Crosstalk between androgen receptor and WNT/β-catenin signaling causes sex-specific adrenocortical hyperplasia in mice

Female bias is highly prevalent in conditions such as adrenal cortex hyperplasia and neoplasia, but the reasons behind this phenomenon are poorly understood. In this study, we show that overexpression of the secreted WNT agonist R-spondin 1 (RSPO1) leads to ectopic activation of WNT/β-catenin signaling and causes sex-specific adrenocortical hyperplasia in mice. Although female adrenals show ectopic proliferation, male adrenals display excessive immune system activation and cortical thinning. Using a combination of genetic manipulations and hormonal treatment, we show that gonadal androgens suppress ectopic proliferation in the adrenal cortex and determine the selective regulation of the WNT-related genes Axin2 and Wnt4. Notably, genetic removal of androgen receptor (AR) from adrenocortical cells restores the mitogenic effect of WNT/β-catenin signaling. This is the first demonstration that AR activity in the adrenal cortex determines susceptibility to canonical WNT signaling-induced hyperplasia.</p

Crosstalk between androgen receptor and WNT/β-catenin signaling causes sex-specific adrenocortical hyperplasia in mice

Female bias is highly prevalent in conditions such as adrenal cortex hyperplasia and neoplasia, but the reasons behind this phenomenon are poorly understood. In this study, we show that overexpression of the secreted WNT agonist R-spondin 1 (RSPO1) leads to ectopic activation of WNT/β-catenin signaling and causes sex-specific adrenocortical hyperplasia in mice. Although female adrenals show ectopic proliferation, male adrenals display excessive immune system activation and cortical thinning. Using a combination of genetic manipulations and hormonal treatment, we show that gonadal androgens suppress ectopic proliferation in the adrenal cortex and determine the selective regulation of the WNT-related genes Axin2 and Wnt4. Notably, genetic removal of androgen receptor (AR) from adrenocortical cells restores the mitogenic effect of WNT/β-catenin signaling. This is the first demonstration that AR activity in the adrenal cortex determines susceptibility to canonical WNT signaling-induced hyperplasia

GREB1L (growth regulation by estrogen in breast cancer 1-like) regulates fatty acid cellular metabolism and pluripotency exit during axial progenitor maturation

L'épiblaste latéral caudal (ELC) est une population de progéniteurs multi-lignages présente aux stades précoces du développement embryonnaire, et importante pour l'élongation axiale des amniotes. Les processus de spécification de l'ELC depuis l'épiblaste embryonnaire et de différenciation de l'ELC en différents lignages sont actuellement mal compris. GREB1L est un gène haploinsuffisant qui, lorsque muté, provoque une série de malformations congénitales chez l'humain, telles que l'agénésie rénale, l'agénésie du canal müllérien, des déficiences auditives, des anomalies cardiaques et des déficiences surrénaliennes. Les premières études réalisées chez les souris Greb1l knock-out (KO) nous ont permis d'émettre l'hypothèse que ce gène pourrait jouer un rôle essentiel lors de la maturation des progéniteurs axiaux. L'objectif principal de ce projet de thèse était d'étudier les mécanismes développementaux et moléculaires de GREB1L pendant la maturation de ces progéniteurs.Des études récentes ont démontré que le métabolisme cellulaire joue un rôle vital dans l'embryogenèse, mais la contribution du métabolisme des lipides au développement de l'embryon est actuellement inconnue. Le deuxième objectif de ce projet était de comprendre le profile métabolique des lipides dans les gastrulae de souris. Nous avons découvert qu'au cours de la maturation de l'ELC, les cellules subissent un remaniement métabolique majeur, passant d'un métabolisme mitochondrial des acides gras à un métabolisme non mitochondrial.Mes analyses d'expression ont révélé que Greb1l est exprimé dans divers progéniteurs au stades précoces, puis dans le mésoderme caudal et les progéniteurs neuraux aux stades ultérieurs. En outre, les embryons KO présentent un phénotype de troncation axiale dès le jour embryonnaire (E) 8.5, qui devient prononcé à partir de E9.0.Pour mieux comprendre les changements moléculaires associés à la perte de fonction de GREB1L, des analyses protéomiques quantitatives et des analyses de single-cell RNA sequencing (scRNAseq) ont été effectuées sur les parties caudales d'embryons E8.25. Ces analyses ont révélé que les embryons KO exprimaient des niveaux plus faibles de protéines impliquées dans la structuration antéro-postérieure, tandis que les protéines jouant un rôle dans la pluripotence, l'adhésion cellulaire, l'organisation du cytosquelette, l'oxydation des acides gras et le métabolisme de l'hexose étaient augmentées. Les analyses scRNAseq ont révélé que la signature transcriptomique de l'ELC et de ses tissus dérivés, tels que le neuroépithélium caudal, le mésoderme paraxial et le mésoderme latéral et intermédiaire, différait entre les embryons sauvages et KO. En accord avec notre analyse protéomique, les transcrits de plusieurs gènes impliqués dans l'oxydation des acides gras (Acads, Eci1, Echdc2, Acat2) étaient enrichis dans les embryons KO. Il est intéressant de noter que les cellules de l'ELC des embryons KO présentent une expression anormalement élevée des marqueurs de pluripotence (Pou5f1, Klf5, Zic3, Wnt5a) et ne parviennent pas augmenter l'expression des marqueurs de maturation (Fgf8, Cyp26a1, Hes5, Hoxa7). De plus, l'ELC des embryons KO prennent des voies de différenciation différentes par rapport au contrôle, expliquant les anomalies de développement associées aux mutations de GREB1L.L'étiquetage GREB1L-V5/3XFLAG dans des cellules ES de souris, suivi d'une différenciation in vitro, a révélé que GREB1L se localise dans les mitochondries et/ou le noyau en fonction du contexte. Les gain/perte de fonction de GREB1L ont entraîné une réorganisation spectaculaire du réseau mitochondrial et des changements dans l'acétylation des protéines, une modification post-traductionnelle majeure régulée par l'oxydation des acides gras.Dans l'ensemble, nous concluons que GREB1L joue un rôle vital pendant la maturation des progéniteurs axiaux, en facilitant la sortie de la pluripotence et en modifiant l'état métabolique requis pour la différenciation.The caudal lateral epiblast (CLE) is a multi-lineage progenitor population found in early stages of embryonic development and is important for embryonic axial elongation of amniotes. The process of CLE specification from the embryonic epiblast and how differentiation of the CLE into subsequent lineages are regulated are presently poorly understood. GREB1L is a haploinsufficient gene that when mutated causes a range of congenital defects in humans. These include renal agenesis, Müllerian duct agenesis, hearing impairments, heart abnormalities, and adrenal defects. Initial studies performed on a Greb1l knock-out (KO) mouse model led us to hypothesize that this gene might play an essential role during axial progenitor maturation. The major aim of this thesis project was to investigate the developmental and molecular mechanisms of GREB1L during axial progenitor maturation.Recent studies have demonstrated that cellular metabolism plays vital roles in embryogenesis, but the contribution of lipid metabolism on the developing embryo is presently unknown. A second focus of this project was to understand the pattern of lipid metabolism in mouse gastrulae. We discovered that during CLE maturations, cells undergo a major metabolic rewiring from mitochondrial fatty acid metabolism to a non-mitochondrial metabolism.In-house RNA-Scope expression analyses revealed that Greb1l is widely expressed in many progenitor tissues of early mouse gastrulae. At later stages, the expression becomes restricted to the caudal mesoderm and neural progenitors. Furthermore, our analysis on Greb1l mutant (KO) embryos showed an axial truncation phenotype as early as embryonic day (E) 8.5, which becomes pronounced by E9.0.To gain insights into the molecular changes associated with the loss of GREB1L function, quantitative proteomic and single cell RNA sequencing (scRNAseq) analyses were performed on the caudal portions of E8.25 embryos. Bioinformatic analysis revealed that KO embryos expressed significantly lower levels of proteins involved in anterior-posterior patterning, while proteins that play active roles in pluripotency, cell adhesion, cytoskeletal organisation, fatty acid oxidation and hexose metabolism, were found to be increased. scRNAseq analyses revealed that the transcriptomic signature of several cell clusters differed between wildtype and KO embryos. Those include the CLE and its derivative tissues, such as the caudal neuroepithelium, the paraxial mesoderm, and the lateral and intermediate mesoderm. In agreement with our proteomic analysis, transcripts of several genes involved in fatty-acid oxidation (Acads, Eci1, Echdc2, Acat2) were enriched in knockout embryos. Interestingly, CLE cells in KO embryos exhibited an abnormally high expression of pluripotency markers (Pou5f1, Klf5, Zic3, Wnt5a) and failed to upregulate maturation markers (Fgf8, Cyp26a1, Hes5, Hoxa7). Furthermore, CLE of KO embryos take different routes of differentiation compared to the control, which explains developmental abnormalities associated with GREB1L/Greb1l mutations.GREB1L-V5/3XFLAG tagging in mouse ES cells followed by in-vitro differentiation revealed GREB1L localises to mitochondria and/or the nucleus in a context-dependent manner. Gain and loss of function of GREB1L resulted in a dramatic reorganisation of the mitochondrial network and changes in protein acetylation, a major post translational modification regulated by fatty acid oxidation.Taken together, we conclude that GREB1L plays a vital role during axial progenitor maturation, facilitating the exit from pluripotency and altering the metabolic state required for differentiation

Single-cell transcriptomic profiling redefines the origin and specification of early adrenogonadal progenitors

Adrenal cortex and gonads represent the two major steroidogenic organs in mammals. Both tissues are considered to share a common developmental origin characterized by the expression of Nr5a1/Sf1. The precise origin of adrenogonadal progenitors and the processes driving differentiation toward the adrenal or gonadal fate remain, however, elusive. Here, we provide a comprehensive single-cell transcriptomic atlas of early mouse adrenogonadal development including 52 cell types belonging to twelve major cell lineages. Trajectory reconstruction reveals that adrenogonadal cells emerge from the lateral plate rather than the intermediate mesoderm. Surprisingly, we find that gonadal and adrenal fates have already diverged prior to Nr5a1 expression. Finally, lineage separation into gonadal and adrenal fates involves canonical versus non-canonical Wnt signaling and differential expression of Hox patterning genes. Thus, our study provides important insights into the molecular programs of adrenal and gonadal fate choice and will be a valuable resource for further research into adrenogonadal ontogenesis

Retinoic acid signaling is directly activated in cardiomyocytes and protects mouse hearts from apoptosis after myocardial infarction

Retinoic acid (RA) is an essential signaling molecule for cardiac development and plays a protective role in the heart after myocardial infarction (MI). In both cases, the effect of RA signaling on cardiomyocytes, the principle cell type of the heart, has been reported to be indirect. Here we have developed an inducible murine transgenic RA-reporter line using CreER(T2) technology that permits lineage tracing of RA-responsive cells and faithfully recapitulates endogenous RA activity in multiple organs during embryonic development. Strikingly, we have observed a direct RA response in cardiomyocytes during mid-late gestation and after MI. Ablation of RA signaling through deletion of the Aldh1a1/a2/a3 genes encoding RA-synthesizing enzymes leads to increased cardiomyocyte apoptosis in adults subjected to MI. RNA sequencing analysis reveals Tgm2 and Ace1, two genes with well-established links to cardiac repair, as potential targets of RA signaling in primary cardiomyocytes, thereby providing novel links between the RA pathway and heart disease