No description available.

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

No description available.

Elongation of the mouse anteroposterior axis depends on stem cell-like axial progenitors including a neuromesodermal (NM) bi-fated population existing in the primitive streak and later in the tail bud. Fate mapping experiments have demonstrated these NM progenitors reside in precise locations of the embryo. At E8.5, these cells are found in the node-streak border (NSB) and anterior epiblast on either side of the primitive streak. At tail bud stages (E10.5-E13.5), these progenitors reside in the chordoneural hinge (CNH). The coexpression of the transcription factors T (brachyury) and Sox2 has been proposed as a good marker to identify NM progenitors in vertebrates. However, this cell signature has never been thoroughly assessed during mouse axis elongation. In this thesis, I performed T and Sox2 double immunofluorescent stainings on different stages of mouse embryos and reconstructed their expression domains in the 3D images to investigate the spatiotemporal dynamics of NM progenitors during axis elongation. The results show the transient existence of T+Sox2+ cells in the posterior progenitor zone, from the headfold stage (E8.0) to the end of axis elongation (E13.5, 65somites). Moreover, the number of T+Sox2+ cells increases between E8.5 and E9.5 but gradually declines afterwards. I then investigated the time points for initiation and loss of NM progenitors by performing a series of heterotopic grafting experiments. It has been previously shown that distal epiblast (Sox2+T- cells) at LS-EB stages (E7.5) are fated to become NSB cells in E8.5 embryos. However, when cells from the distal region of LS-EB stage embryos (E7.5) were grafted to E8.5 NSB, these cells contribute extensity to the notochord but not either neural tissues or paraxial mesoderm. This indicates that NM progenitors may be not yet specified before the onset of T and Sox2 coexpression, while the notochord progenitors are already specified at E7.5. The grafting experiments also show the loss of NM progenitors at E14.5 after the end of axis elongation, which coincides with the disappearance of T+Sox2+ cells in the tail. Collectively, these results indicate that T+Sox2+ cells may represent a distinct cell state that defines NM progenitors. Wnt/β-catenin signalling has been shown to play an important role in maintaining the posterior progenitor zone. However, due to the wide expression of β-catenin and the early lethality of β-catenin null embryos, the exact effect of losing β-catenin in NM progenitors is still unknown. In this study, I took advantage of the Cre-ERT2 system and grafting technique to conditionally delete β-catenin specifically in NM progenitors during ex vivo culture. The results show that Wnt/β-catenin signalling is required cell autonomously for initiating mesoderm fate choice in NM progenitors. In its absence, mesoderm fated NM progenitors convert their fate and differentiate to neural derivatives. Moreover, the interchangeability between neural and mesodermal fate only exists in NM progenitors, as the loss of β-catenin in mesoderm committed progenitors does not affect their fate choice. Using image analysis and quantification software, I also show that Wnt/β-catenin signalling is crucial for the expansion of T+Sox2+ NM progenitors during axis elongation. Due to difficult access and a limited number of NM progenitors in vivo, in vitro generated NM progenitors from pluripotent cells, such as epiblast stem cells (EpiSCs), can offer an insight into the maintenance and differentiation of NM progenitors. Since the in vivo potential of EpiSCs had never been successfully demonstrated before, I first grafted EpiSCs into postimplantation embryos and cultured them ex vivo for 24-48 hours to assess their cell integration. The results show that EpiSCs can integrate successfully in streak stage embryos (E6.5-E7.5), but not at early somite stages (E8.5), when the epiblast has lost its pluripotency. I then further investigated the in vivo potential of EpiSC derivatives. The results show that increasing Wnt signalling in EpiSCs inhibits their ability to generate anterior neural tissues in vivo, which is consistent with the previous in vitro data. Recently, it has been demonstrated that NM progenitors can be derived from EpiSCs. These in vitro derived NM progenitors can incorporate into E8.5 embryos and give rise to both neural and mesodermal derivatives. In this thesis, I show that these in vitro derived NM progenitors do not incorporate successfully in E7.5 embryos. Collectively, by combining grafting experiments with a chimeric embryo formation assay, I can identify the in vivo stage of the in vitro counterparts of the embryonic cell types.

No description available.

ABSTRACT Ethanol is a classic teratogen capable of inducing a wide range of developmental abnormalities that vary in severity, from the barely perceptible to spontaneous abortion. These defects are collectively referred to as foetal alcohol spectrum disorders (FASD). Foetal alcohol syndrome (FAS) lies at the extreme end of this spectrum and is associated with three broad domains: prenatal and/or postnatal growth retardation, distinctive facial features and brain damage. Epidemiological and animal studies clearly indicate that the clinical variability of FASD is related to four distinct window periods: preconception, preimplantation, gastrulation and postorganogenesis. These developmental windows are correlated with peak periods of epigenetic reprogramming, suggesting a common mechanism of ethanol teratogenesis. Together with experimental evidence that ethanol inhibits DNA-methyltransferase, as well as folate metabolism, this suggests an ‘epigenetic model of FASD’. The aim of the present study was to explore the validity of this model by investigating the relationship between ethanol-induced growth retardation and imprinting, following ethanol exposure during the preimplantation period. Employing an experimental study design, together with a hybrid mouse model, embryos and placentae were harvested at 10.5 days post coitus (dpc). The weights of embryos and placentae, as well as methylation profiles at the H19 imprinting control region (ICR) – an important regulator of growth - were measured. It was found that ethanol-treated embryos and placentae were severely growth retarded in comparison to controls: r=-0.760 (p<0.01, one-tailed) and r=-0.816 (p<0.05, two-tailed), respectively. Bisulphite genomic sequencing revealed that the methylation profile at the H19 ICR was unaffected in ethanol-treated embryos, in comparison to saline-treated controls. Conversely, methylation at the paternal and maternal alleles in placentae was found to be reduced and increased, respectively, in comparison to embryos. These results imply that mechanisms for the maintenance of imprinting in the embryo are more robust than in the placenta. This is consistent with the relatively longlived nature of the embryo, which must maintain imprinting for a considerably longer period of time than the placenta. Bisulphite sequencing also revealed that the paternal allele of the H19 ICR had significantly decreased levels of methylation, while the maternal allele had increased levels of methylation, in ethanol treated-placentae, in comparison to saline controls. The changes observed at the paternal allele were localized to the CTCF1 DNA-binding site, while a trend for increased methylation at the maternal allele was observed at the CTCF2 site. A partial correlation further revealed that demethylation at the paternal allele in placentae partly mediated the effect of ethanol on placental weight. An ‘epigenetic switch model’, whereby paternal Igf2 is downregulated by the epigenetic switching of the paternal allele to the maternal epigenotype, is proposed to explain this relationship. However, partial correlations also indicated that demethylation at the paternal allele of the H19 ICR, as well as placental growth retardation, did not mediate the effect of ethanol on embryo growth. Collectively, these data suggest that imprinting at the H19 ICR is not a mechanism of embryo growth retardation prior to 10.5 dpc. In explaining these results, it is proposed that the growth retarded placenta was able to meet the nutritional demands of the similarly growth retarded embryo up until 10.5 dpc. However, an important question for future research would be to examine the relationship between ethanol-induced growth retardation and imprinting during late gestation. During the final growth spurt (>14.5 dpc) the growth retarded placenta may become unable to meet the increased demands for nutrition, which would exacerbate foetal growth restriction. In sum, the present study revealed a novel mechanism of ethanol-induced growth retardation in the placenta but indicated that imprinting at the H19 ICR does not mediate the effect of ethanol on the early embryo. Further research is required to resolve the relationship between imprinting and ethanol-induced growth retardation.

No description available.

Les facteurs de transcription SIM1, OTP, POU3F2 et ARNT2 interagissent ensemble en orchestrant le développement complexe de l’hypothalamus, une région du cerveau contenant plusieurs petites populations circonscrites de neurones, dont le noyau paraventriculaire (PVN). Ce noyau est un important centre intégrateur et l’haploinsuffisance du facteur de transcription Sim1, essentiel au développement du PVN, mène à l’hyperphagie autant chez la souris que l’homme. Différentes souris ont été générées par génie génétique afin de nous aider à trouver d’autres gènes essentiels qui participent à cette cascade transcriptionnelle. Premièrement, la partie antérieure de l’hypothalamus a été récoltée chez des embryons (E12.5) de souris qui surexprimaient le gène Pou3f2 sous le promoteur de Otp7. L’analyse transcriptionnelle de cette partie a été comparée avec les embryons (E12.5) wt de la même portée, de sorte qu’il a été possible de constater que différents gènes ont été régulés à la hausse et d’autres à la baisse. Les gènes en question ont été choisis selon leur pertinence au développement de la région d’intérêt, l’hypothalamus, et de trois autres critères : un accroissement de plus de 1.5, une expression minimale dans l’embryon d’au moins 1000 lectures et le rang le plus haut. Cette méthode discriminatoire a permis d’identifier les gènes les plus affectés dont Lgi2, Fezf2, Sema3c, Six6, Sox14, Lmo4, Nwd2 et Nkx2.1. Les gènes régulés à la baisse étaient Six6, Sox14 et Nkx2.1, tandis que tous les autres étaient à la hausse. Afin de confirmer les résultats obtenus, une validation par hybridation in situ a été utilisée sur des tranches d’hypothalamus d’embryons E12.5. Nous avons pu confirmer la surexpression des marqueurs Lgi2, Fezf2, Sema3c, Lmo4 et de Pou3f2 dans le domaine du PVN. La diminution de l’expression des marqueurs Sox14 et Nkx2.1 a pu être détectée dans el domaine basal de l’hypothalamus, ce qui suggère que l’effet d’une surexpression de Pou3f2 dans le domaine du PVN ait un effet cellulaire non autonome. La diminution de l’expression de Six6 dans le domaine basal n’a pas pu être confirmer de façon reproductible. Deuxièmement, il semblerait qu’il y ait une redondance de rôle entre les facteurs de transcription Otp et Sim1, tous les deux agissant en amont de Pou3f2. Afin de comparer leur impact dans le programme transcriptionnel, la technologie CrisPR-cas9 a été utilisée pour faire un KO du 2e exon d’Otp. Nous avons pu confirmer notre modèle de mutation en le comparant aux autres mutants de la littérature par une réduction de l’expression d’OT, d’OTP, d’AVP et de TRH. Troisièmement, les souris hétérozygotes pour Otp et Sim1 seront croisées, de sorte d’obtenir quatre génotypes : wt, Otp+/-, Sim1+/tlz+ et Otp+/-Sim1+/tlz+. Puisqu’une redondance des rôles de Sim1 et Otp est soupçonnée, le phénotype du double mutant devrait présenter une obésité par hyperphagie plus importante que celle des souris hétérozygotes pour le gène Sim1 ou Otp. Les souris sont pesées à partir de la 5e semaine de vie jusqu’à l’âge de 6 mois à une fréquence d’une fois par semaine. L’apport calorique est également mesuré une fois par semaine sur période de 24h. La double mutation (Otp+/-Sim1+/tlz+) chez les souris mâles causait un phénotype d’obésité plus important que la singularité des mutations, mais ce n’était pas le cas chez les souris femelles. Les souris portant la mutation d’Otp étaient tout de même plus obèses que les souris sauvages pour les deux sexes. Plus de souris seront nécessaire pour déterminer si un apport calorique sans changement au niveau des dépenses énergétiques est la cause de ce gain pondéral. / The transcription factors SIM1, OTP and POU3F2 interact together to orchestrate the complex development of the hypothalamus, a region of the brain containing several small, circumscribed populations of neurons, including the paraventricular nucleus (PVN). This nucleus is an important integrating center, and the haploinsufficiency of the transcription factor Sim1, essential for the development of PVN, leads to overeating in both mice and humans. Different mice have been genetically engineered to help us find other essential genes that participate in this transcriptional cascade. Firstly, the anterior part of the hypothalamus was collected from mice embryos (E12.5) which overexpressed the Pou3f2 gene under the OTP7 promotor. The transcriptional analysis was compared to wt embryos from the same litter to see the different upregulated and downregulated genes. These genes were chosen according to their relevance to the development of the anterior hypothalamus. Three criteria were used to discriminate genes from one another: 1) an increase of more than 1.5, 2) a minimal expression in the embryo (E12.5) of at least 1000 reads and 3) the highest rank. This discriminatory method allowed us to identify the genes Lgi2, Fezf2, Sema3c, Six6, Sox14, Lmo4, Nwd2, Nkx2.1. To have a visuospatial idea of these affected genes, the validation of these results was done by in situ hybridization on E12.5 embryo hypothalamus. We have been able to see an overexpression in the PVN domain for the Lgi2, Fezf2, Sema3c, Lmo4 and Pou3f2 markers. The reduction of expression for Sox14 and Nkx2.1 markers were visible in the basal domain of the hypothalamus, which suggest a non cell autonomous effect of Pou3f2 being overexpressed. The reduction of Six6 couldn’t be consistently visible with repetition. Secondly, a redundant role of OTP and SIM1 seems to occur in the development of the hypothalamus. We created a KO line of the Otp gene by deleting the second exon with CrispR-cas9 and characterized it. We then compared it to the Sim1+/tlz+ line that we already generated in the lab. We were able to confirm our mutation model by seeing a reduction in the expression of crucial markers such as OT, OTP, AVP and TRH. Thirdly, we crossed Otp+/- with Sim1+/tlz+ mice to obtain four different genotypes: wt, Otp+/-, Sim1+/tlz+ and Otp+/- Sim1+/tlz+. Since a redundant aspect has been observed for SIM1 and OTP transcription factors, we were wondering if the obesity phenotype would be worsened by carrying both mutation or not. These mice were weighted every week from 5-week-old up to 6 months old. Food intake has also been measured since the obesity has been reported to be caused by hyperphagia in Sim1 mutant mice. The male mice carrying the double mutation (Otp+/-Sim1+/tlz+) showed a more important weight gain than only Sim1+/tlz+ or Otp+/- mutants, but it was not the case for the female mice. The mice carrying the Otp mutation still got a more important weight gain than the wt mice (females and males). More mice would be necessary to determine if this weight gain is caused by hyperphagia only or if unbalance energy cost is part of the cause.