleus, MnR: median raphe nucleus, DR: dorsal raphe nucleus.

Scale bars: 300 µm (b-c), 200 µm (d-h).

Thanks to presence of strong eGFP fluorescence, it has been possible to highlight serotonergic neurons and axons irrespective of 5-HT immunoreactivity (Fig. 3.11 g,h). Thus, Tph2::eGFP knockin line represents a suitable genetic tool for in vivo tracing of Tph2-expressing cells and projections allowing the study of the consequences of lack of brain 5-HT on serotonergic neuronal circuitry formation. Interestingly, despite mutant animals did not display any evident brain malformation and also the distribution and number of serotonergic somata resulted comparable to that of heterozygous mice, eGFP fluorescence highlighted that lack of serotonin produced severe abnormalities in the serotonergic circuitry formation with a brain region-specific effect.

Serotonergic innervation was highlighted by immunofluorescence on brain coronal sections of both Tph2::eGFP^(FRT) +/- and Tph2::eGFP^(FRT)(FRT-

neo-FRT) -/- adult animals and quantitative and qualitative evaluation of fiber density was performed using densitometric measurements and confocal microscopy analysis, respectively. In the Tph2-null mice, we were able to identify three distinct scenarios in which the density of serotonergic fibers appeared unaffected (e.g. cerebral cortex), dramatically reduced (e.g.

suprachiasmatic nucleus of the hypothalamus and thalamic paraventricular nucleus), or strongly increased (e.g. nucleus accumbens and hippocampus).

The cerebral cortex of Tph2::eGFP^(FRT) heterozygous mice appeared densely innervated by serotonergic fibers, as highlighted by eGFP- immunoreactivity, from layer I to VI, with a little fluctuant density in distinct layers of specific cortical regions. In the parietal cortex (primary somatosensory, S1) eGFP-positive fibers are particularly abundant in layers I and IV-V. Densitometric analysis of eGFP-immunoreactivity resulted in an overlapping curve of the measured ROD throughout the cortical depth in both Tph2::eGFP^(FRT) +/- and Tph2::EGFP(FRT)/(FRT-neo-FRT) -/- mice, demonstrating no alteration in the density of 5-HT axons in the cerebral cortex of Tph2-null mice (Fig. 3.12 a). Confocal analysis of the same cortical regions showed similar path arrangement of serotonergic innervation in the two genotypes (Fig 3.12 b), and further confirmed no variation of serotonergic projections to the cerebral cortex of 5-HT depleted mice.

Figure 3.12 Normal development of serotonergic fibers in the cerebral cortex of Tph2::eGFP(FRT)/(FRT-neo-FRT) null mice. (a) Comparison of relative optical density of eGFP+ serotonergic axons measured through the primary somatosensory cortex of

Tph2::eGFP^(FRT) +/- and Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice (n=3 per genotype). (b,c) Representative composit confocal analysis on coronal section of Tph2::eGFP^(FRT) +/- and Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- primary somatosensory cortex displaying the distribution of serotonergic fibers throughout the cortical layers (I-VI). Data are presented as mean ± SEM. Scale bars: 100 µm.

The suprachiasmatic nucleus (SChN) of the hypothalamus and the paraventricular nucleus (PVN) of the thalamus represent one of the most prominent areas containing serotonergic fibers in the rostral brain.

Serotonergic fibers innervating the SChN are mainly distributed within the ventral and ventromedial aspect of the pear-like shaped nucleus, forming a highly stereotyped topographic arrangement as highlighted by eGFP fluorescence (Fig. 3.13 a-f). Despite morphological features of SChN and PVN DAPI staining to assess anatomical coordinates allowing to discern raphe-projecting fibers to the SChN (Fig. 3.13 a-b, d-e). In the SChN of Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice, eGFP-immunofluorescence was dramatically reduced in intensity as compared to heterozygous mice (Fig.

3.13 e).

Figure 3.13. Altered serotonergic innervation to the suprachiasmatic (SChN) and talamic paraventricular nuclei (PVN) in Tph2::eGFP(FRT)/(FRT-neo-FRT) mutant mice.

(a-f) Representative confocal images of coronal sections immunostained for eGFP through the SChN of Tph2::eGFP^(FRT) +/- (a-c) and Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- (d-f) adult mice. (a,d) DAPI staining showing normal SChN morphology. (b,e) Distribution of eGFP+ serotonin fibers around the pear-like shaped SChN in Tph2::eGFP^(FRT) heterozygous and Tph2::eGFP(FRT)/(FRT-neo-FRT) mutant animals. (c,f) High magnification images showing the dramatic reduction of the eGFP+ fiber density in mutant mice. In the insets, a digital magnification (5x) showing a remarkable reduction of varicosities in Tph2::eGFP(FRT)/(FRT-neo-FRT) mutants as compared to the Tph2::eGFP^(FRT) controls. (g-l)

Representative images of coronal sections through the PVN of Tph2::eGFP^(FRT) +/- (g- i) and Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- (j-l) adult mice. (g,j) Nissl staining showing the characteristic morphology of PVN, which appear normally developed in Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice as compared to Tph2::eGFP^(FRT)) +/- mice. A dashed line is drawn around the PVN. (h,k) Confocal immunostaining analysis showing eGFP+ serotonergic innervation to the PVN. Fibers in Tph2::eGFP(FRT)/(FRT-neo-FRT) mutants are present in a more restricted area with a significantly reduced density. (i,l) The higher magnification further illustrates the reduction in axonal arborizations in Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice. Scale bars: 200 µm (a-b,d-e,g-h,j-k), 15 µm (c,f,i,l).

Although barely visible, eGFP-positive fibers left in the SChN region depicted a pattern resembling the distribution of serotonergic innervation observed in normal mice. High magnification confocal images in the medial region of the SChN revealed that the reduction of fluorescence signal was due to a remarkable reduction in the number of varicosities of the remaining serotonergic fibers (Fig 3.13 c,f).

In the ventral striatum the nucleus accumbens subdivides into two structures, the nucleus accumbens core (NAc) and the nucleus accumbens shell (NAs), both receiving profuse serotonergic innervation. The NAs, in comparison to NAc and dorsal striatum, shows a higher density of 5-HT axons forming a paired crescent-shaped structure located ventromedially the anterior commissure (Fig 3.14 a-b). In animals devoid of brain 5-HT, the organization of serotonergic fibers to the NAs resulted profoundly altered, as highlighted by the distribution of the eGFP immunoreactivity. In Tph2::EGFP(FRT)/(FRT-neo-FRT) -/- mice, the intensity of eGFP immunofluorescence appeared remarkably increased and the crescent- shaped fluorescent territory resulted enlarged on its medio-lateral extent as compared to control Tph2::EGFP^(FRT) +/- animals (Fig. 3.14 a,d). Confocal image analysis showed that the observed phenotype in Tph2-null mice was due to serotonergic hyperinnervation in the NAs as in this region eGFP- positive fibers were considerably augmented in their density and they spread on a wider extent (Fig. 3.14 b,e). To reveal morphological features of the

serotonergic axons detected in mutant animals, higher magnification images of immunofluorescences obtained from the two genotypes were compared.

Analysis further confirmed the presence of highly packed axons forming a dense tangle of eGFP-positive fibers. Moreover, higher magnification images further demonstrated the hyperinnervation of serotonergic fibers in Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice and showed that supernumerary eGFP- positive axons were mainly represented by fibers with thick axonal diameter and characterized by the presence of large varicosities (Fig. 3.14 c,f).

Serotonergic innervation has been described to span all over the hippocampal formation though showing specific density of fibers within distinct layers (Lidov and Molliver, 1982) (Fig 3.14 a,g). Stratum lacunosum- moleculare showed a higher density of 5-HT axons whereas, fainter bands of immunofluorescent signal, revealed a lower density of eGFP positive fibers in the stratum oriens, in the stratum radiatum and in the molecular layer of dentate gyrus of Tph2::eGFP^(FRT) +/- animals (Fig 3.14 a-b,g-h). As a result of Tph2 enzymatic activity disruption, a marked hyperinnervation was observed throughout the rostro-caudal extent of the hippocampus of Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice. The magnitude of the observed hyperinnervation was quantitatively evaluated for each of the four layers in both the dorsal and ventral hippocampal regions. Comparison of measured ROD of eGFP-immunoreactivity between the two genotypes, Tph2::eGFP^(FRT) +/- and Tph2::eGFP(FRT)/(FRT-neo-FRT) -/-, revealed an overall significant (p<0,001) increase of the density of 5-HT axons in all the hippocampal layers analysed, with a general trend of higher density in the ventral hippocampal region of each layer as compared to the dorsal counterpart.

Figure 3.14 Serotonergic hyperinnervation in the nucleus accumbens (NA) and in the hippocampus of Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice. (a-r) Fluorescent microscopy images of coronal sections immunostained for eGFP showing serotonergic fiber distribution through the nucleus accumbens (a-f) and hippocampus (g-r) of adult animals. (a) eGFP-positive serotonergic fibers in the NA are mainly distributed in the

shell (dashed line). (d) In Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice the density of eGFP+

fibers results remarkably increased and the stereotyped crescent-shaped structure in the NA shell appears enlarged. (b-c,e-f) Confocal high magnification images confirm the hyperinnervation of serotonergic fibers in Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice and show an increase of the number of serotonergic varicosities containing strong eGFP fluorescence. (g,m) Epifluorescence images of coronal sections showing the laminar arrangement of eGFP+ serotonergic fibers in dorsal and ventral hippocampus in Tph2::eGFP^(FRT) +/- mice. (j,p) In Tph2::eGFP(FRT)/(FRT-neo-FRT) mutants an overall increase in the density of serotonergic fibers is observed with a higher distribution of eGFP-positive axons within the stratum lacunosum-moleculare. (h-i,k-l,n-o,q-r) Confocal higher magnification of the boxed region of stratum lacunosum-moleculare.

Co: nucleus accumbens core, Sh: nucleus accumbens shell, Or: stratum oriens, Rad:

stratum radiatum, LMol: stratum lacunosum-moleculare, Mol: stratum moleculare.

Scale bars: 300 µm (a,d,g,j,m,p), 50 µm (b,e,h,k,n,q), 15 µm (c,f,i,l,o,r).

Specifically, the density of the serotonergic fibers in the stratum oriens of the dorsal and the ventral hippocampus of Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice was +25% and +47%, respectively, of that measured in heterozygotes.

Similarly, the average densities of the observed hyperinnervation in the dorsal and the ventral hippocampus of 5-HT depleted mice were +40% and +41% in the stratum radiatum, +39% and +44% in the stratum lacunosum- moleculare, +32% and +49% in the molecular layer of dentate gyrus. These data suggest that in the hippocampus serotonin plays a key role in the control of the serotonergic innervation development providing a signal for proper sprouting of serotonergic fibers.

Thus, we conclude that in the Tph2::EGFP(FRT)/(FRT-neo-FRT) -/- mice, lack of brain serotonin affects CNS development leading to different effects, ranging from a dramatic depletion to a marked hyperinnervation of serotonergic axons, in a district specific manner.

3.5 5-HT regulates serotonergic system via a cell- and non-cell- autonomous mechanism

We sought to identify the cues that changed specifically in response to the lack of brain serotonin and that might be at the origin of the observed abnormalities of serotonergic axon development found in Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- animals. We wanted to address the question whether serotonin exerts its action on serotonergic system development in either a cell- or non-cell-autonomous manner. A number of experimental evidences suggests brain derived neurotrophic factor (BDNF) as a candidate given its role in promoting the development and the establishment of neural circuitry of the serotonergic system. We identified an up-regulation of BDNF in the ippocampal region of Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice, which correlates with the increased serotonergic fiber sprouting in the hippocampus (Migliarini et al., submitted). This result suggests that in this district 5-HT acts through a non-cell-autonomous mechanism. In parallel, I asked whether 5-HT might also act through a cell-autonomous mechanism, acting as a trophic factor on serotonergic neurons themselves, modulating the expression of genes crucial for axonal outgrowth. To this purpose I took advantage of the possibility to isolate pure populations of serotonergic (eGFP-positive) neurons from both heterozygous and mutant mice. Pools of 200 dorsal raphe neurons per animal were used to purify mRNA and labelled cDNA were used for microarray analysis of gene expression to identify specific factors whose expression level was modulated as a consequence of serotonin depletion. Bioinformatic analysis showed a number of genes that were significantly either up- or down-regulated in mutant animals, and fold- changes of gene expression levels were quickly identified with Volcano plot visualisation. Volcano plot showed the change-fold versus significance on the x- and y-axes, respectively. Tph2 resulted, as expected, the most down- regulated gene. In Fig. 3.15 c-f, representative Volcano plots of expression level variations of genes belonging to specific family proteins can be visualised. An example of down-regulated genes was Arfagap2 gene, while

Fgf9 and S100β were among those up-regulated as a consequence of serotonin depletion. Interestingly, S100β, a member of the S100 family calcium binding protein B, has been shown to be directly involved in promoting serotonergic system development. The analysis of the huge number of gene whose expression is modulated by lack of serotonin is currently in progress.

Figure 3.15 Microarray transcriptome analysis of serotonergic neurons in Tph2^eGFP -/- mice. (left) Schematic representation of the experimental strategy for the isolation of pure populations of fluorescent Tph2::eGFP cells for gene expression profiling by means of microarray analysis. Brains are collected from both heterozygous and mutant Tph2::eGFP adult mice and cells from dorsal raphe nuclei are obtained, as described in Material and Methods. Use of a combination of diascopic and epifluorescence illumination fluorescent cells (yellow arrows) are discriminated from cell debris and non-fluorescent cells (white arrow) (a).

Pure population of fluorescent cells appearing healthy as preserve their processes (b). (c-f) Representative Volcano plots of four distinct protein families showing genes whose expression is significantly different between Tph2::eGFP heterozygous and mutant animals.

The x-axis corresponds to the log2 of the fold change between heterozygotes and mutants and the y-axis corresponds to the log10(p-value).The higher the log10(p-value) for each gene, the higher the probability that the gene is differentially expressed and not a false positive.

Analysis shows that Tph2 is, as expected, the most down-regulated gene (c). Arfagap2 is one of the down-regulated genes (d), while Fgf9 (e) and S100β (f) were among those up- regulated.