Novel Transgenic Mice for Inducible Gene Overexpression in Pancreatic Cells Define Glucocorticoid Receptor-Mediated Regulations of Beta Cells

Conditional gene deletion in specific cell populations has helped the understanding of pancreas development. Using this approach, we have shown that deleting the glucocorticoid receptor (GR) gene in pancreatic precursor cells leads to a doubled beta-cell mass. Here, we provide genetic tools that permit a temporally and spatially controlled expression of target genes in pancreatic cells using the Tetracycline inducible system. To efficiently target the Tetracycline transactivator (tTA) in specific cell populations, we generated Bacterial Artificial Chromosomes (BAC) transgenic mice expressing the improved Tetracycline transactivator (itTA) either in pancreatic progenitor cells expressing the transcription factor Pdx1 (BAC-Pdx1-itTA), or in beta cells expressing the insulin1 gene (BAC-Ins1-itTA). In the two transgenic models, itTA-mediated activation of reporter genes was efficient and subject to regulation by Doxycycline (Dox). The analysis of a tetracycline-regulated LacZ reporter gene shows that in BAC-Pdx1-itTA mice, itTA is expressed from embryonic (E) day 11.5 in all pancreatic precursor cells. In the adult pancreas, itTA is active in mature beta, delta cells and in few acinar cells. In BAC-Ins1-itTA mice tTA is active from E13.5 and is restricted to beta cells in fetal and adult pancreas. In both lines, tTA activity was suppressed by Dox treatment and re-induced after Dox removal. Using these transgenic lines, we overexpressed the GR in selective pancreatic cell populations and found that overexpression in precursor cells altered adult beta-cell fraction but not glucose tolerance. In contrast, GR overexpression in mature beta cells did not alter beta-cell fraction but impaired glucose tolerance with insufficient insulin secretion. In conclusion, these new itTA mouse models will allow fine-tuning of gene expression to investigate gene function in pancreatic biology and help us understand how glucocorticoid signaling affects on the long-term distinct aspects of beta-cell biology

Myogenesis modelled by human pluripotent stem cells uncovers Duchenne muscular dystrophy phenotypes prior to skeletal muscle commitment

Duchenne muscular dystrophy (DMD) causes severe disability of children and death of young men, with an incidence of approximately 1/5,000 male births. Symptoms appear in early childhood, with a diagnosis made around 4 years old, a time where the amount of muscle damage is already significant, preventing early therapeutic interventions that could be more efficient at halting disease progression. In the meantime, the precise moment at which disease phenotypes arise - even asymptomatically - is still unknown. Thus, there is a critical need to better define DMD onset as well as its first manifestations, which could help identify early disease biomarkers and novel therapeutic targets. In this study, we have used human induced pluripotent stem cells (hiPSCs) from DMD patients to model skeletal myogenesis, and compared their differentiation dynamics to healthy control cells by a comprehensive multi-omics analysis. Transcriptome and miRnome comparisons combined with protein analyses at 7 time points demonstrate that hiPSC differentiation 1) mimics described DMD phenotypes at the differentiation endpoint; and 2) homogeneously and robustly recapitulates key developmental steps - mesoderm, somite, skeletal muscle - which offers the possibility to explore dystrophin functions and find earlier disease biomarkers. Starting at the somite stage, mitochondrial gene dysregulations escalate during differentiation. We also describe fibrosis as an intrinsic feature of skeletal muscle cells that starts early during myogenesis. In sum, our data strongly argue for an early developmental manifestation of DMD whose onset is triggered before the entry into the skeletal muscle compartment, data leading to a necessary reconsideration of dystrophin functions during muscle development

Myogenesis modelled by human pluripotent stem cells uncovers Duchenne muscular dystrophy phenotypes prior to skeletal muscle commitment

Duchenne muscular dystrophy (DMD) causes severe disability of children and death of young men, with an incidence of approximately 1/5,000 male births. Symptoms appear in early childhood, with a diagnosis made around 4 years old, a time where the amount of muscle damage is already significant, preventing early therapeutic interventions that could be more efficient at halting disease progression. In the meantime, the precise moment at which disease phenotypes arise - even asymptomatically - is still unknown. Thus, there is a critical need to better define DMD onset as well as its first manifestations, which could help identify early disease biomarkers and novel therapeutic targets. In this study, we have used human induced pluripotent stem cells (hiPSCs) from DMD patients to model skeletal myogenesis, and compared their differentiation dynamics to healthy control cells by a comprehensive multi-omics analysis. Transcriptome and miRnome comparisons combined with protein analyses at 7 time points demonstrate that hiPSC differentiation 1) mimics described DMD phenotypes at the differentiation endpoint; and 2) homogeneously and robustly recapitulates key developmental steps - mesoderm, somite, skeletal muscle - which offers the possibility to explore dystrophin functions and find earlier disease biomarkers. Starting at the somite stage, mitochondrial gene dysregulations escalate during differentiation. We also describe fibrosis as an intrinsic feature of skeletal muscle cells that starts early during myogenesis. In sum, our data strongly argue for an early developmental manifestation of DMD whose onset is triggered before the entry into the skeletal muscle compartment, data leading to a necessary reconsideration of dystrophin functions during muscle development

Myogenesis modelled by human pluripotent stem cells: a multi‐omic study of Duchenne myopathy early onset

International audienceBackground Duchenne muscular dystrophy (DMD) causes severe disability of children and death of young men, with an incidence of approximately 1/5000 male births. Symptoms appear in early childhood, with a diagnosis made mostly around 4 years old, a time where the amount of muscle damage is already significant, preventing early therapeutic interventions that could be more efficient at halting disease progression. In the meantime, the precise moment at which disease phenotypes arise-even asymptomatically-is still unknown. Thus, there is a critical need to better define DMD onset as well as its first manifestations, which could help identify early disease biomarkers and novel therapeutic targets. Methods We have used both human tissue-derived myoblasts and human induced pluripotent stem cells (hiPSCs) from DMD patients to model skeletal myogenesis and compared their differentiation dynamics with that of healthy control cells by a comprehensive multi-omic analysis at seven time points. Results were strengthened with the analysis of isogenic CRISPR-edited human embryonic stem cells and through comparisons against published transcriptomic and proteomic datasets from human DMD muscles. The study was completed with DMD knockdown/rescue experiments in hiPSC-derived skeletal muscle progenitor cells and adenosine triphosphate measurement in hiPSC-derived myotubes. Results Transcriptome and miRnome comparisons combined with protein analyses demonstrated that hiPSC differentiation (i) leads to embryonic/foetal myotubes that mimic described DMD phenotypes at the differentiation endpoint and (ii) homogeneously and robustly recapitulates key developmental steps-mesoderm, somite, and skeletal muscle. Starting at the somite stage, DMD dysregulations concerned almost 10% of the transcriptome. These include mitochondrial genes whose dysregulations escalate during differentiation. We also describe fibrosis as an intrinsic feature of DMD skeletal muscle cells that begins early during myogenesis. All the omics data are available online for exploration through a graphical interface at https://muscle-dmd.omics.ovh/. Conclusions Our data argue for an early developmental manifestation of DMD whose onset is triggered before the entry into the skeletal muscle compartment, data leading to a necessary reconsideration of dystrophin roles during muscle development. This hiPSC model of skeletal muscle differentiation offers the possibility to explore these functions as well as find earlier DMD biomarkers and therapeutic targets

Dp412e : a novel human embryonic isoform of dystrophin

La dystrophie musculaire de Duchenne (DMD) est une myopathie dévastatrice récessive liée au chromosome X. Cette pathologie est due à la présence de mutations dans le gène DMD codant pour la dystrophine. Le rôle de cette protéine est encore l’objet de nombreuses interrogations et elle pourrait être impliquée dans le développement de la DMD in utero. Les cellules souches pluripotentes humaines (hPSCs) traitées par le BMP4 (bone morphogenetic protein 4) nous ont permis d’étudier les premières étapes de la myogenèse dans un contexte normal et DMD.De façon inattendue, les hPSCs traitées par le BMP4 expriment un transcrit long DMD à un niveau similaire à celui observé dans le muscle squelettique adulte. Ce transcrit possède un exon 1 spécifique et non traduit identifié par 5’RACE PCR dont la séquence est conservée uniquement dans un sous-groupe d’anthropoïdes comprenant l’Homme. L’isoforme de la dystrophine correspondante est caractérisée par un domaine tronqué de fixation à l’actine à l’extrémité N-terminale. Cette protéine de 412 kD a été détectée par Western blot dans des hPSCs normales traitées par le BMP4 ainsi que dans des corps embryoïdes. Suite à des analyses extensives démontrant que son expression était restreinte aux toutes premières étapes de la différenciation, cette nouvelle isoforme a été nommée ‘Dp412e’.Cette étude valide l’utilisation des hPSCs pour analyser les étapes précoces du développement humain dans un contexte normal et pathologique et a conduit à la découverte d’une nouvelle isoforme embryonnaire humaine de la dystrophine de 412 kD. L’étude de la régulation et des fonctions associées à cette nouvelle isoforme contribuera à mieux comprendre la physiopathologie de la DMD et les défauts développementaux potentiels. Le modèle inductible par le BMP4, simple, rapide et robuste, procurant une grande quantité de transcrits longs DMD et la protéine correspondante, est déjà bien adapté aux approches de criblage à haut débit et à haut contenu. Une première preuve d’efficacité de ce modèle a d’ailleurs été réalisée avec succès grâce à une modification par saut d’exon du transcrit Dp412e. La disponibilité de cette plateforme cellulaire performante accéléra le développement, la validation et l’amélioration de thérapies géniques DMD.Duchenne Muscular Dystrophy (DMD) is a devastating X-linked recessive genetic myopathy. This pathology is caused by mutations in the DMD gene encoding dystrophin. Many questions remain about the role of this protein and it could be implicated in the DMD onset in utero. Human pluripotent stem cells (hPSCs) together with the bone morphogenetic protein 4 (BMP4), allowed us to study the early steps of myogenesis in normal and DMD contexts. Unexpectedly, a new long DMD transcript was detected in BMP4-treated hPSCs at levels similar to that found in adult skeletal muscle. This novel transcript had a specific untranslated first exon identified by 5’RACE PCR which was conserved only in a sub-group of anthropoids including Human. The corresponding novel dystrophin protein is characterized by a truncated N-terminal actin-binding domain. This isoform of 412 kD was detected by Western blot in normal BMP4-treated hPSCs and also in embryoid bodies. Following extensive analyses demonstrating that its expression is restricted to the first stages of differentiation, this transcript was named 'Dp412e’. This study validates the use of hPSCs to analyze early phases of human development in normal and pathological contexts and has led to the discovery of a new human embryonic 412 kD dystrophin isoform. Deciphering the regulation process(es) and the function(s) associated to this new isoform will contribute to a better understanding of the DMD physiopathology and potential developmental defects. The simple, fast and robust BMP4-inducible model highlighted here providing large amount of a long DMD transcript and the corresponding protein, is already well-adapted to high-throughput and high-content screening approaches. As a first proof of concept, an exon skipping modification of Dp412e transcript was successfully done with this model. Availability of this powerful cell platform will accelerate the development, validation and improvement of DMD genetic therapies

Consequences of GR overexpression on beta-cell mass and glucose homeostasis in Pdx1-itTA/LacZtetOhGR or Ins1-itTA/LacZtetOhGR mice.

<p>(A) Immunohistochemistry for the glucocorticoid receptor (GR, brown nuclear staining) on pancreatic sections from a control LacZtetOhGR, (B) a Pdx1-itTA/LacZtetOhGR mouse and (C) a Ins1-itTA/LacZtetOhGR mouse. (D) Beta-cell fraction, (E) beta-cell mass, (F) blood glucose during an intra-peritoneal glucose tolerance test and (G) corresponding Area Under the Curve (AUC) in adult Pdx1-itTA/LacZtetOhGR mice. (H) Beta-cell fraction, (I) beta-cell mass, (J) blood glucose during an intra-peritoneal glucose tolerance test and (K) corresponding Area Under the Curve (AUC) in adult Ins1-itTA/LacZtetOhGR mice. Results are expressed as means ± SD for n = 3–5 animals per group. * p<0.05 when comparing double transgenic mice (Pdx1-itTA/LacZtetOhGR or Ins1-itTA/LacZtetOhGR) to control (LacZtetOhGR) mice using a Mann-Whitney non parametric test. Scale bar = 100 µm.</p

The expression of LacZ can be extinguished or induced in adult Pdx1-itTA/LacZtetOhGR mice.

<p>(A) Xgal staining on a pancreatic section of an adult Pdx1-itTA/LacZtetOhGR mouse without Dox treatment. (B) Absence of Xgal staining after 8 weeks of Dox administration (1 mg/ml) in the drinking water of an adult Pdx1-itTA/LacZtetOhGR animal. An islet is outlined. (C) Absence of Xgal staining in a 28 days-old Pdx1-itTA/LacZtetOhGR mouse treated with Dox (0.1 mg/ml) from E0. An islet is outlined. (D) Eight weeks after Dox removal, Xgal staining is observed on a pancreatic section from a Pdx1-itTA/LacZtetOhGR mouse. Note that in contrast with A, Xgal staining is observed in islet cells and not in acinar cells. Scale bar = 50 µm.</p

The Pdx1-itTA is active early during fetal development in pancreatic precursors.

<p>(A) LacZ expression revealed by Xgal staining in pancreatic buds at E11.5 and in the entire pancreas at E13.5 (B) and E15.5 (C) from Pdx-itTA/LacZtetOhGR fetuses. d = dorsal; v = ventral; st = stomach.</p

The Pdx1-itTA activates lacZ expression in beta, delta and in acinar cells.

<p>(A) (E) (I) (M) and (Q) LacZ expression revealed by Xgal staining (blue) on adult pancreatic sections from a Pdx1-itTA/LacZtetOhGR mouse. (B) Immunofluorescence for insulin (green), (C) merge of A and B and (D) magnified view of inset in C. (F) Immunofluorescence for glucagon (green), (G) merge of E and F and (H) magnified view of inset in G. (J) Immunofluorescence for somatostatin (green), (K) merge of I and J and (L) magnified view of inset in K. (N) Immunofluorescence for PP (green), (O) merge of M and N and (P) magnified view of inset in O. (R) Immunofluorescence for amylase (green), (S) merge of Q and R and (T) magnified view of inset in S. Scale bar = 50 µm except for D, H, L, P and T where scale bar = 10 µm.</p