The vertebrate inner ear is a sensory organ responsible for auditory and vestibular function. Since its complex structure and cell types arise from a simply structured group of ectodermal cells, called the otic placode, the development of the inner ear has been a popular subject in embryology and developmental biology for decades. To date, many regulatory molecules and their functions have been identified in inner ear development showing considerable conservation among vertebrates. In vertebrates, Fgfs (fibroblast growth factors) from surrounding tissues are the main otic inducer and regulate various otic genes’ expression. Under the control of Fgf signals, pax2/5/8 genes are expressed in the otic region in the critical stages of otic development suggesting their function in otic development. In order to understand the function of pax2/5/8 genes and their interactions in the developing ear, we utilize zebrafish as a model system. Among zebrafish pax2/5/8 genes, pax8 is the earliest gene expressed in the preotic region while pax2a and pax2b are expressed slightly later. We found that pax8 is initially required for normal otic induction. Subsequently, pax8, pax2a and pax2b function redundantly to maintain otic fate. After otic placodes are induced by an Fgf signaling network, expression of Fgf3, one of otic inducers in zebrafish, persists in the hindbrain rhombomere 4. To investigate the function of the persistant Fgf3 expression, we examined a mutant with expanded Fgf3 expression in the hindbrain. Together with fgf3 knockdown results, we discovered that Fgf3 has later roles in specifying the anteroposterior (A-P) axis in the otic vesicle and regulating hair cell formation. We further identified pax5 as one of the genes regulated by the hindbrain Fgf3 activity, and pax5 function to be required for utricular hair cell survival.

PAX2 is a transcription factor that is essential for development. Aberrant expression of PAX2 in adult tissues is associated with carcinogenesis and experimental evidence shows that PAX2 generally exhibits oncogenic properties. Although PAX2 is not expressed in normal ovaries, it is highly expressed in low malignant potential and low-grade epithelial ovarian tumors, suggesting that PAX2 induction in ovarian surface epithelium (OSE) may contribute to transformation. Herein, we provide evidence that expression of PAX2 in normal murine OSE (mOSE) cells enhances their proliferation and survival and, when combined with loss of P53, induces tumorigenicity. In addition, OSE cells are known to gain an epithelial phenotype and express epithelial markers prior to their transformation. This study revealed that PAX2 induction in mOSE cells results in an enhanced epithelial phenotype associated with reduction of the epithelial-mesenchymal transition markers, SMA-α and COX-2. Furthermore, PAX2 inhibits the mesenchymal phenotype induced by TGF-β and reverses the TGF-β-mediated induction of both SMA-α and COX-2, in mOSE cells. Toward tumor progression, we found that when PAX2 was expressed in murine ovarian cancer cells, it enhanced or inhibited their aggressiveness, depending on the model system. In OSE cells transformed by K-RAS and MYC, PAX2 inhibited P53 accumulation and increased the level of pERK1/2 and COX-2. In addition, PAX2 inhibited apoptotic induction in these tumors, while increasing angiogenesis, both of which are enhancers of tumor aggressiveness. However, in a murine model of high-grade serous ovarian cancer, PAX2 expression reduced tumor mass and improved animal survival, likely via reduced proliferation and metastasis. Mechanistic studies showed that PAX2 increased Htra1 and decreased COX-2 in those tumors. Both HTRA1 and COX-2 are novel downstrream targets for PAX2 that are identified in the current study. These results suggest that PAX2 may not act as a classical oncogene or tumor suppressor in ovarian cancer; rather, it modulates tumorigenesis differently, depending on the tumor context. The observation that PAX2 targets distinct biological and molecular pathways might help to guide future studies to different therapeutic targets in low-grade vs. high-grade cancers.

Several studies have proposed the fallopian tube epithelium as a site of origin of ovarian cancer. The discovery of precursor lesions in the fallopian tube in patients at risk for ovarian cancer supports a probable origin for high-grade serous ovarian carcinoma in this tissue. While the fallopian tube epithelium consists of three distinct cell types, the paired box protein 2 (PAX2) positive cells and potentially the CD44 positive stem-like cells are most relevant to ovarian cancer. Loss of PAX2 expression in the fallopian tube cells is considered to be an early event in epithelial transformation, but the specific role of PAX2 in this transition is unknown. The aim of this study was to define the role of PAX2 in oviductal epithelial cells (OVE) cells and in mouse ovarian surface epithelial cells (MOSE), and to understand its contribution to the formation of serous precursor lesions in the fallopian tubes. Herein, we studied the OVE response to transforming growth factor β (TGFβ, a cytokine found in follicular fluid) and provide evidence of its potential involvement in the regulation of stem cell-like behaviors that may contribute to formation of cancer-initiating cells. Treatment of primary cultures of OVE cells with TGFβ at concentrations found in ovulatory follicular fluid induced an epithelial-mesenchymal transition (EMT) with expected changes in proliferation, cell morphology and expression of SNAIL, Vimentin and E-cadherin. EMT was also associated with decreased expression of PAX2 and an increase in the fraction of cells expressing CD44. Pax2 knockdown in OVE cells and overexpression in ovarian epithelial cells confirmed that PAX2 inhibits CD44 expression and regulates the degree of epithelial differentiation of OVE cells. These results suggest that the loss of PAX2 seen in serous tubal intraepithelial carcinomas (STIC) leads to a shift to a more mesenchymal phenotype associated with stem-like features. Pax2 overexpression in MOSE cells also induced the formation of vascular channels both in vitro and in vivo, which indicate a possible contribution of PAX2 to ovarian cancer progression by increasing the vascular channels to supply nutrients to the tumor cells. Furthermore, since loss of PAX2 in STIC was found associated with P53 and BRCA1 mutations, OVE cells with mutations of the tumor suppressor genes Trp53 and Brca1 were studied. We found that loss of Trp53 with or without loss of Brca1 increased cell proliferation and colony formation in vitro. In addition, loss of Trp53 induced OVE cells to undergo EMT and induced the expression of stem cell–associated genes. We therefore suggest a potential contribution of stem cells in initiating the precursor lesions in the fallopian tubes in combination with tumor suppressor gene mutation.

Loss of hearing affects more than 10 % of the population, and one newborn in a thousand is born with defects of the inner ear. Transcriptional factors involved in the development of inner ear are important in our understanding of the causes of inner ear defects. ISLET1 is one of these factors. ISLET1 expression is detected in the sensory and neuronal cells of the inner ear. It participates in otocyst formation, and the specification and differentiation of cells of cochlea and vestibular system. The functional role of ISLET1 during inner ear development was investigated. Its role was studied by using Pax2-Isl1 transgenic mice that overexpress Islet1 under the control of the Pax2 promoter. Two transgenic lines were generated, Pax2-Isl1/300 and Pax2- Isl1/52. Two copies of the Pax2-Isl1 transgene were inserted to Pax2-Isl1/300 genome and one copy was inserted to the Pax2-Isl1/52 genome. Defects in sense of hearing were detected in both lines and circling behavior, a defect of balance, was detected in the Pax2-Isl1/300 transgenic mice. We observed high postnatal lethality in heterozygote transgenic mice. Pax2-Isl1/52 homozygote mutation is lethal at embryonic day 10 (E10,5). Pax2-Isl1/300 homozygote letality couldn't be detected because of the inability to breed heterozygote mutated mice of this line....

Hindsight (HNT) is a zinc finger transcription factor that is required for morphogenesis of the Drosophila embryo, having roles in germ band retraction (GBR) as well as dorsal closure (DC). HNT expression is also found in sensory organ precursors (SOP) of the developing pupal peripheral nervous system, and muscle progenitor cells, but the role of HNT in neurogenesis and myogenesis during embryogenesis has not been investigated in any depth. Microarray analysis of embryos over-expressing HNT during GBR and DC identified 1290 genes with significant changes in expression. This data set included many potential HNT targets, including genes associated with myogensis, and a disruption of muscle development was observed in embryos over-expressing HNT. It is possible that HNT may function to repress muscle identity genes in muscle founder cells. In addition, HNT over expressing embryos were found to resemble the neurogenic class of mutants. Among the potential target genes, D-Pax2 (shaven, sparkling, CG11049) expression, which is known to be expressed in the developing peripheral nervous system, was confirmed to be up-regulated following HNT over-expression. Interestingly, D-Pax2 and HNT expression were found to co-localize at the onset of their expression at stages 10-12 in embryos, but were not co-localized in later stages of embryogenesis. The up-regulation of D-Pax2 by HNT over-expression was further characterized and was found to be associated with strong ectopic HNT expression. The relevance of HNT to the regulation of D-Pax2 during normal development remains to be determined, but it is possible that endogenous expression of HNT is involved in D-Pax2 repression.

Hindsight (HNT) is a zinc finger transcription factor that is required for morphogenesis of the Drosophila embryo, having roles in germ band retraction (GBR) as well as dorsal closure (DC). HNT expression is also found in sensory organ precursors (SOP) of the developing pupal peripheral nervous system, and muscle progenitor cells, but the role of HNT in neurogenesis and myogenesis during embryogenesis has not been investigated in any depth. Microarray analysis of embryos over-expressing HNT during GBR and DC identified 1290 genes with significant changes in expression. This data set included many potential HNT targets, including genes associated with myogensis, and a disruption of muscle development was observed in embryos over-expressing HNT. It is possible that HNT may function to repress muscle identity genes in muscle founder cells. In addition, HNT over expressing embryos were found to resemble the neurogenic class of mutants. Among the potential target genes, D-Pax2 (shaven, sparkling, CG11049) expression, which is known to be expressed in the developing peripheral nervous system, was confirmed to be up-regulated following HNT over-expression. Interestingly, D-Pax2 and HNT expression were found to co-localize at the onset of their expression at stages 10-12 in embryos, but were not co-localized in later stages of embryogenesis. The up-regulation of D-Pax2 by HNT over-expression was further characterized and was found to be associated with strong ectopic HNT expression. The relevance of HNT to the regulation of D-Pax2 during normal development remains to be determined, but it is possible that endogenous expression of HNT is involved in D-Pax2 repression.

In vertebrates, the otic placode forms inner ear and epibranchial placodes produce sensory ganglia within branchial clefts. Fibroblast growth factor (FGF) family of protein ligands from the surrounding tissues are responsible for otic and epibranchial placode induction. Members of pax2/5/8 family of transcription factors function as mediators during otic induction. To understand the temporal and spatial requirements of Fgf and their interaction with pax2/8 for otic induction, we used heat shock inducible transgenic lines of zebrafish to misexpress fgf3/8 and pax2a/8 under the control of hsp70 promoter. Loss of function studies were done to examine the functions of pax2/8 genes in regulating otic and epibranchial development. We show that global transient activation of hs:fgf3 or hs:fgf8 at mid-late gastrula stages (7-8 hpf) severely impairs otic induction, in part by disrupting formation of the principal signaling centers in the hindbrain. Additionally, mosaic studies show that high-level misexpression blocks otic fate cell-autonomously, whereas low to moderate levels promote otic development. At later stages high-level Fgf misexpression, both globally and locally does not inhibit otic fate, but rather causes a dramatic expansion of endogenous otic domains. Misexpression of hs:pax2a or hs:pax8 also expands endogenous otic domains but is not sufficient to bypass the requirement for Fgf signaling. Co-misexpression of Fgf with pax2a or pax8 leads to production of ectopic otic tissue in a broad range of cranial ectoderm. These data show that changes in timing, distribution and level of Fgf signaling and its downstream effectors influences otic induction. We show that otic and epibranchial placodes are induced at different times and by distinct mechanisms. Initially, Fgf from surrounding tissues induces otic expression of pax8 and sox3, which cooperate synergistically to establish otic fate. Subsequently, pax8 along with pax2a/pax2b downregulate foxi1 expression in otic cells, which is necessary for further otic development. Additionally, pax2/8 activate otic expression of fgf24, which induces epibranchial expression of sox3. Blocking functions of fgf24 or sox3 causes severe epibranchial deficiencies but has little effect on otic development. These results support the model whereby the otic placode forms first and induces epibranchial placodes through pax2/8-dependent Fgf24 signaling.

Le diabète maternel est un facteur de risque majeur pour le développement de malformations congénitales. Dans le syndrome de l’embryopathie diabétique, l’exposition prolongée du fœtus à de hautes concentrations ambientes de glucose induit des dommages qui peuvent affecter plusieurs organes, dont les reins. Les malformations rénales sont la cause de près de 40 pourcent des cas d’insuffisance rénale infantile. L’hyperglycémie constitue un environnement utérin adverse qui nuit à la néphrogenèse et peut causer l’agenèse, la dysplasie (aplasie) ou l’hypoplasie rénale. Les mécanismes moléculaires par lesquels les hautes concentrations ambientes de glucose mènent à la dysmorphogenèse et aux malformations demeurent toutefois mal définis. Le diabète maternel prédispose aussi la progéniture au développement d’autres problèmes à l’âge adulte, tels l’hypertension, l’obésité et le diabète de type 2. Ce phénomène appelé ‘programmation périnatale’ a suscité l’intérêt au cours des dernières décennies, mais les mécanismes responsables demeurent mal compris. Mes études doctorales visaient à élucider les mécanismes moléculaires par lesquels le diabète maternel ou un environnement in utero hyperglycémique affecte la néphrogenèse et programme par la suite la progéniture a développer de l’hypertension par des observations in vitro, ex vivo et in vivo. Nous avons utilisé les cellules MK4, des cellules embryonnaires du mésenchyme métanéphrique de souris, pour nos études in vitro et deux lignées de souris transgéniques (Tg) pour nos études ex vivo et in vivo, soient les souris HoxB7-GFP-Tg et Nephrin-CFP-Tg. Les souris HoxB7-GFP-Tg expriment la protéine fluorescente verte (GFP) dans le bourgeon urétérique (UB), sous le contrôle du promoteur HoxB7. Les souris Nephrin-CFP expriment la protéine fluorescente cyan (CFP) dans les glomérules, sous le contrôle du promoteur nephrin spécifique aux podocytes. Nos études in vitro visaient à déterminer si les hautes concentrations de glucose modulent l’expression du gène Pax2 dans les cellules MK4. Les cellules MK4 ont été traitées pendant 24h avec du milieu contenant soit 5mM D-glucose et 20mM D-mannitol ou 25mM D-glucose et avec ou sans antioxydants ou inhibiteurs de p38 MAPK, p44/42 MAPK, PKC et NF-kB. Nos résultats ont démontré que le D-glucose élevé (25mM) augmente la génération des espèces réactives de l’oxygène (ROS) dans les cellules MK4 et induit spécifiquement l’expression du gène Pax2. Des analogues du glucose tels le D-mannitol, L-glucose ou le 2-Deoxy-D-glucose n’induisent pas cette augmentation dans les cellules MK4. La stimulation de l’expression du gène Pax2 par le D-glucose dans les cellules MK4 peut être bloquée par des inhibiteurs des ROS et de NF-kB, mais pas par des inhibiteurs de p38 MAPK, p44/42 MAPK ou PKC. Ces résultats indiquent que la stimulation de l’expression du gène Pax2 par les concentrations élevées de glucose est due, au moins en partie, à la génération des ROS et l’activation de la voie de signalisation NF-kB, et non pas via les voies PKC, p38 MAPK et p44/42 MAPK. Nos études ex vivo s’intéressaient aux effets d’un milieu hyperglycémique sur la morphogenèse de la ramification du bourgeon urétérique (UB). Des explants de reins embryonnaires (E12 à E18) ont été prélevés par micro-dissection de femelles HoxB7-GFP gestantes. Les explants ont ensuite été cultivés dans un milieu contenant soit 5mM D-glucose et 20mM D-mannitol ou 25mM D-glucose et avec ou sans antioxydants, catalase ou inhibiteur de PI3K/AKT pour diverses durées. Nos résultats ont démontré que le D-glucose stimule la ramification du UB de manière spécifique, et ce via l’expression du gène Pax2. Cette augmentation de la ramification et de l’expression du gène Pax2 peut être bloquée par des inhibiteurs des ROS et de PI3K/AKT. Ces études ont démontré que les hautes concentrations de glucose altèrent la morphogenèse de la ramification du UB via l’expression de Pax2. L’effet stimulant du glucose semble s’effectuer via la génération des ROS et l’activation de la voie de signalisation Akt. Nos études in vivo visaient à déterminer le rôle fondamental du diabète maternel sur les défauts de morphogenèse rénale chez la progéniture. Dans notre modèle animal, le diabète maternel est induit par le streptozotocin (STZ) chez des femelles HoxB7-GFP gestantes (E13). Les souriceaux ont été étudiés à différents âges (naissants et âgés de une, deux ou trois semaines). Nous avons examiné leurs morphologie rénale, nombre de néphrons, expression génique et les événements apoptotiques lors de cette étude à court terme. La progéniture des mères diabétiques avait un plus faible poids, taille et poids des reins, et possédait des glomérules plus petits et moins de néphrons par rapport à la progéniture des mères contrôles. La dysmorphogenèse rénale observée est peut-être causée par l’augmentation de l’apoptose des cellules dans la région du glomérule. Nos résultats ont montré que les souriceaux nés de mères diabétiques possèdent plus de podocytes apoptotiques et plus de marquage contre la caspase-3 active dans leurs tubules rénaux que la progéniture des mères contrôles. Les souriceaux des mères diabétiques montrent une augmentation de l’expression des composants du système rénine angiotensine (RAS) intrarénal comme l’angiotensinogène et la rénine, ainsi qu’une augmentation des isoformes p50 et p65 de NF-kB. Ces résultats indiquent que le diabète maternel active le RAS intrarénal et induit l’apoptose des glomérules, menant à une altération de la morphogenèse rénale de la progéniture. En conclusion, nos études ont permis de démontrer que le glucose élevé ou l’environnement in utero diabétique altère la morphogenèse du UB, qui résulte en un retard dans la néphrogenèse et produit des reins plus petits. Cet effet est dû, au moins en partie, à la génération des ROS, à l’activation du RAS intrarénal et à la voie NF-kB. Nos études futures se concentreront sur les mécanismes par lesquels le diabète maternel induit la programmation périnatale de l’hypertension chez la progéniture adulte. Cette étude à long terme porte sur trois types de progénitures : adultes nés de mères contrôles, diabétiques ou diabétiques traitées avec insuline pendant la gestation. Nous observerons la pression systolique, la morphologie rénale et l’expression de divers gènes et protéines. Nous voulons de plus déterminer si la présence d’un système antioxydant (catalase) peut protéger la progéniture des effets néfastes des ROS causés par l’environnement in utero hyperglycémique. Les souris Catalase-Tg expriment la catalase spécifiquement dans les tubules proximaux et nous permettrons d’explorer notre hypothèse sur le rôle des ROS dans notre modèle expérimental de diabète maternel. / Maternal diabetes is a major risk factor for congenital malformations. When the fetus is exposed to high, sustained, ambient glucose levels, widespread fetal damage may affect multiple organs, including the kidneys, evoking diabetic embryopathy syndrome. Renal malformations account for up to 40% of childhood renal failure cases. Hyperglycemia constitutes an adverse in utero environment that dynamically impairs nephrogenesis, resulting in renal agenesis, dysplasia, aplasia or hypoplasia. However, the molecular mechanisms by which high, ambient glucose levels lead to renal dysmorphogenesis and birth defects have not yet been delineated. Maternal diabetes also programs the offspring to develop other problems later in life, such as hypertension, obesity and type 2 diabetes. This phenomenon, called ‘perinatal programming’, has attracted worldwide attention in recent decades, yet the mechanisms by which it occurs are incompletely understood. My PhD studies are designed to elucidate the underlying molecular pathways by which maternal diabetes or hyperglycemic environments in utero impair nephrogenesis and subsequently make the offspring develop perinatal programming of hypertension in vitro, ex vivo and in vivo. We employed mouse embryonic metanephric mesenchyme cells, namely MK4 cells, for our in vitro experiments, and 2 transgenic (Tg) mouse lines, Hoxb7-GFP-Tg and Nephrin-CFP-Tg mice, for ex vivo and in vivo investigations. Hoxb7-GFP-Tg mice specifically express green fluorescent protein (GFP) in ureteric buds (UB), driven by the Hoxb7 promoter. Nephrin-CFP-Tg mice express cyan fluorescent protein (CFP) in glomeruli, driven by the podocyte-specific nephrin promoter. In our in vitro studies, we examined whether high glucose alters Pax2 gene expresson in MK4 cells. The cells were treated with either 5 mM D-glucose plus 20 mM D-mannitol or 25 mM D-glucose media with or without reactive oxygen species (ROS) blockers (DPI, rotenone), and inhibitors of p38 mitogen-activated protein kinase (MAPK) (SB203580), p44/22 MAPK (PD98059), protein kinase C (PKC) (GF109203X), or nuclear factor kappa B (NK-kB) (PDTC) for 24-hr incubation. Our data showed that high D-glucose (25 mM) increased ROS generation and specifically induced Pax2 gene expression, but not other glucose analogs such as D-mannitol, L-glucose or 2-deoxy-D-glucose in MK4 cells. The stimulatory effect of high D-glucose on Pax2 gene expression could be blocked by ROS and NF-kB inhibitors in MK4 cells but not by inhibitors of p38 MAPK (SB203580), p44/22 MAPK (PD98059), and PKC (GFX) in MK4 cells. These data indicated that the stimulatory effect of high glucose on Pax2 gene expression is mediated, at least in part, via ROS generation and activation of NF-κB, but not via the PKC, p38 MAPK and p44/42 MAPK signalling pathways. In our ex vivo studies, we investigated the influence of a high-glucose milieu on UB branching morphogenesis. Kidney explants (E12 to E18) were microdissected from timed-pregnant Hoxb7-GFP mice and cultured with either 5 mM D-glucose plus 20 mM D-mannitol or 25 mM D-glucose media with or without ROS blockers (DPI, rotenone), catalase and phosphoinositide-3-kinase (PI3K)/AKT inhibitor at different time points, depending on the experiment. We found that high D-glucose specifically stimulated UB branching in a time-dependent manner. High D-glucose stimulation of UB branching morphogenesis was mediated via Pax2 gene expression. High D-glucose-induced UB branching and Pax2 gene expression could be blocked by ROS and PI3K/AKT inhibitors. These studies demonstrated that high glucose alters UB branching morphogenesis via Pax2 gene and protein expression. The stimulatory effect of high glucose seems to be mediated via ROS generation and activation of the AKT signalling pathway. In our in vivo studies, we explored the fundamental role of maternal diabetes on renal morphogenesis impairment in offspring. In our experimental model, maternal diabetes was induced by streptozotocin in pregnant Hoxb7-GFP mice at embryonic day 13. The offspring were examined at several time points after birth (neonatal, 1 week, 2 weeks, and 3 weeks) with follow-up of kidney morphology, nephron number, gene expression, and apoptotic events in this short-term postnatal experiment. We observed that the offspring of diabetic mice had lower body weight, body size, kidney weight, small volume of glomeruli and a reduced number of nephrons in comparison to non-diabetic control offspring. Renal dysmorphogenesis may have been the result of increased cell apoptosis in glomeruli. Our findings showed that the offspring of diabetic mice displayed significantly more apoptotic podocytes as well as augmented active caspase-3 immunostaining in renal tubules compared to control mice offspring. Diabetic mice offspring presented heightened expression of intrarenal renin-angiotensin system (RAS) components, such as angiotensinogen and renin, with upregulation of p50 and p65 NF-kB isoforms. These data indicated that maternal diabetes activates the intrarenal RAS and induces glomerular apoptosis, resulting in impairment of renal morphogenesis in diabetic offspring. In conclusion, our findings indicated that a high-glucose milieu in utero or maternal diabetic alters UB morphogenesis, culminating in retardation of nephrogenesis with smaller kidney size. The underlying mechanism(s) is mediated, at least in part, via ROS generation and activation of the intrarenal RAS and NF-kB pathways. In the future, we aim to investigate the underlying mechanism(s) of how maternal diabetes induces perinatal programming of adult hypertension in offspring in vivo. This long-term postnatal study will be undertaken in 3 groups: adult offspring (20 weeks) of control mice, adult offspring of diabetic pregnant mice, and adult offspring of insulin-treated, diabetic, pregnant mice. We will follow-up by tracking hypertension, kidney morphology, and gene expression. Furthermore, we also plan to determine whether an antioxidant system (catalase) can protect against an hyperglycemic environment in utero that affects embryonic organogenesis via an increase in ROS generation. Catalase-Tg mice that specifically overexpress catalase in proximal tubules will be tested. Such Tg mice with catalase overexpression represent a model for exploring our hypothesis on the role of ROS in gestational diabetes.

Le diabète maternel est un facteur de risque majeur pour le développement de malformations congénitales. Dans le syndrome de l’embryopathie diabétique, l’exposition prolongée du fœtus à de hautes concentrations ambientes de glucose induit des dommages qui peuvent affecter plusieurs organes, dont les reins. Les malformations rénales sont la cause de près de 40 pourcent des cas d’insuffisance rénale infantile. L’hyperglycémie constitue un environnement utérin adverse qui nuit à la néphrogenèse et peut causer l’agenèse, la dysplasie (aplasie) ou l’hypoplasie rénale. Les mécanismes moléculaires par lesquels les hautes concentrations ambientes de glucose mènent à la dysmorphogenèse et aux malformations demeurent toutefois mal définis. Le diabète maternel prédispose aussi la progéniture au développement d’autres problèmes à l’âge adulte, tels l’hypertension, l’obésité et le diabète de type 2. Ce phénomène appelé ‘programmation périnatale’ a suscité l’intérêt au cours des dernières décennies, mais les mécanismes responsables demeurent mal compris. Mes études doctorales visaient à élucider les mécanismes moléculaires par lesquels le diabète maternel ou un environnement in utero hyperglycémique affecte la néphrogenèse et programme par la suite la progéniture a développer de l’hypertension par des observations in vitro, ex vivo et in vivo. Nous avons utilisé les cellules MK4, des cellules embryonnaires du mésenchyme métanéphrique de souris, pour nos études in vitro et deux lignées de souris transgéniques (Tg) pour nos études ex vivo et in vivo, soient les souris HoxB7-GFP-Tg et Nephrin-CFP-Tg. Les souris HoxB7-GFP-Tg expriment la protéine fluorescente verte (GFP) dans le bourgeon urétérique (UB), sous le contrôle du promoteur HoxB7. Les souris Nephrin-CFP expriment la protéine fluorescente cyan (CFP) dans les glomérules, sous le contrôle du promoteur nephrin spécifique aux podocytes. Nos études in vitro visaient à déterminer si les hautes concentrations de glucose modulent l’expression du gène Pax2 dans les cellules MK4. Les cellules MK4 ont été traitées pendant 24h avec du milieu contenant soit 5mM D-glucose et 20mM D-mannitol ou 25mM D-glucose et avec ou sans antioxydants ou inhibiteurs de p38 MAPK, p44/42 MAPK, PKC et NF-kB. Nos résultats ont démontré que le D-glucose élevé (25mM) augmente la génération des espèces réactives de l’oxygène (ROS) dans les cellules MK4 et induit spécifiquement l’expression du gène Pax2. Des analogues du glucose tels le D-mannitol, L-glucose ou le 2-Deoxy-D-glucose n’induisent pas cette augmentation dans les cellules MK4. La stimulation de l’expression du gène Pax2 par le D-glucose dans les cellules MK4 peut être bloquée par des inhibiteurs des ROS et de NF-kB, mais pas par des inhibiteurs de p38 MAPK, p44/42 MAPK ou PKC. Ces résultats indiquent que la stimulation de l’expression du gène Pax2 par les concentrations élevées de glucose est due, au moins en partie, à la génération des ROS et l’activation de la voie de signalisation NF-kB, et non pas via les voies PKC, p38 MAPK et p44/42 MAPK. Nos études ex vivo s’intéressaient aux effets d’un milieu hyperglycémique sur la morphogenèse de la ramification du bourgeon urétérique (UB). Des explants de reins embryonnaires (E12 à E18) ont été prélevés par micro-dissection de femelles HoxB7-GFP gestantes. Les explants ont ensuite été cultivés dans un milieu contenant soit 5mM D-glucose et 20mM D-mannitol ou 25mM D-glucose et avec ou sans antioxydants, catalase ou inhibiteur de PI3K/AKT pour diverses durées. Nos résultats ont démontré que le D-glucose stimule la ramification du UB de manière spécifique, et ce via l’expression du gène Pax2. Cette augmentation de la ramification et de l’expression du gène Pax2 peut être bloquée par des inhibiteurs des ROS et de PI3K/AKT. Ces études ont démontré que les hautes concentrations de glucose altèrent la morphogenèse de la ramification du UB via l’expression de Pax2. L’effet stimulant du glucose semble s’effectuer via la génération des ROS et l’activation de la voie de signalisation Akt. Nos études in vivo visaient à déterminer le rôle fondamental du diabète maternel sur les défauts de morphogenèse rénale chez la progéniture. Dans notre modèle animal, le diabète maternel est induit par le streptozotocin (STZ) chez des femelles HoxB7-GFP gestantes (E13). Les souriceaux ont été étudiés à différents âges (naissants et âgés de une, deux ou trois semaines). Nous avons examiné leurs morphologie rénale, nombre de néphrons, expression génique et les événements apoptotiques lors de cette étude à court terme. La progéniture des mères diabétiques avait un plus faible poids, taille et poids des reins, et possédait des glomérules plus petits et moins de néphrons par rapport à la progéniture des mères contrôles. La dysmorphogenèse rénale observée est peut-être causée par l’augmentation de l’apoptose des cellules dans la région du glomérule. Nos résultats ont montré que les souriceaux nés de mères diabétiques possèdent plus de podocytes apoptotiques et plus de marquage contre la caspase-3 active dans leurs tubules rénaux que la progéniture des mères contrôles. Les souriceaux des mères diabétiques montrent une augmentation de l’expression des composants du système rénine angiotensine (RAS) intrarénal comme l’angiotensinogène et la rénine, ainsi qu’une augmentation des isoformes p50 et p65 de NF-kB. Ces résultats indiquent que le diabète maternel active le RAS intrarénal et induit l’apoptose des glomérules, menant à une altération de la morphogenèse rénale de la progéniture. En conclusion, nos études ont permis de démontrer que le glucose élevé ou l’environnement in utero diabétique altère la morphogenèse du UB, qui résulte en un retard dans la néphrogenèse et produit des reins plus petits. Cet effet est dû, au moins en partie, à la génération des ROS, à l’activation du RAS intrarénal et à la voie NF-kB. Nos études futures se concentreront sur les mécanismes par lesquels le diabète maternel induit la programmation périnatale de l’hypertension chez la progéniture adulte. Cette étude à long terme porte sur trois types de progénitures : adultes nés de mères contrôles, diabétiques ou diabétiques traitées avec insuline pendant la gestation. Nous observerons la pression systolique, la morphologie rénale et l’expression de divers gènes et protéines. Nous voulons de plus déterminer si la présence d’un système antioxydant (catalase) peut protéger la progéniture des effets néfastes des ROS causés par l’environnement in utero hyperglycémique. Les souris Catalase-Tg expriment la catalase spécifiquement dans les tubules proximaux et nous permettrons d’explorer notre hypothèse sur le rôle des ROS dans notre modèle expérimental de diabète maternel. / Maternal diabetes is a major risk factor for congenital malformations. When the fetus is exposed to high, sustained, ambient glucose levels, widespread fetal damage may affect multiple organs, including the kidneys, evoking diabetic embryopathy syndrome. Renal malformations account for up to 40% of childhood renal failure cases. Hyperglycemia constitutes an adverse in utero environment that dynamically impairs nephrogenesis, resulting in renal agenesis, dysplasia, aplasia or hypoplasia. However, the molecular mechanisms by which high, ambient glucose levels lead to renal dysmorphogenesis and birth defects have not yet been delineated. Maternal diabetes also programs the offspring to develop other problems later in life, such as hypertension, obesity and type 2 diabetes. This phenomenon, called ‘perinatal programming’, has attracted worldwide attention in recent decades, yet the mechanisms by which it occurs are incompletely understood. My PhD studies are designed to elucidate the underlying molecular pathways by which maternal diabetes or hyperglycemic environments in utero impair nephrogenesis and subsequently make the offspring develop perinatal programming of hypertension in vitro, ex vivo and in vivo. We employed mouse embryonic metanephric mesenchyme cells, namely MK4 cells, for our in vitro experiments, and 2 transgenic (Tg) mouse lines, Hoxb7-GFP-Tg and Nephrin-CFP-Tg mice, for ex vivo and in vivo investigations. Hoxb7-GFP-Tg mice specifically express green fluorescent protein (GFP) in ureteric buds (UB), driven by the Hoxb7 promoter. Nephrin-CFP-Tg mice express cyan fluorescent protein (CFP) in glomeruli, driven by the podocyte-specific nephrin promoter. In our in vitro studies, we examined whether high glucose alters Pax2 gene expresson in MK4 cells. The cells were treated with either 5 mM D-glucose plus 20 mM D-mannitol or 25 mM D-glucose media with or without reactive oxygen species (ROS) blockers (DPI, rotenone), and inhibitors of p38 mitogen-activated protein kinase (MAPK) (SB203580), p44/22 MAPK (PD98059), protein kinase C (PKC) (GF109203X), or nuclear factor kappa B (NK-kB) (PDTC) for 24-hr incubation. Our data showed that high D-glucose (25 mM) increased ROS generation and specifically induced Pax2 gene expression, but not other glucose analogs such as D-mannitol, L-glucose or 2-deoxy-D-glucose in MK4 cells. The stimulatory effect of high D-glucose on Pax2 gene expression could be blocked by ROS and NF-kB inhibitors in MK4 cells but not by inhibitors of p38 MAPK (SB203580), p44/22 MAPK (PD98059), and PKC (GFX) in MK4 cells. These data indicated that the stimulatory effect of high glucose on Pax2 gene expression is mediated, at least in part, via ROS generation and activation of NF-κB, but not via the PKC, p38 MAPK and p44/42 MAPK signalling pathways. In our ex vivo studies, we investigated the influence of a high-glucose milieu on UB branching morphogenesis. Kidney explants (E12 to E18) were microdissected from timed-pregnant Hoxb7-GFP mice and cultured with either 5 mM D-glucose plus 20 mM D-mannitol or 25 mM D-glucose media with or without ROS blockers (DPI, rotenone), catalase and phosphoinositide-3-kinase (PI3K)/AKT inhibitor at different time points, depending on the experiment. We found that high D-glucose specifically stimulated UB branching in a time-dependent manner. High D-glucose stimulation of UB branching morphogenesis was mediated via Pax2 gene expression. High D-glucose-induced UB branching and Pax2 gene expression could be blocked by ROS and PI3K/AKT inhibitors. These studies demonstrated that high glucose alters UB branching morphogenesis via Pax2 gene and protein expression. The stimulatory effect of high glucose seems to be mediated via ROS generation and activation of the AKT signalling pathway. In our in vivo studies, we explored the fundamental role of maternal diabetes on renal morphogenesis impairment in offspring. In our experimental model, maternal diabetes was induced by streptozotocin in pregnant Hoxb7-GFP mice at embryonic day 13. The offspring were examined at several time points after birth (neonatal, 1 week, 2 weeks, and 3 weeks) with follow-up of kidney morphology, nephron number, gene expression, and apoptotic events in this short-term postnatal experiment. We observed that the offspring of diabetic mice had lower body weight, body size, kidney weight, small volume of glomeruli and a reduced number of nephrons in comparison to non-diabetic control offspring. Renal dysmorphogenesis may have been the result of increased cell apoptosis in glomeruli. Our findings showed that the offspring of diabetic mice displayed significantly more apoptotic podocytes as well as augmented active caspase-3 immunostaining in renal tubules compared to control mice offspring. Diabetic mice offspring presented heightened expression of intrarenal renin-angiotensin system (RAS) components, such as angiotensinogen and renin, with upregulation of p50 and p65 NF-kB isoforms. These data indicated that maternal diabetes activates the intrarenal RAS and induces glomerular apoptosis, resulting in impairment of renal morphogenesis in diabetic offspring. In conclusion, our findings indicated that a high-glucose milieu in utero or maternal diabetic alters UB morphogenesis, culminating in retardation of nephrogenesis with smaller kidney size. The underlying mechanism(s) is mediated, at least in part, via ROS generation and activation of the intrarenal RAS and NF-kB pathways. In the future, we aim to investigate the underlying mechanism(s) of how maternal diabetes induces perinatal programming of adult hypertension in offspring in vivo. This long-term postnatal study will be undertaken in 3 groups: adult offspring (20 weeks) of control mice, adult offspring of diabetic pregnant mice, and adult offspring of insulin-treated, diabetic, pregnant mice. We will follow-up by tracking hypertension, kidney morphology, and gene expression. Furthermore, we also plan to determine whether an antioxidant system (catalase) can protect against an hyperglycemic environment in utero that affects embryonic organogenesis via an increase in ROS generation. Catalase-Tg mice that specifically overexpress catalase in proximal tubules will be tested. Such Tg mice with catalase overexpression represent a model for exploring our hypothesis on the role of ROS in gestational diabetes.

Gene regulatory networks, underlying the molecular regulation of eye development are conserved across many animal phyla. Genes from the Pax family of transcription factors are one of the most conserved members through the evolution, regulating the development of crucial parts of eye, including the photoreceptor cells. Pax transcription factors are considered to be regulators of opsins, molecules providing the conversion of the light stimulus into the electrochemical signalisation in the photoreceptors cells. In this thesis, pax6 and pax2/5/8 transcription factors are investigated as potential regulators of eye development in Platynereis dumerilii. pax6 and pax2/5/8 transcription factors are tested as potential regulators of the r-opsin in Platynereis, based on the observed early expression onsets of these genes. Wild-type expression analysis of pax6 and pax2/5/8 using the whole mount RNA in-situ hybridization is provided, accompanied by the initial analysis of the Platynereis pax6 knockout line. pax6 heterozygote mutants are shown to be viable and able to reproduce, however, homozygote mutation of pax6 in Platynereis is lethal. Our data suggest that transcription factors pax2/5/8, otx and six3 are not regulated by the pax6 in Platynereis. Concerning the r-opsin present in the Platynereis eyes, pax6...