Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Les Wnts représentent une famille de glycoprotéines de signalisation qui sont connues pour les nombreux rôles qu'ils jouent durant le développement embryonnaire et dans la cancerogénèse. Plusieurs Wnts, leurs récepteurs (Fzd) et d'autres composants des voies de signalisation des Wnt sont exprimés dans l’ovaire postnatal, et il a été démontré que l’expression de certains de ces gènes est régulée pendant le développement et l'ovulation/luteinization folliculaires. Toutefois, leurs rôles physiologiques dans l’ovaire demeurent mal définis. Pour étudier le rôle de WNT4 dans le développement folliculaire, nous avons entrepris d’identifier ses cibles transcriptionnels dans les cellules de la granulosa. Pour ce faire, nous avons employé la souris Catnbflox(ex3)/flox(ex3), chez laquelle une activation constitutive de la voie de Wnt/β-catenin a lieu suite à l’action de la recombinare Cre. Des cellules de la granulosa de ces souris ont été mises en culture et infectées avec un adenovirus pour causer la surexpression de WNT4 ou l’expression de Cre. L’ARN a alors été extrait de ces cellules et analysé par micro-puce. Les résultats ont démontré qu’une forte proportion des gènes induits par WNT4 étaient des gènes impliqués dans la réponse cellulaire au stress. Presque tous gènes induits par WNT4 ont également été induits par Cre, indiquant que WNT4 signale via la voie Wnt/β-catenin dans ces cellules. Nos résultats suggèrent donc que WNT4 favorise la survie des follicules par l’induction de gènes de réponse au stress dans les cellules de la granulosa, augmentant ainsi la résistance cellulaire à l'apoptose. / The Wnts comprise a large family of local-acting, secreted glycoprotein signaling molecules that are known mostly for the numerous roles they play in embryonic development and cancer. Several Wnts, their cognate receptors of the Frizzled (Fzd) family and other components of the Wnt signaling pathways are expressed in the postnatal ovary, and several have been shown to exhibit specific patterns of regulation in response to gonadotropin stimulation. Nonetheless, their role(s) in ovarian physiology remain poorly defined. To study the role of WNT4 in follicle development, we endeavoured to identify its transcriptional targets in granulosa cells. To this end, we used the Catnbflox(ex3)/flox(ex3) mouse model, in which constitutive activation of the Wnt/β-catenin pathway is obtained following Cre-mediated genetic recombination. Cultured granulosa cells from these mice were infected with adenoviruses to either overexpress WNT4 or to express Cre. RNA from these cells was then extracted and subjected to microarray analysis. Results revealed that a large proportion of the genes induced by WNT4 were genes previously shown to mediate cellular stress responses. Nearly all genes that were up-regulated by WNT4 were also induced by the Cre, indicating that WNT4 signals via the Wnt/β-catenin pathway in these cells. Our findings suggest that WNT4 mediates ovarian follicle survival by inducing a stress response in granulosa cells, thereby increasing their resistance to apoptosis.

Les Wnts représentent une famille de glycoprotéines de signalisation qui sont connues pour les nombreux rôles qu'ils jouent durant le développement embryonnaire et dans la cancerogénèse. Plusieurs Wnts, leurs récepteurs (Fzd) et d'autres composants des voies de signalisation des Wnt sont exprimés dans l’ovaire postnatal, et il a été démontré que l’expression de certains de ces gènes est régulée pendant le développement et l'ovulation/luteinization folliculaires. Toutefois, leurs rôles physiologiques dans l’ovaire demeurent mal définis. Pour étudier le rôle de WNT4 dans le développement folliculaire, nous avons entrepris d’identifier ses cibles transcriptionnels dans les cellules de la granulosa. Pour ce faire, nous avons employé la souris Catnbflox(ex3)/flox(ex3), chez laquelle une activation constitutive de la voie de Wnt/β-catenin a lieu suite à l’action de la recombinare Cre. Des cellules de la granulosa de ces souris ont été mises en culture et infectées avec un adenovirus pour causer la surexpression de WNT4 ou l’expression de Cre. L’ARN a alors été extrait de ces cellules et analysé par micro-puce. Les résultats ont démontré qu’une forte proportion des gènes induits par WNT4 étaient des gènes impliqués dans la réponse cellulaire au stress. Presque tous gènes induits par WNT4 ont également été induits par Cre, indiquant que WNT4 signale via la voie Wnt/β-catenin dans ces cellules. Nos résultats suggèrent donc que WNT4 favorise la survie des follicules par l’induction de gènes de réponse au stress dans les cellules de la granulosa, augmentant ainsi la résistance cellulaire à l'apoptose. / The Wnts comprise a large family of local-acting, secreted glycoprotein signaling molecules that are known mostly for the numerous roles they play in embryonic development and cancer. Several Wnts, their cognate receptors of the Frizzled (Fzd) family and other components of the Wnt signaling pathways are expressed in the postnatal ovary, and several have been shown to exhibit specific patterns of regulation in response to gonadotropin stimulation. Nonetheless, their role(s) in ovarian physiology remain poorly defined. To study the role of WNT4 in follicle development, we endeavoured to identify its transcriptional targets in granulosa cells. To this end, we used the Catnbflox(ex3)/flox(ex3) mouse model, in which constitutive activation of the Wnt/β-catenin pathway is obtained following Cre-mediated genetic recombination. Cultured granulosa cells from these mice were infected with adenoviruses to either overexpress WNT4 or to express Cre. RNA from these cells was then extracted and subjected to microarray analysis. Results revealed that a large proportion of the genes induced by WNT4 were genes previously shown to mediate cellular stress responses. Nearly all genes that were up-regulated by WNT4 were also induced by the Cre, indicating that WNT4 signals via the Wnt/β-catenin pathway in these cells. Our findings suggest that WNT4 mediates ovarian follicle survival by inducing a stress response in granulosa cells, thereby increasing their resistance to apoptosis.

<p>The divergence of distinct cell populations from multipotent progenitors is poorly understood, particularly <italic>in vivo</italic>. The gonad is an ideal place to study this process because it originates as a bipotential primordium where multiple distinct lineages acquire sex-specific fates as the organ differentiates as a testis or an ovary. The early gonad is composed of four lineages: supporting cells, interstitial/stromal cells, germ cells, and endothelial cells. Each lineage in the early gonad consists of bipotential progenitors capable of adopting either a male or female fate, which they do in a coordinated manner to form a functional testis or ovary. The supporting cell lineage is of particular interest because the decision of these cells to adopt the male or female fate dictates the fate of the gonad as a whole. </p><p><p>To gain a more detailed understanding of the process of gonadal differentiation at the level of the individual cell populations, we conducted microarrays on sorted cells of the four lineages from XX and XY mouse gonads at three time points spanning the period when the gonadal cells transition from sexually undifferentiated progenitors to their respective sex-specific fates. Our analysis identified genes specifically depleted and enriched in each lineage as it underwent sex-specific differentiation. We also determined that the sexually undifferentiated germ cell and supporting cell progenitors showed lineage priming. Multipotent progenitors that show lineage priming express markers of the various fates into which they can differentiate and subsequently silence genes associated with the fate not adopted as they differentiate. We found that germ cell progenitors were primed with a bias toward the male fate. In contrast, supporting cell progenitors were primed with a female bias. This yields new insights into the mechanisms by which different cell types in a single organ adopt their respective fates. </p><p><p>We also used a genetic approach to investigate how individual factors contribute to the adoption of the male supporting cell fate. We previously demonstrated that <italic>Fgf9</italic> and <italic>Wnt4</italic> act as mutually antagonistic factors to promote male or female development of the bipotential mammalian gonad. <italic>Fgf9</italic> is necessary to maintain <italic>Sox9</italic> expression, which drives male development. However, whether FGF9 acted directly on <italic>Sox9</italic> or indirectly through repression of <italic>Wnt4</italic>, was unknown. <italic>Wnt4</italic> is a female-primed gene, and is therefore repressed during male development. To determine how <italic>Fgf9</italic> functioned, we generated double <italic>Fgf9/Wnt4</italic> and <italic>Fgfr2/Wnt4</italic> mutants. While single XY <italic>Fgf9</italic> and <italic>Fgfr2</italic> mutants showed partial or complete male-to-female sex reversal, loss of <italic>Wnt4</italic> in an <italic>Fgf9</italic> or <italic>Fgfr2</italic> mutant background rescued normal testis development. We also found that <italic>Wnt4</italic> and another female-associated gene (<italic>Rspo1</italic>) were derepressed in <italic>Fgf9</italic> mutants prior to the down-regulation of <italic>Sox9</italic>. Thus, the primary function of <italic>Fgf9</italic> is the repression of female genes, including <italic>Wnt4</italic>. We also tested the reciprocal possibility: that de-repression of <italic>Fgf9</italic> was responsible for the aspects of male development observed in XX <italic>Wnt4</italic> mutants. However, we show that loss of <italic>Fgf9</italic> in XX <italic>Wnt4<super>-/-</super></italic> gonads does not rescue the partial female-to-male sex reversal. </p><p><p>Based on the <italic>Fgf9/Wnt4</italic> double mutant studies, we propose a two part model of male sex determination in which both the activation of male genes and repression of female genes is required. Also, this work demonstrates that the repression of the female-primed gene <italic>Wnt4</italic> is required for male development, and <italic>Fgf9</italic> is one factor that leads to the repression of female-primed genes.</p> / Dissertation

Components of the molecular pathway underlying gonadogenesis in organisms with temperature-dependent sex determination (TSD) have been retained from genetic sex determination. Furthermore, although much of this network has been conserved, new functions for these genes have evolved in this different mode of sex determination. We find that the transcription factors Sox9 and Dmrt1 and the hormone Mis are involved in the formation of a testis and/or the repression of an ovary at a male-producing temperature. While Mis expression may be maintained by Sox9, the initial upregulation of Mis in the developing testis is most likely modulated by some other upstream factor. Dmrt1 appears to play an upstream role in testis sex determination. We provide evidence that the transcription factor Dax1 and the signaling molecule Wnt4, cloned for the first time in an organism with TSD, play roles in gonadogenesis in both sexes. Finally, we show that the transcription factor FoxL2 and the signaling molecule Rspo1 are involved in the formation of an ovary and/or the repression of a testis at a female-producing temperature. In the first investigation of Rspo1 in any organism exhibiting TSD, we demonstrate it is involved upstream in ovarian sex determination. Complementary to descriptive studies, we optimize a whole organ culture system in which gonad explants develop in vitro for up to three weeks. We show that expression of the sex-determining network in isolated gonads mimics in ovo patterns, revealing an endogenous temperature-sensing mechanism that does not require other embryonic tissues. Ectopic expression of Sox9 reveals a possible positive feedback regulation of Dmrt1. The use of this culture system opens the door to functional manipulation of the gonad at the molecular level and is suitable for a myriad of future studies. This work makes strides in elucidating the molecular network underlying gonadogenesis in an organism exhibiting TSD, and invites investigation of the evolution of gene function. The data lend insight into the changing roles of molecules in sex determination across diverse taxa, and into the evolution of developmental pathways in general. / text

Transforming growth factor-β (TGF-β) superfamily of cytokines comprises of several members, which can broadly be sub-divided into three classes [TGF-βs, Activin/Nodal, and Bone morphogenetic proteins (BMPs)]. Most members of this family play critical roles during embryo development differentiation and regulation of homeostasis. In mammals there are three TGF-β isoforms, TGF-β1, 2 and 3. All the three TGF-β isoforms have important roles in embryo development as revealed by mouse knock-out models. TGF-β has also been associated with several pathological conditions such as inflammation, Fibrosis, and cancer. In cancers, TGF-β plays both tumor suppressive and tumor promoting roles depending upon the context. TGF-β has growth inhibitory effect on epithelial cells which is essential to maintain tissue homeostasis. TGF-β induces the expression of several cyclin dependent kinase inhibitors such as p21Cip1, p15Ink4b while down-regulating the expression of cMYC in the epithelial cells. In lieu of its tumor suppressive role, several cancers harbor mutations in the components of the TGF-β signaling axis such as receptors and effector molecules called SMADs. Interestingly various cancers also show hyper activation of TGF-β signaling. It has been suggested that cancer cells become unresponsive to the growth inhibitory effects of TGF-β by losing the expression of p21Cip1, and p15INK4b. Oncogenic transformation of cancer cells can override the growth inhibitory effects of TGF-β. While the loss of growth inhibitory effects by TGF-β are seen in the tumor cells, several tumor promoting actions are also observed in these cells such as induction of EMT. TGF-β activates mesenchymal cells leading to the formation of a reactive stroma in tumors and TGF-β suppresses almost all types of cells of the immune system causing a local immune-suppressive environment. TGF-β also recruits mesenchymal stem cells into the stroma which secrete several cytokines. The sum total of all these effects is pro-angiogenic, pro-infiltrative and pro-metastatic. In the canonical TGF-β signaling pathway, ligands bind to the hetero-tetrameric receptor complex of TGFβR1 and TGFβR2 leading to activation of the TGFβR1 by TGFβR2. Activated TGFβR1 then phosphorylates and activates R-SMAD molecules (SMAD2, SMAD3) which complexes with the co-SMAD (SMAD4) and translocate into the nucleus to effect transcriptional changes. Non-canonical TGF-β signals are many and almost all the known signaling pathways like MAPK, WNT, PI3K-AKT, NOTCH, Integrin, Hedgehog, Hippo etc. have been shown to be activated by TGF-β in different contexts. The canonical TGF-β/SMAD pathway has been shown to be essential for both tumor suppressive and tumor promoting actions of TGF-β. Although the non-canonical signalling pathways have been shown to be context dependent, the exact mechanisms have not been elucidated. In previous studies, we have shown the importance of non-canonical TGF-β signaling in normal vs. carcinoma cells. However, there has been no study that addressed the differential effects of TGF-β on cells of connective tissue origin. To throw light on such questions we have undertaken this study with the following objectives: 1) Whole genome expression profiling of TGF-β targets in normal fibroblasts, transformed fibroblasts and sarcoma cells 2) Elucidation of non-canonical signaling pathways differentially regulated by TGF-β 3) Identification and characterization of novel TGF-β targets The cell-lines chosen for the study are: 1) hFhTERT (human foreskin fibroblasts immortalized with human terminal telomerase); 2) hFhTERT-LTgRAS (hFhTERT transformed with SV40 large T antigen and activated RAS); and 3) HT1080 (fibrosarcoma). We performed whole genome expression profiling using 4×44K Agilent Human Whole Genome Oligonucleotide Arrays. Analysis of the microarray results revealed that TGF-β regulated a large number of genes in all the three cell-lines but few targets were found to be commonly regulated between any two or all the three cell-lines. 5291 genes were differentially regulated by TGF-β between hFhTERT and hFhTERT-LTgRAS and 2274 genes were differentially regulated by TGF-β between hFhTERT and HT1080 cells. Gene set enrichment analysis (GSEA) of these two gene lists revealed enrichment of similar gene sets in the HT1080 and hFhTERT-LTgRAS cells compared to the hFhTERT cells. MAPK signaling pathway components were enriched in the hFhTERT cells. Closer inspection revealed that several upstream regulators of the MAPK pathway were in fact down-regulated by TGF-β in these cells compared to both hFhTERT-LTgRAS and HT1080 cells suggesting a depression of the MAPK pathway by TGF-β in the hFhTERT cells. Assessment of the phosphorylation status of ERK1/2 and p38 MAPK proteins after TGF-β treatment showed that both ERK1/2 and p38 MAPK pathways were not activated in response to TGF-β in the hFhTERT cells. On the other hand in hFhTERT-LTgRAS and HT1080 cells, both ERK1/2 and p38 MAPK were activated post TGF-β treatment. Activity of the AP1 and SMAD responsive p3TP-lux reporter plasmid was dependent on only the SMAD pathway in hFhTERT cells while in the hFhTERT-LTgRAS and HT1080 cells both MAPK and SMAD pathway were found to regulate the expression of the p3TP-lux reporter. This suggests activation of MAPK and SMAD pathways in transformed and tumor cells while there is no activation of MAPK in normal cells of mesenchymal origin. Components of the WNT signaling pathway such as WNT ligands WNT4, and WNT11, frizzled receptors, FZD4, FZD8 and FZD9, regulators like SFRP1, SFRP2, AXIN2 and several targets of the WNT-β-catenin pathway were regulated by TGF-β in the hFhTERT cells but not in the hFhTERT-LTgRAS and HT1080 cells suggesting a positive regulation of the pathway by TGF-β in the hFhTERT cells. Indeed, TGF-β induced the activity of the WNT responsive reporter, pTOP-FLASH in the hFhTERT cells but not in the hFhTERTLTgRAS and HT1080 cells. WNT4 and WNT11 were two of the novel targets of TGF-β identified in hFhTERT cells. Further experiments suggested that TGF-β conferred regulation of these genes was specific to the fibroblast cells since induction of these genes by TGF-β was not observed in any of the cancer cell lines or in HaCaT cells. Some recent studies have demonstrated remodelling of cytoskeleton in epithelial cells by the non-canonical WNT ligands such as WNT5a, WNT4 and WNT11. WNT4 has also been shown to be required for the maintenance of α-SMA levels in smooth muscle cells. In this study we have shown that WNT4 can induce α-SMA in the hFhTERT cells leading to their activation. TGF-β conferred activation of these cells was also found to be dependent on the presence of WNT4. In brief, our study identified differentially activated pathways by TGF-β in immortal and transformed fibroblasts. WNT4 was identified as a crucial molecule required for the TGF-β conferred activation of fibroblasts.

Abstract In mice, gene targeting has become a useful tool for resolving the functions of proteins and for creating new animal models. Cre/loxP technology has been used broadly for generation of genetically modified mice. The Cre recombinase recognizes a specific DNA sequence, called loxP, and removes any DNA fragment between two loxP-sites. The activity of the Cre recombinase can be controlled spatially and temporally with cell- or tissue-specific promoters and synthetic inducing agents, such as tamoxifen or tetracycline. In this thesis, we employed tamoxifen-induced Cre recombination in vitro. Cre-ERTM mice were crossed to ROSA26LacZ reporters and Cre-recombination induced by 4OH-TM was monitored by LacZ staining. 0.5 μM 4OH-TM treatment was competent for tamoxifen-induced Cre-mediated activation of LacZ both in kidney cultures and in experimentally induced kidney mesenchymes. Wnt4 is a secreted signaling molecule, which is necessary for the development of several organs including kidney, ovary, adrenal gland, mammary and pituitary glands. Wnt4 is crucial for kidney development and conventional Wnt4-/- mice die soon after birth, likely due to renal failure. In this thesis, two different Wnt4 alleles, Wnt4EGFPCre and floxed Wnt4, were generated and analyzed to learn more about the Wnt4 functions and to apply these mouse lines to renal functional genomics. In the Wnt4EGFPCre, the EGFPCre fusion cDNA was targeted into exon I of the Wnt4 locus. EGFP-derived fluorescence was observed in the pretubular aggregates from E12.5 embryonic kidney onwards. Further characterization by crossing with the floxed ROSA26LacZ and yellow fluorescent protein (YFP) reporter lines demonstrated that in addition to the kidney, reporter expression was observed in the gonad, spinal cord, lung and the adrenal gland. The expression pattern of the Cre activity recapitulates well the known pattern of the Wnt4 gene. Time-lapse analysis in organ culture settings showed that the Wnt4 expressing cells contributed to the nephrons, some cells near the stalk of the developing ureter, as well as a number of positive supposed medullary stromal cells. In the conditional Wnt4 knock-out, loxP sites were placed to flank exons 3 to 5. The Wnt4 gene was specifically inactivated with CAGCre and Wnt4EGFPCre lines. In both of these crosses deletion of Wnt4 gene function led to impaired kidney development. In conclusion, we identified the culture conditions that can be used for the tamoxifen-induced conditional mutagenesis in tissue cultures. In addition, the created Wnt4 mouse lines serve as new useful tools for addressing the roles of Wnt4 function in diverse tissues and in different stages of development. / Tiivistelmä Hiirillä geenikohdennuksesta on muodostunut hyödyllinen väline proteiinien tehtävien selvittämisessä ja uusien eläinmallien luomisessa. Cre/loxP -tekniikkaa on käytetty laajasti muuntogeenisten hiirien tuottamisessa. Cre-rekombinaasi tunnistaa spesifisen DNA-jakson, niin kutsutun loxP:n, ja poistaa kaikki DNA-jaksot kahden loxP-sekvenssin väliltä. Cre-rekombinaasin aktiivisuutta voidaan säädellä paikallisesti ja ajallisesti solu- tai kudosspesifisillä promoottoreilla ja synteettisillä indusoivilla kemikaaleilla, kuten tamoksifeenillä tai tetrasykliinillä. Tässä väitöskirjassa hyödynsimme tamoksifeenin aiheuttamaa Cre-rekombinaatiota in vitro -kudosviljelmissä. Cre-ERTM-hiirilinja risteytettiin ROSA26LacZ-reportterilinjan kanssa, ja 4-hydroksitamoksifeenin indusoima Cre-rekombinaasin aktiivisuutta monitoroitiin LacZ–värjäyksellä. 0.5&#160;µM:n 4OH-TM konsentraatiolla LacZ-reportterigeeni saatiin aktivoitua tehokkaasti Cre-rekombinaasin avulla sekä munuaisviljelmissä että munuaismesenkyymiviljelmissä. Wnt4 on erittyvä signalointimolekyyli, jolla on keskeinen rooli useiden elinten, kuten munuaisen, munasarjan, lisämunuaisen, rintarauhasen ja aivolisäkkeen kehittymisessä. Wnt4-geenillä on ratkaisevan tärkeä rooli munuaisen kehityksessä, ja poistogeeninen Wnt4-/-hiiri kuolee pian syntymän jälkeen, todennäköisesti munuaisen vajaatoimintaan. Tässä väitöskirjatyössä tuotettiin kaksi eri Wnt4 alleelia, Wnt4EGFPCre ja konditionaalinen Wnt4. Nämä hiirilinjat analysoitiin, jotta saisimme lisää tietoa Wnt4-geenin toiminnasta ja pystyisimme soveltamaan kyseisiä hiirikantoja munuaisten toiminnan selvittämisessä. Wnt4EGFPCre-alleelissa EGFPCre-fuusio -cDNA kohdennettiin osaksi endogeenisen Wnt4-geenin ykköseksonia. Vihreän fluoresoivan proteiinin (EGFP) aktiivisuus havaittiin varhaisen munuaisen kehityksen aikana. Wnt4EGFPCre-alleelin lisäkarakterisointi reportterilinjoilla (Rosa26LacZ ja Rosa26YFP) osoitti, että Wnt4-geenin ilmentyminen havaittiin munuaisen lisäksi sukurauhasissa, selkäytimessä, keuhkoissa sekä lisämunuaisessa. Wnt4EGFPCre-alleeli ilmentyi niissä kudoksissa, joissa endogeenisen Wnt4-geenin tiedetään olevan aktiivinen. Time-lapse -analyysin avulla osoitettiin, että Wnt4-geeniä ilmentävät solut muodostavat tiettyjä rakenteita munuaisen kehityksen aikana. Wnt4-geeni ilmentyi nefroneissa, kehittyvän virtsajohtimen soluissa sekä useissa medullaarisissa stroomasoluissa. Konditionaalisessa (ehdollisessa) Wnt4 knock-out-hiirilinjassa loxP-sekvenssit sijoitettiin eksonien kolme sekä viisi ympärille. Wnt4-geenin toiminta inaktivoitiin CAGCre- ja Wnt4EGFPCre-hiirilinjojen avulla. Näissä molemmissa tapauksissa Wnt4-geenin toiminnan poistaminen johti munuaisen kehityshäiriöön. Yhteenvetona voimme todeta, että olemme tunnistaneet ne kasvatusolosuhteet, joita voidaan hyödyntää, kun halutaan aktivoida reportterigeenejä tai kehityksen kannalta tärkeitä geenejä tamoksifeenin aiheuttamaa Cre/loxP -rekombinaatiota hyväksikäyttäen kudosviljelmissä. Samoja olosuhteita ja menetelmää käyttäen voidaan myös poistaa jonkun kehityksen kannalta tärkeän geenin toiminta ja tutkia sitä kudosviljelmässä. Tuotetut Wnt4-hiirikannat ovat lisäksi uusia hyödyllisiä työkaluja, kun halutaan tutkia Wnt4-geenin toimintaa erilaisissa kudoksissa ja eri kehitysvaiheiden aikana.

Abstract Wnt4, a member of the Wnt family of secreted factors, is essential for kidney organogenesis since the kidney fails to develop in its absence. Besides the kidney, Wnt4 signaling is involved in the control of development of several other organs such as the gonads, adrenal glands and pituitary gland. In the context of the embryonic kidney, Wnt4 signaling induces mesenchymal to epithelial transition of the progenitor cells in the metanephric mesenchyme, an early step in nephrogenesis. Wnt4 signaling may also be relevant in the development of a childhood kidney tumor, the Wilms’ tumor, that involves the function of Wilms’ tumor suppressor protein 1 (WT1). Wilms’ tumor is thought to arise from the early metanephric mesenchymal cells of the embryonic kidney, but the detailed mechanisms are not known. The main aim of this project was to study the mechanisms that regulate expression of the Wnt4 gene by using immortalized embryonic kidney mesenchyme-derived mK4 cells as a model. The Wnt4 gene expression was also analyzed in vivo in the frog embryonic pronephros. Through the use of reporter assays and a two-hybrid screen, Sox11, a member of the SoxC family of transcription factors, was identified as a synergistic protein that interacts with WT1. Immunoprecipitation studies provided further evidence that Sox11 and WT1 may physically interact with each other in the developing embryonic kidney. Indeed, Sox11 and WT1 may regulate the Wnt4 gene expression in vivo since the morpholino-based knock-down of either WT1 or Sox11 led to notable downregulation of the Wnt4 gene expression in the frog embryonic pronephros. The other general aim of this thesis was to develop novel tissue targeting and therapy tools to the cell lineages regulated by the Wnt4 signals, including the podocytes. For this purpose, we utilized mice carrying a floxed expression cassette for the avidin-LDL receptor fusion protein, Lodavin, in the constitutively active Rosa-26 locus. Three Cre driver mice, including the Wnt4-Cre knock-in line, were used to activate Lodavin expression in the respective cells of the embryonic kidney. Moreover, we generated a podocyte injury model by expressing the human receptor for diphtheria toxin specifically in the podocytes. This was achieved by crossing mice containing a floxed expression cassette for this receptor in the Rosa-26 locus with those expressing the Cre recombinase under the nephrin promoter. Administration of diphtheria toxin led initially to podocyte damage only, followed by a progression to glomerular sclerosis. As a summary, Sox11 and WT1 serve as synergistic transcription factors that may regulate expression of the Wnt4 gene in vivo. The transgenic mouse models generated and used provide the basis to generate acute and chronic kidney disease models and the potential to purify the respective cells for developing cell-based therapy avenues for the kidney. Moreover, the Lodavin-based approaches may enable targeted delivery of biotinylated small compounds, proteins, viruses or even cells and novel means for in vivo imaging and functional studies. / Tiivistelmä Wnt-4 kuuluu signaloivien proteiinien Wnt-perheeseen ja sen toiminta on välttämätöntä munuaisen kehityksessä. Ilman Wnt-4 proteiinia munuainen ei kehity. Munuaisen lisäksi Wnt4-signalointi on mukana useiden muiden elinten, kuten sukurauhasten, lisämunuaisen ja aivolisäkkeen säätelyssä. Alkion munuaisessa Wnt4-signalointi saa aikaan mesenkymaalisen kantasolukon epitelisoitumisen, edustaen näin ollen nefronin kehityksen varhaisia vaiheita. Wnt4-signaloinnilla on myös merkittävä asema lapsuusiän munuaiskasvaimen, niin kutsutun Wilmsin kasvaimen kehittymisessä. Tämän tyyppisessä kasvaimessa keskeisenä on Wilmsin tuumoriproteiinin WT1:n toiminta, mutta myös Wnt4:n toiminnalla voi olla merkitystä. Wilmsin kasvaimen arvellaan saavan alkunsa varhaisista sikiöaikaisista jälkimunuaisen soluista, mutta yksityiskohtaisia mekanismeja ei vielä tunneta. Tämän projektin tarkoituksena oli tutkia Wnt4-geenin ilmentymistä sääteleviä mekanismeja käyttäen mallina mK4-soluja eli alkion munuaisesta saatuja, immortalisoituja soluja. Wnt4-geenin ilmentymistä analysoitiin myös in vivo sammakon alkion alkumunuaisessa. Tuplahybridi-analysoinnin avulla tunnistettiin transkriptiotekijäperhe SoxC:n jäsen Sox11 samantoimiseksi proteiiniksi transkriptiotekijä WT1:n kanssa Wnt4-geenin ilmentymisen säätelyssä. Immunopresipitaatiotutkimukset tukivat ajatusta, että Sox11 ja WT1 voisivat olla fyysisessä vuorovaikutuksessa säädellessään nefroninmuodostuksen alullepanossa ratkaisevan Wnt4-geenin ilmentymistä. Sox11 ja WT1 voivat mahdollisesti säädellä Wnt4-geenin ilmentymistä myös in vivo, sillä morfoliineihin perustuvissa kokeissa sekä WT1:n että Sox11:n hiljennys laski Wnt4-geenin ilmentymistasoa sammakon alkumunuaisessa. Tämän väitöstutkimuksen toinen yleinen tavoite oli kehittää uusia kudoskohdennus- ja terapiakeinoja Wnt4-signaloinnin säätelemille solulinjoille, kuten podosyyteille. Tätä tarkoitusta varten kloonattiin siirtogeeninen hiiri, jossa floksattu avidiini-LDL -reseptorifuusioproteiini, Lodavin, kohdennettiin jatkuvasti aktiiviseen Rosa-26 -lokukseen. Kolmea eri Cre-hiirilinjaa käytettiin aktivoimaan Lodavinin ilmentyminen kussakin tietyssä alkion munuaisen solupopulaatiossa. Yksi näistä Cre-linjoista oli Wnt4-Cre. Jotta kyettäisiin vahingoittamaan samoja soluja, jotka ilmentävät Lodavinia, hyödynnettiin difteriamyrkyn ihmisen reseptoria (iDTR). IDTR:n ilmentäminen tietyissä hiiren soluissa tekee ne alttiiksi tappavalle difteriamyrkylle. IDTR-perusteisen munuaisvauriomallin kehittämiseksi käytettiin floksattua iDTR-hiirimallia, ja geenin ilmentyminen aktivoitiin Wnt4-indusoiduissa munuaissolulinjoissa, erityisesti podosyyteissä Nephrin Cre -välitteisesti. NephrinCre;R26RiDTR hiiriä altistettiin difteriamyrkylle ja niiden munuaiskerästen muutoksia seurattiin. Tutkimukset antavat viitteitä siitä, että R26R-floksatut iDTR-hiiret toimivat hyvänä mallina kehitettäessä sekä akuutteja että kroonisia munuaistautimalleja. Yhteenvetona voidaan todeta, että Sox-11 ja WT-1 ovat samantoimisia transkriptiotekijöitä, jotka voivat säädellä Wnt4-geenin ilmentymistä in vivo. Tutkimuksessa kehitetyt ja käytetyt siirtogeeniset hiirimallit tarjoavat perustan kehittää sekä akuutteja että kroonisia munuaistautimalleja. Samalla ne mahdollistavat kulloistenkin solujen eristämisen uusien soluperusteisten hoitomenetelmien kehittelemiseksi. Lisäksi Lodavin-perusteiset lähestymistavat voivat mahdollistaa biotinyloitujen pienten yhdisteiden, proteiinien, virusten tai jopa solujen kuljetuksen kohdennetusti sekä avata uusia mahdollisuuksia in vivo -kuvantamiselle ja toiminnallisille tutkimuksille.

Le développement du tractus reproducteur femelle est issu de la coordination minutieuse de nombreuses voies de signalisation régulant les processus de prolifération, différenciation et d’apoptose cellulaire durant l’embryogenèse. Les voies Wnt et Hippo se démarquent à cet égard. L’activation de la voie Wnt, via des ligands spécifiques, participe à la stabilisation et l’augmentation de l’activité transcriptionnelle du coactivateur de transcription β-catenin. La voie Hippo, pour sa part, ne possède aucun de ligand spécifique. L’inactivation de la voie Hippo (via les kinases Lats1 et Lats2) entraine la stabilisation des coactivateurs de la transcription YAP/TAZ et l’augmentation de leur activité transcriptionnelle. Plusieurs évidences suggèrent notamment la possibilité de redondance fonctionnelle entre certains ligands de la voie Wnt, dont Wnt4 et Wnt5a, dans le développement du tractus reproducteur femelle. Cette avenue demeure toutefois peu étudiée. L’implication de la voie Hippo n’a pas été rapportée dans le développement du tractus reproducteur femelle. Toutefois, les nombreuses interactions rapportées dans la littérature entre les deux voies suggèrent un rôle méconnu de la voie Hippo. L’objectif de ce projet était donc d’élucider les rôles de Wnt4, Wnt5a, Lats1 et Lats2 dans le mésenchyme de Müller et le développement de l’utérus. Les résultats de notre première étude ont confirmé la fonction partiellement redondante de Wnt4 et Wnt5a dans le développement de l’utérus. Notre modèle est notamment caractérisé par des anomalies développementales ainsi qu’une perte de fonction utérine associée à des anomalies de décidualisation in vivo et une diminution de la viabilité des concepti. Les résultats de notre seconde étude ont confirmé les rôles redondants de Lats1 et Lats2 dans le maintien de la multipotentialité des cellules mésenchymateuses müllériennes. Une différenciation hâtive des cellules mésenchymateuses müllériennes en myofibroblastes via, entre autres, l’expression du gène cible Ctgf, a été observée. Nos résultats additionnels n’ont pu mettre en évidence une interaction potentielle entre les voies Wnt et Hippo pouvant expliquer l’apparition des phénotypes. Ces deux études permettent de confirmer certains rôles connus et d’établir de nouveaux rôles de ces voies dans le développement des canaux de Müller. Ils pourront aussi établir les fondements de modèles permettant l’étude de différentes pathologies utérines et l’identification de cibles thérapeutiques. / The development of the female reproductive tract arises from the coordination of numerous signaling pathways regulating processes such as proliferation, differentiation and apoptosis during embryogenesis. The Wnt and Hippo pathways are known to be involved in these processes. Wnt pathway activation, via its specific ligands, results in the stabilisation and increased transcriptional activity of β-catenin. The Hippo pathway does not possess any specific ligands. In contrast to Wnt, inactivation of the Hippo pathway (via Lats1 and Lats2 kinases) is required for the stabilization and increased activity of the transcriptional coactivators YAP and TAZ. The Wnt pathway is known to be involved in the development of the female reproductive tract. Further evidence also suggests the possibility of functional redundancy amongst certain WNT ligands such as Wnt4 and Wnt5a. The Hippo pathway is not known to be implicated in the development of the female reproductive tract. However, numerous interactions have been reported between both pathways, suggesting a possible unknown role of Hippo in that context. The objective of this project was to elucidate the roles of Wnt4, Wnt5a, Lats1 and Lats2 in the Müllerian mesenchyme and the development of the uterus. Results from our first study confirmed the partially redundant roles of Wnt4 and Wnt5a in the development of the uterus. Our model was notably characterized by developmental abnormalities and loss of uterine functions resulting in in vivo decidualization defects and loss of conceptus viability. Results from our second study confirmed the redundant roles of Lats1 and Lats2 in the maintenance of Müllerian mesenchymal cell multipotency. We observed premature differentiation of Müllerian mesenchymal cells into myofibroblasts in absence of both Lats1 and Lats2. These changes were in part due to the increased expression of the target gene Ctgf. Our additional results could not demonstrate any potential interactions between the Wnt and Hippo pathways that could explain the phenotypic changes. In conclusion, our studies confirmed and further discovered novel roles of these pathways in the development of the Müllerian ducts. These models could also lead to better understanding of the pathophysiology of certain uterine diseases and the discovery of potential therapeutic approaches.