Prof. François Cuzin

Prof Ahmed Mansouri

Dr Miriam Cnop

Dr Jacob Hecksher

Ana Filippo

Pierre Sandrine

Seb Anthony

Mes premiers remerciements s'adressent à mon directeur de thèse Patrick Collombat, sans qui ce joli projet n'aurait pas vu le jour. Merci de m'avoir fait confiance et de m'avoir accueillie dans ton équipe !

FIGURE INDEX

INTRODUCTION I. Diabetes Mellitus

Affecting approximately 422 million people worldwide ("Worldwide trends in diabetes since 1980," 2016), diabetes mellitus figures among the most common metabolic conditions. The prevalence of diabetes mellitus is increasing and predicted to rise to 552 million affected people by 2030 [START_REF] Whiting | IDF Diabetes Atlas: Global estimates of the prevalence of diabetes for 2011 and 2030[END_REF]. Despite therapies and prevention strategies, diabetes is one of the four main chronic diseases and was the direct cause of approximately 1.5 million deaths in 2012 ("WHO | Global report on diabetes").

Due to a failure of insulin production or action, diabetes is responsible for longlasting elevated blood glucose levels, such chronic hyperglycemia being associated with various complications. Indeed, diabetes is reported to be the main cause of blindness and increases the risk of cardiovascular diseases, nephropathy, stroke and amputation [START_REF] Alwan | Global status report on noncommunicable diseases[END_REF][START_REF] Morrish | Mortality and causes of death in the WHO Multinational Study of Vascular Disease in Diabetes[END_REF][START_REF] Pascolini | Global estimates of visual impairment: 2010[END_REF].

Diabetes patients are also at risk of dying prematurely [START_REF] Manuel | Health-Related Quality of Life and Health-Adjusted Life Expectancy of People with Diabetes on Ontario, Canada, 1996-1997[END_REF];

Type 1 Diabetes Mellitus (T1DM)

Formerly known as juvenile diabetes or insulin-dependent diabetes, Type 1

Diabetes Mellitus (T1DM) represents approximately 10% of all diabetic patients. Two peaks of disease onset have been reported: the first between 6 and 15 years of age and the second later in adolescence, typically before 30 years [START_REF] Herold | Type 1 diabetes: translating mechanistic observations into effective clinical outcomes[END_REF].

T1DM is a polygenic disorder characterized by a cytolytic T-cell mediated autoimmune-destruction of the insulin-producing -cells [START_REF] Bottazzo | Islet-cell antibodies in diabetes mellitus with autouimmne polyendocrine deficiencies[END_REF][START_REF] Gepts | Pathologic anatomy of the pancreas in juvenile diabetes mellitus[END_REF][START_REF] Kolb | Autoimmune versus inflammatory type I diabetes: a controversy?[END_REF], such selective loss leading to a lack of insulin secretion subsequently resulting in high blood glucose levels (Figure 1A-B).

While the exact causes for this autoimmune-mediated cell destruction are not yet known, a genetic component in the development of T1DM is well documented.

The first described susceptibility locus associated with T1DM was the human leukocyte antigen (HLA) gene [START_REF] Nerup | HL-A antigens and diabetes mellitus[END_REF][START_REF] Singal | Histocompatibility (HL-A) Antigens, Lymphocytotoxic Antibodies and Tissue Antibodies in Patients with Diabetes Mellitus[END_REF].

Other genes outside the HLA locus and strongly associated with T1DM have also been identified. Indeed, polymorphisms in the insulin [START_REF] Rotwein | Genetic analysis of the hypervariable region flanking the human insulin gene[END_REF], PTPN22 [START_REF] Bottini | A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes[END_REF], CTLA-4 [START_REF] Ueda | Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease[END_REF] and IL-2RA [START_REF] Maier | IL2RA genetic heterogeneity in multiple sclerosis and type 1 diabetes susceptibility and soluble interleukin-2 receptor production[END_REF] genes are frequently associated with T1DM. In addition to genetic influences, the rapid rise in T1DM incidence suggests the contribution of environmental [START_REF] Atkinson | Cellular immunity to a determinant common to glutamate decarboxylase and Coxsackie virus in insulin-dependent diabetes[END_REF][START_REF] Gillespie | The rising incidence of childhood type 1 diabetes and reduced contribution of high-risk HLA haplotypes[END_REF][START_REF] Ziegler | Accelerated progression from islet autoimmunity to diabetes is causing the escalating incidence of type 1 diabetes in young children[END_REF] or viral [START_REF] Ginsberg-Fellner | Diabetes mellitus and autoimmunity in patients with the congenital rubella syndrome[END_REF][START_REF] Honeyman | Association between rotavirus infection and pancreatic islet autoimmunity in children at risk of developing type 1 diabetes[END_REF] stimuli in the development of this metabolic disease.

The progressive destruction of the -cells by the immune system of T1DM patients leads to the production of autoantibodies directed towards several antigens, including GAD65 [START_REF] Baekkeskov | Identificationof the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase[END_REF][START_REF] Solimena | Autoantibodies to glutamic acid decarboxylase in a patient with stiff-man syndrome, epilepsy, and type I diabetes mellitus[END_REF][START_REF] Towns | GAD65 autoantibodies and its role as biomarker of Type 1 diabetes and Latent Autoimmune Diabetes in Adults (LADA)[END_REF], insulin [START_REF] Palmer | Insulin antibodies in insulin-dependent diabetics before insulin treatment[END_REF][START_REF] Wegmann | Insulin-specific T cells are a predominant component of islet infiltrates in pre-diabetic NOD mice[END_REF] and proinsulin [START_REF] Kuglin | Evidence of IgG autoantibodies against human proinsulin in patients with IDDM before insulin treatment[END_REF], ICA512 [START_REF] Rabin | Cloning and expression of IDDM-specific human autoantigens[END_REF] and ZNT8 [START_REF] Wenzlau | The cation efflux transporter ZnT8 (Slc30A8) is a major autoantigen in human type 1 diabetes[END_REF].

Easily detectable in the serum, autoantibodies represent robust hallmarks for T1DM

diagnosis [START_REF] Bingley | Prediction of IDDM in the general population: Strategies based on combinations of autoantibody markers[END_REF][START_REF] Riley | A prospective study of the development of diabetes in relatives of patients with insulin-dependent diabetes[END_REF][START_REF] Verge | Prediction of type I diabetes in first-degree relatives using a combination of insulin, GAD, and ICA512bdc/IA-2 autoantibodies[END_REF]. insulin is released from pancreatic -cells, leading to glucose uptake by target tissues and normoglycemia (A). In type 1 diabetic patients, the loss of -cells in pancreatic islets induces a deficiency in insulin secretion and a lack of glucose uptake, further resulting in high blood glucose levels (B). In type 2 diabetic patients, insulin is secreted but a defect in its action on glucose uptake by target tissues results in hyperglycemia (C).

Even though symptoms usually do not appear before at least 80% of the -cell mass has been destroyed [START_REF] Gillespie | Type 1 diabetes: pathogenesis and prevention[END_REF], the final absolute destruction of these cells leads to the dependence on exogenous insulin administration for T1DM patient survival.

Type 2 Diabetes Mellitus (T2DM)

Type 2 Diabetes Mellitus (T2DM), previously known as adult-onset diabetes or non-insulin dependent diabetes, is the most common type of diabetes. Traditionally associated with adults, T2DM is a progressive condition characterized by insulin resistance due to defective insulin action on peripheral target tissues (liver, adipose tissue, muscles -Figure 1A,C). This sustained insulin resistance is accompanied by a -cell compensation in order to increase insulin secretion. However, such adjustments can result, in time, in a failure in -cell function [START_REF] Kahn | The importance of the beta-cell in the pathogenesis of type 2 diabetes mellitus[END_REF], further contributing to the loss of -cells and impairment of insulin secretion. The subsequent reduced -cell mass in T2DM patients highlights such failure in -cell compensation [START_REF] Klöppel | Islet pathology and the pathogenesis of type 1 and type 2 diabetes mellitus revisited[END_REF].

T2DM is a complex disease caused by genetic variations acting in concert with environmental factors. Indeed, the IRS-1 and PPAR genes are highly polymorphic in patients suffering from T2DM [START_REF] Andrulionytè | Common polymorphisms of the PPAR-gamma2 (Pro12Ala) and PGC-1alpha (Gly482Ser) genes are associated with the conversion from impaired glucose tolerance to type 2 diabetes in the STOP-NIDDM trial[END_REF][START_REF] Kahn | Genetics of non-insulin-dependent (type-II) diabetes mellitus[END_REF]. Furthermore, several environmental factors influence T2DM prevalence [START_REF] Hu | Television watching and other sedentary behaviors in relation to risk of obesity and type 2 diabetes mellitus in women[END_REF][START_REF] Murea | Genetic and environmental factors associated with type 2 diabetes and diabetic vascular complications[END_REF], among which obesitytriggered inflammation, which was proposed to be the major risk factor in this disease [START_REF] Haffner | Epidemiology of type 2 diabetes: risk factors[END_REF]. In addition, endoplasmic reticulum stress, due to high fat diet, has been shown to have an important impact in insulin signaling impairment and in -cell dysfunction [START_REF] Cnop | Endoplasmic reticulum stress, obesity and diabetes[END_REF][START_REF] Eizirik | ER stress in pancreatic beta cells: the thin red line between adaptation and failure[END_REF][START_REF] Eizirik | The role for endoplasmic reticulum stress in diabetes mellitus[END_REF].

Treatment of T2DM mostly focuses on lowering blood glucose levels by lifestyle adjustments, such as an adapted diet combined with increased physical activity [START_REF] Eriksson | Prevention of type 2 (non-insulin-dependent) diabetes mellitus by diet and physical exercise. The 6-year Malmö feasibility study[END_REF][START_REF] Pan | Effects of Diet and Exercise in Preventing NIDDM in People with Impaired Glucose Tolerance[END_REF]. Although less effective than lifestyle changes, oral hypoglycemic agents can also be used to manage T2DM hyperglycemia [START_REF] Knowler | Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin[END_REF][START_REF] Ripsin | Management of blood glucose in type 2 diabetes mellitus[END_REF]. Insulin therapy can eventually be considered, if previously mentioned strategies are not effective or in advanced stages of this disease.

Gestational diabetes

Gestational diabetes is a metabolic disorder with a prevalence of 9.2% [START_REF] Desisto | Prevalence estimates of gestational diabetes mellitus in the United States, Pregnancy Risk Assessment Monitoring System (PRAMS), 2007-2010[END_REF] during the 3 rd trimester of pregnancy. Reported risk factors include maternal age and obesity [START_REF] Solomon | A prospective study of pregravid determinants of gestational diabetes mellitus[END_REF]. During gestation, the action of human placental lactogen and other hormones, such as prolactin, may interfere with insulin signaling [START_REF] Carr | Gestational Diabetes: Detection, Management, and Implications[END_REF][START_REF] Sorenson | Adaptation of islets of Langerhans to pregnancy: betacell growth, enhanced insulin secretion and the role of lactogenic hormones[END_REF] leading to insulin resistance. Moreover, although insulin does not pass through the placental barrier, glucose does and can stimulate the fetus to produce the required insulin. This excess of insulin can result in neonatal hypoglycemia while glucose surplus can lead to macrosomia [START_REF] Wendland | Gestational diabetes and pregnancy outcomes--a systematic review of the World Health Organization (WHO) and the International Association of Diabetes in Pregnancy Study Groups (IADPSG) diagnostic criteria[END_REF][START_REF] Wong | The clinical significance of overt diabetes in pregnancy[END_REF]. In addition, the incidence of obesity and T2DM is increased in these children [START_REF] Dabelea | Association of intrauterine exposure to maternal diabetes and obesity with type 2 diabetes in youth: The SEARCH case-control study[END_REF].

Although gestational diabetes is reversed within 48 hours after delivery for 90% of women [START_REF] Kahn | Type 2 diabetes: When insulin secretion fails to compensate for insulin resistance[END_REF], it increases the risk of the mother developing T2DM [START_REF] Bellamy | Type 2 diabetes mellitus after gestational diabetes: a systematic review and meta-analysis[END_REF]. Moderate physical exercise, improved diet and eventually, exogenous insulin injections can be used to prevent and control gestational diabetes [START_REF] Alwan | Treatments for gestational diabetes[END_REF].

Monogenic diabetes

As described above, Type 1, Type 2 and gestational diabetes are polygenic diseases. However, rare monogenic forms of diabetes exist and represent 1-2% of diabetes cases [START_REF] Eide | Prevalence of HNF1A (MODY3) mutations in a Norwegian population (the HUNT2 Study)[END_REF]. MODY (Maturity Onset Diabetes of the Young)

represents the main subtype of monogenic diabetes and generally occurs before 25 years of age [START_REF] Tattersall | A difference between the inheritance of classical juvenile-onset and maturity-onset type diabetes of young people[END_REF]. This condition can be due to inherited or spontaneous mutations in different genes, such as HNF-4, HNF-1 or HNF-1 [START_REF] Ellard | Hepatocyte nuclear factor 1 alpha (HNF-1a) mutations in maturity-onset diabetes of the young[END_REF].

Neonatal diabetes mellitus (NDM), another monogenic form of diabetes, commonly occurs before 6 months of age [START_REF] Schwitzgebel | Many faces of monogenic diabetes[END_REF]. It can be transient or permanent and is generally due to mutations in the ABCC8 and KCNJ11 genes, involved in insulin signaling pathway [START_REF] Flanagan | Mutations in ATP-sensitive K+ channel genes cause transient neonatal diabetes and permanent diabetes in childhood or adulthood[END_REF].

II. Pancreas anatomy and function

Apparently initially discovered by Herophilus, a Greek anatomist, in approximately 300 BC, the organ was named "pancreas" by Ruphos of Ephesus around the first century AD. Translated "all flesh" from the Greek [pan: all and kreas: flesh], probably due to the consistency of the tissue [START_REF] Busnardo | History of the pancreas[END_REF], the pancreas has also been called "the hermit organ" because of its hidden location [START_REF] Gavaghan | The Pancreas-Hermit of the Abdomen[END_REF].

Gross anatomy of the pancreas

The pancreas is a deep abdominal gland, located behind the stomach and connected to the duodenum of the small intestine (Figure 2).

The human pancreas is divided into different anatomic parts: the head, the body and the tail [START_REF] Bockman | Anatomy of the pancreas[END_REF]. While the head of the pancreas is situated near the duodenum, the tail part lies close to the hilum of the spleen. The body segment stretches beneath the stomach [START_REF] Dolensek | Structural similarities and differences between the human and the mouse pancreas[END_REF]-Figure 2). Of note, the mouse pancreas is more diffuse within the abdomen and is segmented into the duodenal, splenic and gastric lobes [START_REF] Dolensek | Structural similarities and differences between the human and the mouse pancreas[END_REF]. The pancreas is an amphicrine gland, thus composed of two compartments: the exocrine tissue and the endocrine tissue, each with distinct structures and functions (Figure 2). Responsible for the secretion of various enzymes and hormones, it has a preponderant role in food digestion and glucose homeostasis [START_REF] Cano | Pancreatic Development and Disease[END_REF][START_REF] Slack | Developmental biology of the pancreas[END_REF].

Organization and function of the exocrine tissue

Representing the major component of the gland, the exocrine compartment accounts for approximately 95% of the total organ [START_REF] Rahier | Cell populations in the endocrine pancreas of human neonates and infants[END_REF]. The exocrine pancreas is involved in two physiological processes: the regulation of gastric acid and chemical digestion. It is mostly composed of serous acinar cells and ductal cells forming a branched network throughout the gland [START_REF] Edlund | Organogenesis: Pancreatic organogenesis -developmental mechanisms and implications for therapy[END_REF].

a. Acinar cells

Pancreatic acini are made of pyramidal acinar cells containing zymogen granules within secretory vesicles. These exocrine acinar cells produce and secrete a juice composed of several enzymes (i.e. lipases, amylase, trypsin) involved in food digestion [START_REF] Tan | The pancreas[END_REF]. Combined, these enzymes hydrolyze complex nutrients, such as polysaccharides, triglycerides and proteins [START_REF] Whitcomb | Human pancreatic digestive enzymes[END_REF].

Pancreatic enzymes are released as pancreatic juice into the duodenum through the branched ductal network.

b. Duct cells

Pancreatic ductal cells are organized into a complex network running throughout the entire pancreas. Acinar cell secretions are collected by intercalated ducts and siphoned into intralobular ducts; this pancreatic juice is drained into the duodenum at the papilla of Vater through the main pancreatic duct also called duct of Wirsung [START_REF] Reichert | Pancreatic ductal cells in development, regeneration, and neoplasia[END_REF]-Figure 2).

To regulate duodenal acidity, ductal cells produce water and bicarbonate which is added to the enzyme mixture, thus providing optimal conditions for enzyme activity [START_REF] Githens | The Pancreatic Duct Cell: Proliferative Capabilities, Specific Characteristics, Metaplasia, Isolation, and Culture[END_REF].

Organization and function of the endocrine tissue

The endocrine compartment of the pancreas is composed of small functional units named islets of Langerhans. Originally described by Paul Langerhans in his thesis in 1869 [START_REF] Langerhans | Beitrage zur Mikroskopischen Anatomie der Bauchspeicheldruse[END_REF], the islets represent about 4-5% of the pancreatic mass and are found scattered throughout the exocrine tissue [START_REF] Ionescu-Tirgoviste | A 3D map of the islet routes throughout the healthy human pancreas[END_REF][START_REF] Rahier | Cell populations in the endocrine pancreas of human neonates and infants[END_REF]. However, regional islet distribution in the different parts of the pancreas is subject to controversy. Indeed, while reports suggest the highest number of mouse or human islets in the pancreatic tail [START_REF] Wang | Regional Differences in Islet Distribution in the Human Pancreas -Preferential Beta-Cell Loss in the Head Region in Patients with Type 2 Diabetes[END_REF], other studies point to a similar enrichment in the head of the pancreas [START_REF] Hörnblad | An improved protocol for optical projection tomography imaging reveals lobular heterogeneities in pancreatic islet and β-cell mass distribution[END_REF].

Islets of Langerhans are arranged into clusters containing five hormonesecreting cell types: -cells, -cells, -cells, PP-cells and -cells; respectively responsible for the secretion of insulin, glucagon, somatostatin, pancreatic polypeptide (PP) and ghrelin [START_REF] Adrian | Pancreatic polypeptide, glucagon and insulin secretion from the isolated perfused canine pancreas[END_REF][START_REF] Prado | Ghrelin cells replace insulin-producing beta cells in two mouse models of pancreas development[END_REF][START_REF] Roncoroni | Effect of somatostatin on exocrine pancreas evaluated on a total external pancreatic fistula of neoplastic origin[END_REF])-Figures 23). Interestingly, variations in the microarchitecture of islets have been observed between species. Indeed, while a central core of -cells surrounded by a mantle of non--cells is typical for mouse islets (Figure 3B), human islets appear more unorganized with non--cells dispersed within the -cell mass [START_REF] Brissova | Assessment of Human Pancreatic Islet Architecture and Composition by Laser Scanning Confocal Microscopy[END_REF][START_REF] Cabrera | The unique cytoarchitecture of human pancreatic islets has implications for islet cell function[END_REF][START_REF] Steiner | Pancreatic islet plasticity: interspecies comparison of islet architecture and composition[END_REF]-Figure 3A). Of note, recent reports argue the possibility of finding human islets displaying mouse islet organization and vice versa, depending on physiological variations, such as the increase in insulin demand observed during pregnancy [START_REF] Kharouta | No mantle formation in rodent islets-The prototype of islet revisited[END_REF][START_REF] Kilimnik | Quantification of islet size and architecture[END_REF][START_REF] Kim | Islet architecture: a comparative study[END_REF]. Other variations, such as endocrine cell proportions within the islets, are also observed between organisms. Indeed, while human islets contain approximately 50-70% -cells, 20-40% of -cells and less than 5% of -cells, mouse islets display 60-80%, 10-20% and less than 5% -, -and cells respectively [START_REF] Andralojc | Ghrelin-producing epsilon cells in the developing and adult human pancreas[END_REF][START_REF] Steiner | Pancreatic islet plasticity: interspecies comparison of islet architecture and composition[END_REF]-Figure 4). Critical for glucose homeostasis regulation, pancreatic islets are densely vascularized allowing the direct secretion of hormones into the blood circulation [START_REF] Brissova | Pancreatic islet production of vascular endothelial growth factor-A is essential for islet vascularization, revascularization, and function[END_REF][START_REF] Lifson | Relation between blood flow and morphology in islet organ of rat pancreas[END_REF]. Blood glucose levels are finely tuned to be maintained within normal ranges, corresponding to <6.1 mmol/l in a fasted state (Table1).

a. Insulin-secreting -cells

Pancreatic -cells represent the most abundant cell type in islets with 50-70% in humans and 60-80% in mice [START_REF] Dolensek | Structural similarities and differences between the human and the mouse pancreas[END_REF]-Figure 4). Synthesizing and secreting the hypoglycemic hormone insulin, they play a key role in regulating glucose homeostasis. Insulin is a protein of 51 amino acids, composed of two polypeptide chains linked by disulfide bonds resulting from the cleavage of proinsulin by the action of prohormone convertases PC1/3 and PC2 [START_REF] Steiner | Insulin biosynthesis: evidence for a precursor[END_REF][START_REF] Weiss | Insulin Biosynthesis, Secretion, Structure, and Structure-Activity Relationships[END_REF]. Stored as granules in secretory vesicles, insulin is secreted upon high glucose blood levels. Indeed, glucose enters -cells via the low affinity GLUT2

transporter expressed at the cell membrane, its assimilation leading to an increase in intracellular ATP concentrations. This rise causes the closure of KATP channels, membrane depolarization and the opening of Ca 2+ channels. The influx of Ca 2+ leads to the release of insulin by exocytosis into the blood [START_REF] Ashcroft | Glucose induces closure of single potassium channels in isolated rat pancreatic beta-cells[END_REF][START_REF] Larsson | Oscillations in KATP channel activity promote oscillations in cytoplasmic free Ca2+ concentration in the pancreatic beta cell[END_REF][START_REF] Nichols | Adenosine diphosphate as an intracellular regulator of insulin secretion[END_REF]-Figure 5).

Insulin travels to its target tissues through the bloodstream (liver, adipose tissue, skeletal muscles) where it interacts with its receptor (IR; insulin receptor) present at the membrane of target cells and activates its own signaling pathway.

Activation of the insulin signaling pathway leads to the recruitment to the plasma membrane of the glucose transporters to facilitate uptake and storage of circulating glucose. In this manner, insulin stimulates glucose absorption by muscle and adipose tissue cells but also glycolysis and glycogenesis in the liver to store glucose as glycogen and decrease glycemia. In addition, insulin inhibits hepatic glycogenolysis and gluconeogenesis to block glycogen catabolism and the synthesis of glucose. [START_REF] Ashcroft | K ATP channels and islet hormone secretion : new insights and controversies[END_REF]. Insulin secretion is inhibited at low glucose concentrations. At high glucose levels, glucose enters the -cell through its receptor GLUT2, increasing intracellular ATP concentration further leading to the closure of KATP channels, the depolarization of the plasma membrane and the opening of the Ca 2+ channels. As a result, the intracellular concentration of Ca2 + increases causing the exocytosis of insulin granules.

Although glucose is the main stimulus involved in insulin secretion, other molecules have been shown to induce insulin release, such as amino acids, acetylcholine or cholecystokinin [START_REF] Hermans | Modulation of the effect of acetylcholine on insulin release by the membrane potential of B cells[END_REF][START_REF] Karlsson | Effects of three different cholecystokinin receptor antagonists on basal and stimulated insulin and glucagon secretion in mice[END_REF][START_REF] Zhang | Mechanisms of amino acid-stimulated insulin secretion in congenital hyperinsulinism[END_REF]. Moreover, somatostatin has been shown to inhibit insulin secretion [START_REF] Hauge-Evans | Somatostatin secreted by islet deltacells fulfills multiple roles as a paracrine regulator of islet function[END_REF].

b. Glucagon-secreting -cells

Representing 20-40% and 10-20% of human and mouse total endocrine cell type respectively [START_REF] Dolensek | Structural similarities and differences between the human and the mouse pancreas[END_REF], -cells secrete the hormone glucagon, a critical actor in the regulation of glucose homeostasis. Indeed, as a hyperglycemic hormone, glucagon plays the antagonist role compared to insulin in the maintenance of blood glucose levels within normal ranges [START_REF] Unger | Glucagon physiology and pathophysiology[END_REF]. In accordance with a common feature of hormones, glucagon is originally synthesized as a precursor named proglucagon. This precursor will be processed in pancreatic -cells to produce the 29-amino acid peptide glucagon [START_REF] Furuta | Severe Defect in Proglucagon Processing in Islet A-cells of Prohormone Convertase 2 Null Mice[END_REF], stored as granules in secretory vesicles.

The exocytosis of glucagon is induced when blood sugar levels drop, during fasting or increased energy expenditure, to promote target tissues to mobilize glucose and therefore increase glycemic levels. In the liver, glucagon interacts with its membrane-bound receptor -a G-protein coupled receptor - [START_REF] Authier | Glucagon receptors[END_REF][START_REF] Brubaker | Structure-function of the glucagon receptor family of G protein-coupled receptors: the glucagon, GIP, GLP-1, and GLP-2 receptors[END_REF] to promote hepatic gluconeogenesis and glycogenolysis, therefore stimulating the breakdown of glycogen [START_REF] Habegger | The metabolic actions of glucagon revisited[END_REF].

Regulation of glucagon secretion is driven by various signals. Glucagon release is inhibited by glucose, insulin and somatostatin [START_REF] Hamaguchi | Abnormal glucagon response to arginine and its normalization in obese hyperinsulinaemic patients with glucose intolerance: importance of insulin action on pancreatic Alpha cells[END_REF][START_REF] Hauge-Evans | Somatostatin secreted by islet deltacells fulfills multiple roles as a paracrine regulator of islet function[END_REF][START_REF] Kisanuki | Expression of insulin receptor on clonal pancreatic alpha cells and its possible role for insulin-stimulated negative regulation of glucagon secretion[END_REF]. Moreover, other stimuli such as circulating amino acids [START_REF] Pagliara | Glucose modulation of amino acid-induced glucagon and insulin release in the isolated perfused rat pancreas[END_REF] and fatty acids [START_REF] Bollheimer | Stimulatory short-term effects of free fatty acids on glucagon secretion at low to normal glucose concentrations[END_REF] have been shown to impact the secretion of glucagon.

c. Somatostatin-secreting -cells

Pancreatic -cells make up less than 10% and 5% of endocrine cell types in human and mouse islets, respectively [START_REF] Dolensek | Structural similarities and differences between the human and the mouse pancreas[END_REF]. -cells secrete somatostatin (SST), also known as growth hormone-inhibiting hormone (GHIH) or somatotropin release-inhibiting factor (SRIF).

Somatostatin exists as two isoforms, somatostatin-14 (SST-14) and somatostatin-28 (SST-28), resulting from different cleavage of the prosomatostatin [START_REF] Andrews | Biosynthesis and processing of the somatostatin family of peptide hormones[END_REF]. Binding somatostatin receptors (members of the Gprotein coupled receptor family which comprise five subtypes -SSTR1-5 - [START_REF] Yamada | Cloning, functional expression and pharmacological characterization of a fourth (hSSTR4) and a fifth (hSSTR5) human somatostatin receptor subtype[END_REF], SST-14 and SST-28 are able to exert a wide range of effects.

Typically, somatostatin is described as a paracrine and endocrine peptide as it can induce the inhibition of the secretion of numerous hormones of the gastrointestinal tract, the nervous system and the pancreas [START_REF] Dollinger | Effects of somatostatin on exocrine and endocrine pancreatic function stimulated by intestinal hormones in man[END_REF]. In addition to pancreatic -cells, somatostatin is synthetized by cells in the central nervous and gastrointestinal system (Reichlin, 1983). While tissue distribution of SST-14 and SST-28 is still debated, it has been suggested that the two peptides have different inhibitory potentials. SST-14 strongly inhibits glucagon secretion through SSTR2 while SST-28 mostly blocks insulin release through SSTR5 [START_REF] D'alessio | A physiologic role for somatostatin 28 as a regulator of insulin secretion[END_REF][START_REF] Mandarino | Selective effects of somatostatin-14, -25 and -28 on in vitro insulin and glucagon secretion[END_REF][START_REF] Mitra | Colocalization of somatostatin receptor sst5 and insulin in rat pancreatic beta-cells[END_REF]. However, recent analyses suggested that SSTR5 is not expressed in islet endocrine cells [START_REF] Digruccio | Comprehensive alpha, beta and delta cell transcriptomes reveal that ghrelin selectively activates delta cells and promotes somatostatin release from pancreatic islets[END_REF]. Various stimuli induce somatostatin secretion, such as low concentrations of glucose, fatty acids, HCL and urocortin 3 [START_REF] Rouiller | Release of pancreatic and gastric somatostatin-like immunoreactivity in response to the octapeptide of cholecystokinin, secretin gastric inhibitory polypeptide, and gastrin-17 in dogs[END_REF][START_REF] Schusdziarra | Pancreatic and gastric somatostatin release in response to intragastric and intraduodenal nutrients and HCl in the dog[END_REF][START_REF] Van Der Meulen | Urocortin3 mediates somatostatin-dependent negative feedback control of insulin secretion[END_REF].

d. Pancreatic polypeptide-secreting PP-cells

Representing less than 5 and 1% of human and mouse total endocrine cells respectively, PP-cells are more abundant in the islets located in head region of the pancreas [START_REF] Baetens | Endocrine pancreas: threedimensional reconstruction shows two types of islets of langerhans[END_REF][START_REF] Gingerich | Regional pancreatic concentration and in-vitro secretion of canine pancreatic polypeptide, insulin, and glucagon[END_REF]. PP-cells secrete pancreatic polypeptide (PP), a peptide of 36 amino acids and a member of the neuropeptide Y (NPY) family [START_REF] Holzer | Neuropeptide Y, peptide YY and pancreatic polypeptide in the gut-brain axis[END_REF]. Pancreatic polypeptide is initially synthesized as a precursor named pre-propancreatic polypeptide before further processing to produce the hormone [START_REF] Boel | A cDNA encoding a small common precursor for human pancreatic polypeptide and pancreatic icosapeptide[END_REF][START_REF] Leiter | Structure of a precursor to human pancreatic polypeptide[END_REF].

Principally secreted postprandially [START_REF] Batterham | Pancreatic Polypeptide Reduces Appetite and Food Intake in Humans[END_REF], PP is involved in gastrointestinal motility, food intake, energy metabolism and gastric secretions [START_REF] Asakawa | Characterization of the effects of pancreatic polypeptide in the regulation of energy balance[END_REF][START_REF] Katsuura | Roles of pancreatic polypeptide in regulation of food intake[END_REF][START_REF] Lin | Bovine pancreatic peptide: action on gastric and pancreatic secretion in dogs[END_REF]. The precise role of PP in the pancreas is not yet evident, although its implication in the regulation of pancreatic exocrine secretions is well documented [START_REF] Lonovics | Pancreatic polypeptide. A review[END_REF][START_REF] Louie | Action of pancreatic polypeptide on rat pancreatic secretion: in vivo and in vitro[END_REF][START_REF] Putnam | Inhibitory regulation of rat exocrine pancreas by peptide YY and pancreatic polypeptide[END_REF]. Moreover, it has been suggested a role for PP in the stimulation of insulin secretion via the inhibition of somatostatin release [START_REF] Kim | Pancreatic polypeptide inhibits somatostatin secretion[END_REF].

e. Ghrelin-secreting -cells

Pancreatic -cells are abundantly present in the fetal pancreas [START_REF] Rindi | Characterisation of gastric ghrelin cells in man and other mammals: Studies in adult and fetal tissues[END_REF][START_REF] Wierup | Ghrelin is expressed in a novel endocrine cell type in developing rat islets and inhibits insulin secretion from INS-1 (832/13) cells[END_REF] but constitute less than 1% of the endocrine cells in adult human and mouse islets [START_REF] Andralojc | Ghrelin-producing epsilon cells in the developing and adult human pancreas[END_REF][START_REF] Dolensek | Structural similarities and differences between the human and the mouse pancreas[END_REF]. Ghrelin, the hormone secreted by the pancreatic -cells, is a 28 amino acid peptide resulting from two successive cleavages of the precursor pre-proghrelin and is mainly produced and secreted by gastrointestinal cells upon food intake [START_REF] Cummings | A Preprandial Rise in Plasma Ghrelin Levels Suggests a Role in Meal Initiation in Humans[END_REF]. This hormone is involved in numerous physiological processes, such as regulation of appetite, body weight, gastrointestinal motility, gastric secretions and control of anxiety (Müller et al., 2015).

The role of ghrelin in the pancreas and on insulin secretion is still debated as it has been shown to stimulate or suppress insulin release depending on its plasma concentration [START_REF] Granata | Unraveling the role of the ghrelin gene peptides in the endocrine pancreas[END_REF][START_REF] Salehi | Effects of ghrelin on insulin and glucagon secretion: A study of isolated pancreatic islets and intact mice[END_REF]. Subject to controversy is the hypothesis that ghrelin could influence somatostatin, glucagon and PP secretion [START_REF] Arosio | Stimulatory effects of ghrelin on circulating somatostatin and pancreatic polypeptide levels[END_REF][START_REF] Egido | Inhibitory effect of ghrelin on insulin and pancreatic somatostatin secretion[END_REF][START_REF] Salehi | Effects of ghrelin on insulin and glucagon secretion: A study of isolated pancreatic islets and intact mice[END_REF]. However, a recent report suggested a role of ghrelin in potentiating glucose-stimulated somatostatin secretion in mouse and human islets [START_REF] Digruccio | Comprehensive alpha, beta and delta cell transcriptomes reveal that ghrelin selectively activates delta cells and promotes somatostatin release from pancreatic islets[END_REF].

III. Mouse pancreatic development

Our current knowledge of pancreatic development mostly results from information gleamed using loss-and/or gain-of-function mouse models and genetic lineage-tracing experiments. It is important to mention that, while sharing similarities, rodent and human pancreas development also show divergences [START_REF] Sarkar | Global gene expression profiling and histochemical analysis of the developing human fetal pancreas[END_REF]. Pancreas development is a highly complex process, requiring diverse interactions between tissues, genes and soluble factors. An elegant approach using whole-mount immunofluorescence experiments and global gene expression analyses on endodermal domains suggested that pancreas organogenesis is mostly dependent on combinations of large cohort of transcription factors (TFs) and signaling pathways [START_REF] Sherwood | Transcriptional Dynamics of Endodermal Organ Formation[END_REF]. Of note, epigenetic modifications collaborate with transcription factors to determine pancreatic cell fates [START_REF] Haumaitre | Histone deacetylase inhibitors modify pancreatic cell fate determination and amplify endocrine progenitors[END_REF][START_REF] Xu | Chromatin "prepattern" and histone modifiers in a fate choice for liver and pancreas[END_REF], although this aspect will not be discussed in this manuscript.

Here, we will describe key events and TFs involved in mouse pancreas organogenesis.

Early development a. Endoderm patterning

During embryogenesis, following fertilization, the zygote undergoes multiple cycles of mitotic divisions until the formation of the blastocyst. The blastocyst will implant into the uterine wall at which point the process of gastrulation begins. In vertebrates, gastrulation leads to the formation of three germ layers identified as ectoderm, mesoderm and endoderm, each layer giving rise to distinct organs. In mice, at embryonic day (E) 6-7.5, the endoderm initially appears as a flat single-cell layer epithelium which will then, 2 days later (E8.5), internalize and fold to form the primitive gut tube.

b. Gut tube patterning and pancreas budding

Upon stimulation by soluble factors originating from the adjacent germ layers, particularly the mesoderm, the primitive gut tube will be regionalized into different segments along the anterior-posterior and dorsal-ventral axes, later giving rise to all endoderm-derived organs [START_REF] Zorn | Vertebrate endoderm development and organ formation[END_REF]-Figure 6). This induction appears to be directed by numerous factors, in particular fibroblast growth factor (FGF)-4 [START_REF] Bayha | Retinoic acid signaling organizes endodermal organ specification along the entire antero-posterior axis[END_REF][START_REF] Hebrok | Hedgehog signaling in pancreas development[END_REF][START_REF] Weiss | Insulin Biosynthesis, Secretion, Structure, and Structure-Activity Relationships[END_REF]. [START_REF] Zorn | Vertebrate endoderm development and organ formation[END_REF]. Schematic representation of the gut tube of a mouse embryo at E10.5. The gut tube is divided into Foregut, Midgut and Hindgut segments through anterior-posterior and dorsal-ventral axes and will give rise to digestive and respiratory tracts and associated organs.

In vertebrates, the pancreas emerges from ventral and dorsal domains of the foregut endoderm (E9.5) [START_REF] Slack | Developmental biology of the pancreas[END_REF]. As a result of their localization along the gut tube, the two buds are not surrounded by the same tissues and are therefore subjected to different signals (Figure 7). The dorsal outgrowth of the pancreas receive permissive signals from the notochord and the dorsal aorta [START_REF] Hebrok | Notochord repression of endodermal Sonic hedgehog permits pancreas development[END_REF], whereas the ventral bud is subjected to cardiac mesoderm and septum transversum mesenchyme signals [START_REF] Kumar | Signals from lateral plate mesoderm instruct endoderm toward a pancreatic fate[END_REF].

Signals from the notochord, such as FGF, activin ligands and retinoic acid (RA), direct the adjacent endoderm to develop into the pancreatic dorsal bud [START_REF] Martín | Dorsal pancreas agenesis in retinoic acid-deficient Raldh2 mutant mice[END_REF], by repressing sonic hedgehog (Shh) expression [START_REF] Kim | Notochord to endoderm signaling is required for pancreas development[END_REF], an activator of hedgehog (Hh) pathway (Figure 7A). Repression of the signaling factor Shh results in the expression of Pdx1 [START_REF] Hebrok | Notochord repression of endodermal Sonic hedgehog permits pancreas development[END_REF], a key regulator of pancreas development. In addition, FGFs and BMPs signals from the cardiac mesoderm and the septum transversum mesenchyme are required for Pdx1 expression and ventral pancreatic bud specification [START_REF] Kumar | Signals from lateral plate mesoderm instruct endoderm toward a pancreatic fate[END_REF][START_REF] Wandzioch | Dynamic signaling network for the specification of embryonic pancreas and liver progenitors[END_REF]-Figure 7B).

Pancreas specification

Ventral and dorsal buds are morphologically detectable around E9.5 in mice.

However, as early as E8.5 and prior the outgrowth of these two primordia, the ventral and dorsal prepancreatic regions are specified. Indeed, Wessells and Cohen demonstrated the ability of cells in the foregut endoderm to differentiate into all pancreatic lineages [START_REF] Wessells | Early pancreas organogenesis: Morphogenesis, tissue interactions, and mass effects[END_REF]. It has also been suggested that such prepancreatic epithelium contains multipotent pancreatic progenitors (MPCs)

able to differentiate into all pancreatic cells [START_REF] Gu | Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors[END_REF]. Of note, pancreatic anlage specification is a dynamic process involving several transcription factors acting in coordination (Figure 9).

Pdx1 (pancreatic and duodenal homeobox 1) is a key regulator of pancreas development, its expression in progenitor cells (at E8.5) driving the differentiation of all exocrine and endocrine pancreatic cells. Mice lacking Pdx1 display bud formation but pancreas agenesis, demonstrating that Pdx1 is necessary for bud growth but not for their initial induction [START_REF] Jonsson | Insulin-promoter-factor 1 is required for pancreas development in mice[END_REF][START_REF] Offield | PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum[END_REF][START_REF] Stoffers | Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence[END_REF], suggesting that pancreatic development is initiated before Pdx1 expression.

Motor neuron and pancreas homeobox 1 (Mnx1), also known as Homeobox HB9

(HLXB9) precedes Pdx1 in the dorsal pancreatic endoderm as it is expressed on embryonic day 8 [START_REF] Harrison | Pancreas dorsal lobe agenesis and abnormal islets of Langerhans in Hlxb9 -deficient mice[END_REF][START_REF] Li | Selective agenesis of the dorsal pancreas in mice lacking homeobox gene Hlxb9[END_REF]. Its expression is necessary for dorsal bud induction, as HLXB9-deficient mice displayed dorsal lobe agenesis due to a lack of Pdx1 induction. In addition to Pdx1 and Mnx1 expression, early pancreatic specification requires numerous additional transcription factors, including

Ptf1a [START_REF] Burlison | Pdx-1 and Ptf1a concurrently determine fate specification of pancreatic multipotent progenitor cells[END_REF][START_REF] Kawaguchi | The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors[END_REF][START_REF] Krapp | The bHLH protein PTF1-p48 is essential for the formation of the exocrine and the correct spatial organization of the endocrine pancreas[END_REF], Sox9 [START_REF] Seymour | SOX9 is required for maintenance of the pancreatic progenitor cell pool[END_REF] and Hnf1 [START_REF] De Vas | Hnf1b controls pancreas morphogenesis and the generation of Ngn3+ endocrine progenitors[END_REF][START_REF] Haumaitre | Lack of TCF2/vHNF1 in mice leads to pancreas agenesis[END_REF].

Indeed, the mice deficient for each of these genes display varying degrees of pancreas hypoplasia or agenesis.

Importantly, mouse pancreas organogenesis is separated into two major waves : a primary and a secondary transitions [START_REF] Pictet | An ultrastructural analysis of the developing embryonic pancreas[END_REF][START_REF] Rutter | Regulation of specific protein synthesis in cytodifferentiation[END_REF].

a. Primary transition

The primary transition begins around E9.5 with the thickening of the foregut endoderm, which mainly contains MPCs. The shift from a monolayer to a multilayer epithelium induces the invasion of the surrounding mesenchyme and the formation of morphologically detectable ventral and dorsal buds [START_REF] Pictet | An ultrastructural analysis of the developing embryonic pancreas[END_REF] -Figure 8A). By E10.5, both ventral and dorsal epithelium undergo extensive growth and active proliferation of the MPCs, inducing epithelium stratification further resulting in the formation of microlumens [START_REF] Villasenor | Epithelial dynamics of pancreatic branching morphogenesis[END_REF]-Figure 8B). The following coalescence of these microlumens and branching morphogenesis will give rise to a large tubular network. At E12.5, as the gut rotates, the ventral and the dorsal bud fuse into a single organ [START_REF] Jørgensen | An illustrated review of early pancreas development in the mouse[END_REF]. Following buds fusion (by E12.5), the pancreatic epithelium undergoes dramatic morphogenetic reorganization, known as tip/trunk compartmentalization, leading to the formation of two domains (Figure 8C). Of note, Notch signaling appears to play a key role in the tip/trunk segregation [START_REF] Qu | Notch-mediated posttranslational control of Ngn3 protein stability regulates pancreatic patterning and cell fate commitment[END_REF].

b. Secondary transition

The secondary transition begins around E12.5-E13.5 and is marked by an extensive reorganization due to a massive growth and branching of the pancreatic epithelium, concomitant to a major wave of endocrine and exocrine cell differentiation.

The bipotent "trunk" region contains cells that will give rise to endocrine and ductal cells while the multipotent "tip" domain contains MPCs capable of differentiating into acinar, ductal and endocrine cells. The multipotent tip progenitors undergo a developmental switch at around E13-E14 and are then restricted to acinar progenitors [START_REF] Zhou | A Multipotent Progenitor Domain Guides Pancreatic Organogenesis[END_REF]. The specification of the different cell fates is driven by numerous genes and transcription factors, with Ptf1a and Nkx6.1 as key regulators of tip/trunk segregation (Figure 8D). Ptf1a is expressed in the tip cells while Nkx6.1 is exclusively expressed in trunk bipotent cells. Importantly, these two transcription factors mutually inhibit each other, favoring one or the other domain. It has been suggested that Ptf1a represses Nkx6.1 to block endocrine differentiation at the expense of acinar development [START_REF] Schaffer | Nkx6 transcription factors and Ptf1a function as antagonistic lineage determinants in multipotent pancreatic progenitors[END_REF].

Exocrine specification a. Acinar cell fate

Acinar cells arise from MPCs located in the tip cells of the pancreatic epithelium. This differentiation occurs between E14.5 and E15.5 in mice and acini are morphologically visible by E15.5.

Acinar cell specification is regulated by several transcription factors (Figure 9), including Ptf1a and Nr5a2 [START_REF] Esni | Notch inhibits Ptf1 function and acinar cell differentiation in developing mouse and zebrafish pancreas[END_REF][START_REF] Rose | The Role of PTF1-P48 in Pancreatic Acinar Gene Expression[END_REF]. As previously mentioned, Ptf1a-null mice display a pancreas agenesis. Interestingly, a total loss of the exocrine tissue is observed in these mice, while few endocrine cells remain [START_REF] Krapp | The p48 DNA-binding subunit of transcription factor PTF1 is a new exocrine pancreasspecific basic helix-loop-helix protein[END_REF]. In addition, the loss of Nr5a2 in adult mice results in impaired exocrine enzymes production and secretion [START_REF] Holmstrom | LRH-1 and PTF1-L coregulate an exocrine pancreas-specific transcriptional network for digestive function[END_REF]. Moreover, Mist1 is required for acinar fate maintenance and complete differentiation. Although

Mist1-deficient mice display acinar cells, these cells present defective apical-basal polarity leading to a massive disorganization of the exocrine tissue [START_REF] Direnzo | Induced Mist1 expression promotes remodeling of mouse pancreatic acinar cells[END_REF][START_REF] Pin | The bHLH transcription factor Mist1 is required to maintain exocrine pancreas cell organization and acinar cell identity[END_REF]. Interestingly, carbopeptidase (Cpa) is expressed by the forming acini at E12.5 and is considered to be an early exocrine marker [START_REF] Jørgensen | An illustrated review of early pancreas development in the mouse[END_REF]. Finally, recent studies described c-Myc as another acinar differentiation factor, as its inactivation in pancreatic progenitors leads to acinar cell hypoplasia [START_REF] Bonal | Pancreatic Inactivation of c-Myc Decreases Acinar Mass and Transdifferentiates Acinar Cells Into Adipocytes in Mice[END_REF][START_REF] Nakhai | Conditional inactivation of Myc impairs development of the exocrine pancreas[END_REF].

b. Ductal cell fate

The mechanisms involved in ductal cell fate specification are poorly understood. However, as previously mentioned, the "trunk" region contains bipotent cells able to give rise to endocrine and ductal cells. It has been suggested that the switch that induces trunk cells to derive into duct cells is mostly driven by Notch pathway (Figure 9). The activation of the Notch signaling pathway induces the expression of a target gene Hes1. Importantly, Hes1 has been found to act as a repressor of the proendocrine gene Neurogenin 3 (Neurog3). Indeed, Neurog3-deficiency is proposed to induce ductal cell specification [START_REF] Jensen | Control of endodermal endocrine development by Hes-1[END_REF][START_REF] Magenheim | Ngn3+ endocrine progenitor cells control the fate and morphogenesis of pancreatic ductal epithelium[END_REF] while

Hes1 mutant mice display an augmented differentiation of endocrine cells [START_REF] Jørgensen | An illustrated review of early pancreas development in the mouse[END_REF]. Moreover, the ductal epithelium expresses Sox9 [START_REF] Delous | sox9b is a key regulator of pancreaticobiliary ductal system development[END_REF], Hnf6 [START_REF] Pierreux | The Transcription Factor Hepatocyte Nuclear Factor-6 Controls the Development of Pancreatic Ducts in the Mouse[END_REF] and Hnf1 [START_REF] De Vas | Hnf1b controls pancreas morphogenesis and the generation of Ngn3+ endocrine progenitors[END_REF], these transcription factors forming a transcriptional network that has been involved in duct cell specification. Of note, the deletion of the corresponding genes leads to the impairment of duct morphogenesis.

Endocrine specification

Endocrine allocation and maintenance of the different endocrine cell lineages requires the sequential and stage-dependent activation or repression of specific genes. Indeed, endocrine cell specification is controlled by a network of transcription factors acting in specific combinations (Figure 9). The master gene involved in endocrine fate determination is Neurog3. Neurog3 is a proendocrine gene required for the development of all the endocrine cells, as Neurog3-deficient mice do not display any hormone-expressing endocrine cells [START_REF] Gradwohl | Neurogenin3 Is Required for the Development of the Four Endocrine Cell Lineages of the Pancreas[END_REF]. Both expression [START_REF] Shih | A Notch-dependent molecular circuitry initiates pancreatic endocrine and ductal cell differentiation[END_REF].

Neurog3 expression begins at E8.5 and peaks at E15.5, corresponding to the major wave of endocrine cell differentiation with little or no of this gene in hormonesexpressing cells [START_REF] Schwitzgebel | Expression of neurogenin3 reveals an islet cell precursor population in the pancreas[END_REF]. Previous reports demonstrated that

Neurog3 initiates endocrine specification but acts upstream of numerous transcription factors important for endocrine cell differentiation/maintenance. Indeed, using genedeficient mouse models and further genetic analyses, it has been shown that Insm1, Rfx6, Isl1, NeuroD1, Pax6, Myt1 are required for proper endocrine-cell differentiation.

The deletion of one of these gene impact in different manner final islet-cell differentiation [START_REF] Ahlgren | Independent requirement for ISL1 in formation of pancreatic mesenchyme and islet cells[END_REF][START_REF] Mellitzer | IA1 is NGN3-dependent and essential for differentiation of the endocrine pancreas[END_REF][START_REF] Naya | Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/NeuroD-deficient mice[END_REF][START_REF] Sander | Genetic analysis reveals that PAX6 is required for normal transcription of pancreatic hormone genes and islet development[END_REF][START_REF] Smith | Rfx6 directs islet formation and insulin production in mice and humans[END_REF][START_REF] Soyer | Rfx6 is an Ngn3-dependent winged helix transcription factor required for pancreatic islet cell development[END_REF][START_REF] Wang | Myt1 and Ngn3 form a feed-forward expression loop to promote endocrine islet cell differentiation[END_REF]-Figure 9).

Differentiation of endocrine cells

As previously mentioned, Neurog3-expressing cells generate all the five endocrine cell types: -, -, -, PP-and -cells. While early expression of Neurog3 exclusively gives rise to -cells (E8.5), -, -, PP-and -cells appear at E10.5, E11.5, E12.5 and E14.5 respectively [START_REF] Johansson | Temporal Control of Neurogenin3 Activity in Pancreas Progenitors Reveals Competence Windows for the Generation of Different Endocrine Cell Types[END_REF][START_REF] Prado | Ghrelin cells replace insulin-producing beta cells in two mouse models of pancreas development[END_REF]. Endocrine cells subsequently migrate and emerge from the ductal epithelium and aggregate to eventually form the islets of Langerhans around E18.5 [START_REF] Bouwens | Islet morphogenesis and stem cell markers in rat pancreas[END_REF].

The specification of the different hormone-secreting cells largely depends on the sequential activation/repression of TFs. Importantly, these TFs control the differentiation, as well as the maintenance and the function of endocrine cell subtypes.

Here we will focus on -, -and -cell determination/maintenance.

a. -cellsglucagon

Arx (aristaless-related homeobox) plays a key role in the determination of the -and PP-cell lineages, later being restricted to mature glucagon-expressing cells where it is involved in maintaining their identity. Indeed, Arx mutant mice display severe hypoglycemia caused by a loss of -cells associated with a proportional increase in -and -cell counts [START_REF] Collombat | Opposing actions of Arx and Pax4 in endocrine pancreas development[END_REF].

Although Pax6, NeuroD1 and Nkx2.2 are not specific to -cells, their deletion induces glucagon-expressing cell deficiency [START_REF] Naya | Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/NeuroD-deficient mice[END_REF][START_REF] Sander | Genetic analysis reveals that PAX6 is required for normal transcription of pancreatic hormone genes and islet development[END_REF][START_REF] Sussel | Mice lacking the homeodomain transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic beta cells[END_REF]. Brn4 (also known as POU3F4) is exclusively expressed by cells. Although not necessary for -cell development, Brn4 acts as a transcriptional regulator of glucagon expression [START_REF] Heller | The role of Brn4/Pou3f4 and Pax6 in forming the pancreatic glucagon cell identity[END_REF][START_REF] Hussain | POU domain transcription factor brain 4 confers pancreatic alpha-cell-specific expression of the proglucagon gene through interaction with a novel proximal promoter G1 element[END_REF].

b. -cellsinsulin

A number of TFs involved in early pancreatic development are also found to be important in -cell identity maintenance.

Pax4 is critical for -cell determination and is exclusively expressed in -cells in the adult pancreas. Accordingly, Pax4-deficient mice develop severe hyperglycemia and die shortly after birth. Analysis of their pancreas revealed the absence of -and -cells, concomitant with an increase in -cell numbers [START_REF] Sosa-Pineda | The Pax4 gene is essential for differentiation of insulin-producing beta-cells in the mammalian pancreas[END_REF]. Together with Pax4, Pdx1 and Nkx6.1 are major -cell determinants. Indeed, -cell-specific inactivation of Nkx6.1 or Pdx1 leads to their reprogramming into -and -cells [START_REF] Gao | Pdx1 maintains beta-cell identity and function by repressing an alpha-cell program[END_REF][START_REF] Schaffer | Nkx6.1 Controls a Gene Regulatory Network Required for Establishing and Maintaining Pancreatic Beta Cell Identity[END_REF]. Moreover, the loss of one allele of Pdx1 results in impaired -cell function and glucose intolerance [START_REF] Brissova | Reduction in pancreatic transcription factor PDX-1 impairs glucose-stimulated insulin secretion[END_REF].

MafA is expressed exclusively in -cells and regulates the expression of genes involved in insulin synthesis and secretion. Its deletion causes impaired insulin secretion and abnormal islet architecture [START_REF] Olbrot | Identification of β-cell-specific insulin gene transcription factor RIPE3b1 as mammalian MafA[END_REF][START_REF] Zhang | MafA Is a Key Regulator of Glucose-Stimulated Insulin Secretion[END_REF].

Interestingly, MafA was found to regulate insulin gene expression together with Pdx1

and NeuroD1 [START_REF] Zhao | The Islet Cell-enriched MafA Activator Is a Key Regulator of Insulin Gene Transcription[END_REF]. While not expressed exclusively in -cells, Nkx2.2 plays a role in the differentiation and maturation of insulin-producing cells, as its deletion induces severe hyperglycemia in mice [START_REF] Sussel | Mice lacking the homeodomain transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic beta cells[END_REF]. Similarly, NeuroD1 and Pax6 appear to be involved in establishing and maintaining -cells as their loss results in reduced -cell numbers [START_REF] Gu | Pancreatic β Cells Require NeuroD to Achieve and Maintain Functional Maturity[END_REF][START_REF] Naya | Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/NeuroD-deficient mice[END_REF][START_REF] Sander | Genetic analysis reveals that PAX6 is required for normal transcription of pancreatic hormone genes and islet development[END_REF]. Pdx1-null--cells. The authors therefore suggested that the increased -cell number is due to either their augmented proliferation or the reallocation of endocrine progenitors toward a -cell fate [START_REF] Gannon | pdx-1 function is specifically required in embryonic beta cells to generate appropriate numbers of endocrine cell types and maintain glucose homeostasis[END_REF].

In addition, an increase in somatostatin-positive cells was observed in mice deficient for Arx [START_REF] Collombat | Opposing actions of Arx and Pax4 in endocrine pancreas development[END_REF], as well as in Arx/Pax4 double mutant mice [START_REF] Collombat | The simultaneous loss of Arx and Pax4 genes promotes a somatostatin-producing cell fate specification at the expense of the alpha-and beta-cell lineages in the mouse endocrine pancreas[END_REF]. Moreover, pancreata of mice lacking Arx and Nkx2.2 displayed an increase in -cell numbers, a significant number of such cells expressing the hormone ghrelin [START_REF] Kordowich | Arx and Nkx2.2 compound deficiency redirects pancreatic alpha-and beta-cell differentiation to a somatostatin/ghrelin co-expressing cell lineage[END_REF]. These studies strongly suggest an antagonist effect of Arx on somatostatin-expressing cell development.

The first transcription factor involved in pancreatic somatostatin-cell development has been recently characterized. In addition to its critical role in ventral pancreas organogenesis [START_REF] Bort | Hex homeobox genedependent tissue positioning is required for organogenesis of the ventral pancreas[END_REF], it has been shown that Hhex is essential for the differentiation and the maintenance of -cells [START_REF] Zhang | The diabetes gene Hhex maintains δ -cell differentiation and islet function The diabetes gene Hhex maintains d -cell differentiation and islet function[END_REF]. In this elegant study, Hhex deficiency in mouse pancreas was found to induce a massive loss of -cell numbers concomitant with a decrease in somatostatin secretion.

Interestingly, a recent study showed a -to- cell conversion upon the inactivation of Mnx1 in endocrine precursors. The authors further suggested that Mnx1 acts as a repressor of δ-cell specification through the direct or indirect inhibition of Hhex expression [START_REF] Pan | Inactivating the permanent neonatal diabetes gene Mnx1 switches insulin-producing β-cells to a δlike fate and reveals a facultative proliferative capacity in aged β-cells[END_REF].

Finally, -endosulfine, PCSK9 and Ptch1 receptor were found to be specifically expressed in -cells in rat-, human-and mouse-islets; but no investigation concerning their role in -cell fate specification has been undertaken [START_REF] Grieco | Delta-cell-specific expression of hedgehog pathway Ptch1 receptor in murine and human endocrine pancreas[END_REF][START_REF] Gros | Localization of α-endosulphine in pancreatic somatostatin δ cells and expression during rat pancreas development[END_REF][START_REF] Langhi | PCSK9 is expressed in pancreatic delta-cells and does not alter insulin secretion[END_REF].

IV. T1DM actual therapies & current research

As previously mentioned, diabetes patients are exposed to various cardiovascular complications due to chronic increased blood glucose levels and are at risk of dying prematurely. Thereby, doctors and researchers are acting collectively in an attempt to manage or even reverse this hyperglycemia.

Management of T1DM

The priority in the management of T1DM is to monitor blood glucose levels to strictly maintain glycemia within normal ranges (Table 1), thereby preventing hyperglycemia-and hypoglycemia-associated complications.

a. Exogenous insulin administration

To compensate the inability of the pancreas to secrete insulin, type 1 diabetic patients are dependent on exogenous insulin administration, such compensation being indispensable for their survival. Although efficient, chronic insulin administration through subcutaneous daily injections results in difficulties to strictly regulate glycemic levels (due to fluctuations in diet, physical exercises or age) and induces glycemic variability (alternations between hyper-and hypoglycemia) [START_REF] Group | The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus[END_REF]. The more recent implementation of subcutaneous pumps allows sustained delivery of insulin and appears to be more efficient at maintaining normoglycemia [START_REF] Shetty | Insulin pump use in adults with type 1 diabetes--practical issues[END_REF].

Another or an additive strategy involves the use of a combination of different types of insulin with distinct timing of actions in lowering glucose levels. Mixed preparations with long-, regular or short-acting and rapid-acting insulin can be used for an improved delivery of insulin and therefore glycemic regulation. Long-acting insulin acts several hours after injection and for a period of 24 hours while regular or short-acting insulin acts within 30 minutes and is effective for 3 to 6 hours. Finally, rapid-acting insulin requires only 15 min to lower blood glucose levels and is efficient for up to 4 hours [START_REF] Mccall | Treating type 1 diabetes: from strategies for insulin delivery to dual hormonal control[END_REF]. Such preparations afford a better glycemic control, depending on the need and eating habits of patients, and can be used through injections or via pumps.

Recently developed, the artificial pancreas (AP) is based on a closed-loop control system consisting of a continuous subcutaneous glucose sensor and insulin pump. This allows constant glucose-monitoring and insulin delivery.

Although not yet available, clinical trials demonstrate that this approach offers significant advantages as it manages blood glucose levels with less variations and presents less daily stress for the patients [START_REF] Kovatchev | The Artificial Pancreas in 2016: A Digital Treatment Ecosystem for Diabetes[END_REF][START_REF] Kropff | 2 month evening and night closed-loop glucose control in patients with type 1 diabetes under free-living conditions: A randomised crossover trial[END_REF]. However, hypoglycemia remains a risk that should be dealt prior to a putative application.

b. Total pancreas and islets transplantation

As previously mentioned, exogenous insulin administration represents an imperfect treatment for diabetes due to its inability to finely regulate blood glucose levels. Thus, two alternatives aiming at replacing the damaged -cell mass in diabetic patients exist: whole pancreas or islets transplantation. Whole pancreas transplantation often also includes kidney transplants, as the organs could have been damaged due to hyperglycemia-associated complications. However, pancreas transplantation is a heavy burden for diabetic patients, which is why islets transplantation has been favored for the past decade. The Edmonton protocol [START_REF] Shapiro | Islet transplantation in seven patienrs with Type I diabetes mellitus using a glucocortincoid-free immunosuppressive regimen[END_REF] has played a key role in islet transplantation from cadaveric donors. It is of importance to note that these grafts involve lifelong immunosuppressant treatment to avoid rejection and new -cell destruction, such treatment rendering the patients susceptible to other infections.

Nonetheless, total pancreas or islet transplantation normally result in a rapid glycemia normalization and exogenous insulin independence for up to 5 years, further improving the quality of life of the patients [START_REF] Domínguez-Bendala | The Human Endocrine Pancreas: New Insights on Replacement and Regeneration[END_REF][START_REF] Sureshkumar | Quality of life after organ transplantation in type 1 diabetics with end-stage renal disease[END_REF]. However, these medical procedures are mostly used for type 1 diabetic patients with serious complications.

Although efficient, these therapies face the shortage of organ donors and the associated side-effects of immunosuppressive drugs [START_REF] Bruni | Islet cell transplantation for the treatment of type 1 diabetes: Recent advances and future challenges[END_REF].

Current axes of research

Despite the help offered by the different strategies available to physiologically mimic the action of insulin, the difficulties in strictly regulating glycemic levels, discomfort due to daily injections or pump replacement and financial limitations, have encourage researchers to investigate alternatives. Consequently, current research focuses on the replacement of the injured--cell-mass in diabetic patients using several approaches and cell sources. The common goal of these diverse strategies is to generate cells synthetizing and secreting appropriate amounts of insulin under physiological conditions (Figure 10). of an antibiotic resistance gene under the control of human insulin gene [START_REF] Soria | Insulinsecreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice[END_REF]. Later, D'Amour et al. derived human embryonic stem cells (hESCs) into pancreatic progenitors after a five-step protocol using sequential exposure of various inducing agents and growth factors [START_REF] D'amour | Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells[END_REF]. Although this study was the first demonstration of human-derived--cells, the resulting insulin-secreting cells were glucose unresponsive. Later, the optimization of the previous protocol resulted in hESC-derived-insulin-producing cells able to respond to glucose following their transplantation and further differentiation in mice [START_REF] Kroon | Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo[END_REF]. However, all these protocols led to cell producing too low amounts of secreted insulin mostly due to incomplete maturation. Recent studies have developed protocols that have had more success. Indeed, Rezania et al published a seven-stage protocol leading to the differentiation of hESCs into -like cells, which were responsive to glucose and able to restore chemically-induced diabetes in vivo after transplantation into immunodeficient mice [START_REF] Rezania | Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells[END_REF]. In addition, Pagliuca et al. described a promising protocol allowing the production of large numbers of insulin-secreting cells from human pluripotent stem cells, such cells preventing hyperglycemia following transplantation into Akita immunodeficient mice [START_REF] Pagliuca | Generation of Functional Human Pancreatic β Cells In Vitro[END_REF]. In 2015, a further improved protocol was published, leading the production of glucose responsive -cells from hESCs in vitro and in vivo [START_REF] Russ | Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro[END_REF].

Although representing a promising therapy with clinical trials currently ongoing (Viacyte, Inc.), the use of ESCs faces several obstacles. Indeed, the use of hESCs [START_REF] Takahashi | Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[END_REF][START_REF] Yu | Induced pluripotent stem cell lines derived from human somatic cells[END_REF]. Later, iPSCs were obtained by reprogramming other cell types, such as neuronal progenitor cells [START_REF] Kim | Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors[END_REF], keratinocytes [START_REF] Aasen | Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes[END_REF], hepatocytes and gastric epithelial cells [START_REF] Aoi | Generation of pluripotent stem cells from adult mouse liver and stomach cells[END_REF]. Recently, pluripotent stem cells were induced from mouse somatic cells using chemicals that could improve reprogramming efficiency or replace one or all previously mentioned transcription factors [START_REF] Bar-Nur | Small molecules facilitate rapid and synchronous iPSC generation[END_REF][START_REF] Hou | Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds[END_REF][START_REF] Ye | Pluripotent stem cells induced from mouse neural stem cells and small intestinal epithelial cells by small molecule compounds[END_REF]. Using different strategies, such as chemicallyinduced differentiation, iPSCS can be directed to differentiate into any cell type including -cells [START_REF] Alipio | Reversal of hyperglycemia in diabetic mouse models using induced-pluripotent stem (iPS)derived pancreatic beta-like cells[END_REF][START_REF] Zhang | Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulinproducing cells[END_REF]. The use of iPSCs in diabetes therapy therefore presents advantages when compare to hESCs. Indeed, the reprogrammed cells avoid ethical issues of hESCs and graft rejection as they are obtained from the differentiation of adult patients somatic cells.

Although promising, the use of embryonic stem cells and induced pluripotent stem cells in diabetes therapy still needs further investigation. It is important to note that ESCs and iPSCs possess oncogenic properties, as undifferentiated or not fully differentiated stem cells transplanted can form teratomas (tumors containing cells from 3 germ layers) [START_REF] Hentze | Teratoma formation by human embryonic stem cells: evaluation of essential parameters for future safety studies[END_REF]. In addition, autoimmunity in type 1 diabetic patients can trigger the destruction of newly-implanted -cells.

2) Tissue stem cells

Tissue stem cells are found in various tissues including bone marrow [START_REF] Jiang | Pluripotency of mesenchymal stem cells derived from adult marrow[END_REF], gastric epithelium [START_REF] Bjerknes | Multipotential stem cells in adult mouse gastric epithelium[END_REF], liver [START_REF] Overturf | Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes[END_REF], brain [START_REF] Reynolds | Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system[END_REF], adipose tissue [START_REF] Zuk | Human Adipose Tissue Is a Source of Multipotent Stem Cells[END_REF] and umbilical cord [START_REF] Prindull | Haematopoietic stem cells (CFUc) in human cord blood[END_REF]. Most stem cells found in post-natal tissues are multipotent, therefore able to develop into different cell types. Although highly debated, they represent another putative source for -cell regeneration.

Reports have shown the differentiation of bone marrow stem cells into insulin-producing cells [START_REF] Ianus | In vivo derivation of glucosecompetent pancreatic endocrine cells from bone marrow without evidence of cell fusion[END_REF][START_REF] Karnieli | Generation of insulin-producing cells from human bone marrow mesenchymal stem cells by genetic manipulation[END_REF], whereas a following study failed to reproduce these findings [START_REF] Taneera | Failure of transplanted bone marrow cells to adopt a pancreatic betacell fate[END_REF]. Stem cells from human umbilical cord were also suggested to be able to derive into islet cells [START_REF] Pessina | Pancreas developing markers expressed on human mononucleated umbilical cord blood cells[END_REF][START_REF] Sun | Induction of human umbilical cord blood-derived stem cells with embryonic stem cell phenotypes into insulin producing islet-like structure[END_REF]. In addition, human adipose tissue stem cells were induced to differentiate into pancreatic endocrine cells [START_REF] Timper | Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagon expressing cells[END_REF] and into functional insulin-producing cells in mice [START_REF] Kajiyama | Pdx1-transfected adipose tissue-derived stem cells differentiate into insulin-producing cells in vivo and reduce hyperglycemia in diabetic mice[END_REF]. Liver stem cells have also been suggested to possess the ability to differentiate into insulin-secreting cells [START_REF] Ferber | Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocininduced hyperglycemia[END_REF][START_REF] Yang | In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells[END_REF].

Excluding ethical concerns and risk of immune rejection, the use of tissue stem cells offers promising perspectives for diabetes therapy, although more investigation is required to fully restore -cell mass function in diabetic patients using these cell sources.

b. -cell mass replacement from pancreatic cells

With a rising number of reports, reprogramming/transdifferentiation of adult pancreatic cells into functional -cells is an exciting topic but still highly debated.

Even though pancreas plasticity has mostly been described in experimental diabetes models in mice, several approaches including genetic or chemical manipulations, have been used to induce acinar-, ductal-or endocrine-cell conversion into insulinproducing cells. Also, the development of lineage tracing tools has resulted in large progress in this field.

1) From acinar cells

Abundant in the pancreatic tissue, exocrine acinar cells represent more than 95% of the gland, thus they constitute an interesting source for -cell replacement.

An elegant approach by Zhou et al. demonstrated that adenovirus-mediated ectopic expression of Pdx1, Neurog3 and MafA (all three preponderantly involved in pancreatic development, see section III), resulted in the direct conversion of acinar cells into insulin-secreting cells able to improve hyperglycemia in diabetic mice [START_REF] Zhou | In vivo reprogramming of adult pancreatic exocrine cells to β-cells[END_REF]. However, these cells were not fully efficient as they did not cluster into organized islets. Another study suggested that cytokine stimulation with EGF (epidermal growth factor) and CNTF (ciliary neurotrophic factor) of chemicallyinduced diabetic mice induces the restoration of a functional -cell mass through acinar-to--cell conversion via Neurog3 activation [START_REF] Baeyens | Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice[END_REF].

Interestingly, using genetic manipulation or chemical exposure, recent publications have reproduced this conversion in vitro and ex vivo using human exocrine cells [START_REF] Klein | BMP-7 induces adult human pancreatic exocrine-to-endocrine conversion[END_REF][START_REF] Lemper | Reprogramming of human pancreatic exocrine cells to β-like cells[END_REF][START_REF] Lima | Suppression of epithelial-to-mesenchymal transitioning enhances ex vivo reprogramming of human exocrine pancreatic tissue toward functional insulin-producing β-like cells[END_REF]. However, the study undertaken by Desai et al. invalidates the theory of acinar cells ability to convert into -like cells in mice, leaving the topic contested [START_REF] Desai | Preexisting pancreatic acinar cells contribute to acinar cell , but not islet β cell , regeneration[END_REF].

2) From ductal cells

As previously discussed (see section III), endocrine cells emerge from the ductal epithelium and aggregate into islets of Langerhans during development. Such observations led to several theories, one suggesting that such endocrine cells could arise from pancreatic progenitors in the ducts [START_REF] Bonner-Weir | Transdifferentiation of pancreatic ductal cells to endocrine β-cells[END_REF][START_REF] Bonner-Weir | B-Cell Growth and Regeneration : Replication Is Only Part of the Story[END_REF]. Heimberg and colleagues demonstrated the recruitment of ductal progenitors in the pancreatic injury model of PDL (partial pancreatic duct ligation) in mice. These cells were found able to convert into islet cells, this conversion being mediated by Neurog3 expression [START_REF] Xu | β-cells can be generated from endogenous progenitors in injured adult mouse pancreas[END_REF]. Partial pancreatectomy (Ppx) is another model of pancreatic injury widely used in pancreatic cell conversion studies.

While Lee et al. demonstrated that -cell regeneration does not require the reactivation of Neurog3 expression in the pancreas after 50% Ppx [START_REF] Lee | Regeneration of pancreatic islets after partial pancreatectomy in mice does not involve the reactivation of neurogenin-3[END_REF], a following study suggested transient reexpression of the pro-endocrine gene only after 90% Ppx [START_REF] Li | Activation of pancreatic-duct-derived progenitor cells during pancreas regeneration in adult rats[END_REF]. Despite the observed -cell regeneration, ductal cell plasticity was challenged by two studies affirming that ductal cells can derive into endocrine cells only before birth [START_REF] Kopp | Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas[END_REF][START_REF] Solar | Pancreatic exocrine duct cells give rise to insulin-producing β Cells during embryogenesis but not after birth[END_REF].

Still controversial and non-confirmed, one recent hypothesis suggests the existence of different ductal cell populations with only a fraction of these able to convert into endocrine cells [START_REF] Grün | De Novo Prediction of Stem Cell Identity using Single-Cell Transcriptome Data[END_REF].

3) From endocrine cells

The increase of the -cell mass from the interconversion between the different endocrine cell types or pre-existing -cells expansion is a widely investigated field.

Aside from specific conditions, such as pregnancy or insulin resistance, islet cells are mostly quiescent with a low replication rate of 0.2% per day [START_REF] Teta | Very slow turnover of beta-cells in aged adult mice[END_REF]. Inducing -cell self-replication using specific mitogenic factors, such as exendin-4 [START_REF] Xu | Exendin-4 stimulates both Beta-cell replication and neogenesis, resulting in increased Beta-cell mass and improved glucose tolerance in diabetic rats[END_REF] or IGF-1 [START_REF] Agudo | IGF-I mediates regeneration of endocrine pancreas by increasing beta cell replication through cell cycle protein modulation in mice[END_REF], has been shown to be efficient to increase the -cell mass. Similar results using human islets demonstrated that the modulation of cell cycle genes in human -cells [START_REF] Fiaschi-Taesch | Cytoplasmic-Nuclear Trafficking of G1/S Cell Cycle Molecules and Adult Human β-Cell Replication[END_REF] or the use of HGF (hepatocyte growth factor - [START_REF] Beattie | A novel approach to increase human islet cell mass while preserving beta-cell function[END_REF] It is worth noting that in these different models, in addition to glucagonexpressing cells regeneration, an increase in the number of somatostatin-expressing -like cells was consistently observed (Figure 11). Considering the re-expression of the pro-endocrine gene Neurog3 in the ductal lining of these transgenic mice, one could assume a continuous neogenesis of these somatostatin-expressing cells.

Unexpectedly, while being continuously produced, these -cells were not found to be accumulated over time.

Based on these observations and considering that -cells are closely linked to -cells during development, as they share the same lineage, we sought to determine whether adult -cells could not be induced to adopt a -like cell identity. (5), prior to undergo EMT (6) and adopt an endocrine cell identity (7). Such a continuous cycle of conversion/regeneration results in insulin + cell hyperplasia (8).

I. Animal procedures

All mice were housed on a standard 12:12h light:dark cycle, with standard diet food and water ad libitum. Animal care was conducted according to French ethical regulations. This project received the approval from our local ethics comity (NCE/2011-22). 129/Sv wildtype mice were obtained from Taconic laboratories.

Experiments were performed on transgenic and control males only. Controls include wildtype mice or transgene-negative littermates.

Generation of transgenic mouse line

Using the Cre-LoxP system to investigate -cell plasticity in pancreatic islets, we generated Sst-Cre::Pax4-OE double transgenic animals allowing both the conditional expression of Pax4 in somatostatin + cells and their lineage tracing (Figure 12 -For details, see the results section). Importantly, Sst-Cre::Pax4-OE transgenic mice were found to be viable, fertile, with no premature death being observed.

Genotyping

Genotyping was performed on small ear punch biopsies using the KAPA Express DNA Extraction Kit (KK7103, Kapa Biosystems). Briefly, the digestion of each sample was performed with 100μL of KAPA Express Extract lysis buffer (10μL 10X KAPA Express Extract Buffer; 2.0μL 1 U/μL KAPA Express Extract Enzyme;

88μl PCR-grade water), followed by an incubation in a thermomixer for 10 min at 75°C with shaking at top speed. Heat-inactivation of the thermostable KAPA Express Extract protease was achieved after incubation for 5 min at 95°C with shaking and samples were centrifuged at 14000 rpm for 1 min to pellet debris. The DNAcontaining supernatant was transferred to a fresh tube and Polymerase Chain Reaction (PCR) was then used to assess the genotype of our mice.

PCRs were performed by combining in each reaction tube 15µL of 2X GoTaq® G2

Hot Start Green Master Mix (M7423, Promega); 0.08µL of both forward and reverse primers (Table 2) and 13.84µL H2O.

Induction of streptozotocin-mediated hyperglycemia

Streptozotocin is a glucosamine-nitrosourea compound causing DNA damage.

Its chemical structure is similar to glucose which allows it to be transported specifically into the -cell via the glucose transporter GLUT-2. To induce hyperglycemia, streptozotocin, (STZ; S0130, Sigma) was dissolved in 0.1M sodium citrate buffer (pH 4.5), and a single dose was administered intraperitoneally (115 mg/kg) within 10 min after dissolution. Diabetes progression was assessed by monitoring the blood glucose levels and/or survival rates of mice. Glycemia was measured with a ONETOUCH glucometer (Life Scan, Inc., CA).

III. Tolerance tests and blood glucose level measurements

IV. RNA analyses RNA extraction from total pancreas

Mice were euthanized by cervical dislocation and the pancreatic tissue was isolated and placed in cold RNA later solution (AM7024, Fisher Scientific) for 48 hours at 4°C. A small piece of pancreas was taken and homogenized in lysis buffer (10L of -mercaptoethanol in 10mL of RLT buffer from Qiagen) with a tissue disruptor. Total pancreas RNA was extracted using Rneasy Mini Kit (Qiagen)

according to the manufacturer's instructions.

cDNA synthesis

RNA concentration and quality were determined using an Agilent 2100

Bioanalyzer system (Agilent Technologies). 10µL of H2O containing 1g of RNA was then processed for first strand synthesis mixing 1µL 10mM dNTP and 1µL 50mM oligodT. This reaction was then incubated 5' at 65°C, then on ice for at least 1 min.

For cDNA synthesis, 10µL of DNA synthesis mix (2µL 10x RT buffer; 4µL 25 mM MgCl2; 2µL 0.1 M DTT; 1µL Rnase out; 1µL Superscript III) was added to each sample and incubated for 50 min at 50°C, 5 min at 85°C then chilled on ice. Finally, 1µL of Rnase H was added and samples were incubated for 20 min at 37°C. qPCR reactions were performed in tubes containing 5µL 2x SYBR Green Supermix, 0.5µL Primer Assay, 3µL H2O and 1.5µL of 1/20 diluted cDNA.

OCT embedding -Cryostat sectioning

Mice were euthanized by cervical dislocation and the pancreatic tissue was isolated and placed in cold PBS. Tissue was fixed for 30 min at 4°C in a fixative solution (1.35mL 37% paraformaldehyde; 400L 25% glutaraldehyde; 100L 10X NP40; 5mL 10X PBS; H2O to 50mL) and washed 6 x 20 min in cold PBS. To remove the water and protect the tissue from freezing damage, the sample was incubated overnight at 4°C in a 25% sucrose solution diluted in PBS. Finally, the organ was washed 2 x 1h in Jung freezing medium (Leica Biosystems) and embedded in plastic molds in Jung freezing medium on dry ice. Cryo blocks were stored at -80°C prior to sectioning.

16m sections were cut using a Cryostat (Leica) with the following settings:

object temperature of -14°C and chamber temperature -19°C. Sections were placed on polarized glass slides (Fisher Scientific) and dried for 45 min at 42°C into a heating block. Slides were subsequently stored at -80°C until staining.

VI. Staining of pancreatic sections Immunohistochemistry (IHC)

Paraffin sections were deparaffinized 3 x 3 min in xylene, rehydrated in decreasing ethanol dilutions (5 min in 2 x 95%; 5 min in 80%; 5 min in 60%; 5 min in 30%), and finally rinsed twice for 5 min in ddH2O. To reveal epitopes that could have been masked during the tissue preparation, antigen retrieval was performed by boiling the tissue for 1 min at 140°C in 1.6L ddH2O with 15mL Antigen Unmasking solution (Citric acid based pH6, Vector) in a pressure cooker. Slides were then transferred into tap water for approximately 30 min. Of note, this antigen retrieval step results in the denaturation of GFP, subsequently leading to its quenching.

Tissue sections were then washed 3 x 5 min in PBS, kept in a blocking solution for 45 min at room temperature (PBS-FCS 10% -Gibco) to block any nonspecific binding of the antibodies and incubated overnight in a humid chamber at 4°C with primary antibodies (Table 4) diluted in PBS-FCS 10%.

The samples were rinsed 3 x 5 min in PBS before application of the appropriate secondary antibodies (Table 5) for 45 min at room temperature in a humid chamber. Finally, slides were washed 3 x 5 min in PBS, dried, and mounted with coverslips and Vectashield HardSet Mounting Medium (Vector) with 4', 6-diamidino-2-phenylindole (DAPI) as a nuclear counterstain.

Slides were kept at 4°C prior to observation. Pictures were processed using ZEISS Axioimager Z1 equipped with the appropriate filter sets and a monochrome camera, with Axiovision software from ZEISS.

IHC on cryosections

Cryosections were washed from the embedding Jung freezing medium for 3 x 15 min in PBS and blocked in a PBS-FCS 10% blocking solution for 45 min at room temperature in a humid chamber. The sections were then incubated overnight in a humid chamber at 4°C with primary antibodies (Table 4) diluted in PBS-FCS 10%.

The samples were rinsed 3 x 5 min in PBS before application of the appropriate secondary antibodies (Table 5) for 45 min at room temperature in a humid chamber.

VII. Quantification analyses

Quantification were performed on pictures from Zeiss Axioimager Z1 with Axiovision software and an automatic mosaic acquisition system (Zeiss). The entire pancreata of at least 3 mice per genotype and per condition were serially cut in 8 µm thick sections, applied to glass slides and every 5 to 10 pancreatic sections were processed for immunohistochemistry.

For cre recombinase efficiency and -galactosidase expressing cells, positive cells were counted manually. IdU and BrdU counting was assessed manually counting proliferative cells in the different pancreatic compartments. For hormones quantifications, widefield multi-channels tile scan images treatment and analysis were done using a Fiji [START_REF] Schindelin | Fiji: an open source platform for biological image analysis[END_REF] home-made semi-automated macro.

Briefly, the overall pancreas slice area was determined and measured using a low intensity based threshold on the insulin or glucagon or somatostatin labelling channel. Islets were then segmented and counted on the insulin labelling, applying a high intensity threshold. The density of islets per pancreas section was obtained reporting the number of islets to the pancreatic area. In a second step, the respective area of each segmented islet was measured. The corresponding contours were then transferred to the somatostatin or glucagon images. Finally, the signal coverage of either glucagon or somatostatin was measured and then normalized to the total area of the corresponding islet. showing a dramatic increase in islet number and size. Quantitative analyses of at least 3 animals from each group revealed a ~1.5x increase in islet size (F) and 2x in islet number (E) in 2-month old transgenic pancreata. Quantitative analyses of insulin + area in 2-month old mice outlined a ~4x increase respectively, consistent with the observed increased islet number and counts (G). At 5-and 8-months of age, an average of 3x, 2x, and 8x increase in islet counts (E), size (F), and insulin + area respectively, was quantified in Sst-Cre::Pax4-OE pancreata as compared to age-matched controls. Quantification of insulin + cell proportions demonstrated an increase in 5-month old transgenic animals compared to controls (H). Quantification of islet glucagon + cell content in 2-and 5-month old mutant islets did not reveal any differences compared to controls (I). A significant decrease in somatostatin + proportions was observed in 2-and 5-month old mutant islets compared to controls (J). All values are depicted as mean SEM of n>3 independent animals. Statistics for insulin + area, islet size, glucagon and somatostatin contents were performed with Mann-Whitney test. For islet number p-values were calculated by unpaired t test with Welch's correction. **** p<0.0001, ** p<0.01, * p<0.05. Scale bars, 500m.

RESULTS

I

Additional analyses performed in 5-month old animals ascertained a further increase reaching 3.300.33-fold in the islet count, 2.270.09-fold in islet size and 7.570.08fold in insulin + cell counts as compared to controls (Figure 15E-G). Similar augmentations were also observed in 8-month old animals with a 2.120.09-fold increase in islet size, a 3.450.19-fold increase in islet number and an increase in insulin + area of 7.290.17-fold versus age-/sex-matched controls (Figure 15E-G),

suggestive of a plateau in islet growth and multiplication after 5 months of age.

Focusing our quantitative studies on relative islet cell proportions, the insulin + cell counts were found increased in 5-month old Sst-Cre::Pax4-OE islets (+ 71.09% -Figure 15H), whereas the glucagon + cell proportions were found unchanged (Figure 15I), suggesting an increase in -cell counts following faithfully the overall endocrine cell hyperplasia. Surprisingly, the somatostatin + cell contents was 1.610.34-fold reduced in 2-month old Sst-Cre::Pax4-OE islets and further diminished (3.050.15fold) in their 5-month old counterparts (Figure 15J).

In addition to the evidently altered endocrine cell count observed in Sst-Cre::Pax4-OE pancreata, an abnormal positioning of non--cells was noted within the islets. Indeed, while glucagon-and somatostatin-expressing cells were predictably found uniformly distributed at the periphery of control islets (Figure 16A Together, these results suggest that the misexpression of Pax4 in -cells promotes both an increase in islet numbers and size. Interestingly, the islet hypertrophy appears to result from an insulin + cell hyperplasia and, to a lesser extent, from an increase in glucagon + cell counts, indicative of neogenesis processes.

However, somatostatin + cells are found underrepresented, suggesting that the neogenesis processes observed in Sst-Cre::Pax4-OE pancreata could involve their conversion into alternative cell subtypes.

III. Pax4 misexpression in somatostatin + cells induces their conversion into -like cells

To proportion increasing to 4.70.27% in 5-month old Sst-Cre::Pax4-OE animals (F). For the proportion of supplementary -like cells labeled with the -galactosidase reporter, the following formula was used: (% of labeled -like cells x relative -like cell count)/(-like cell increase -100). A similar proportion of 90.87% of supplementary -like cells appeared galactosidase + was found in the different age groups. Statistics were determined using Mann-Whitney test **** p<0.0001. Scale bars, 50m.

In control Sst-Cre::ROSA26--gal pancreata, -gal + cells were found, as expected, in the mantle of the islets, where somatostatin-expressing cells are normally located (Figure 17C). While, no insulin/-gal double positive cells could be found in control islets (Figure 17C), several insulin + /-galactosidase + cells were observed both in the After 10 days of IdU treatment, a further increase in proliferating cells was noted in the ductal epithelium (inlet in E) and islets (F) of transgenic mice as compared to controls (B, C). Quantitative analyses revealed a 5.730.38-fold increase in proliferating cell counts within the ductal lining or epithelium after short-term IdU (G). Interestingly, a 2.470.13-fold and a 3.340.21-fold increase in IdU + cells is outlined in the endocrine compartment and in ductal lining, respectively, of Sst-Cre::Pax4-OE pancreata after long-term IdU treatment (G). For the purpose of clarity, in selected photographs, islets are outlined with dashed white lines. All values are depicted as mean SEM of n>3 independent animals. Statistics were performed Mann-Whitney test or unpaired t test with Welch's correction. **** p<0.0001, ns p>0.05. Scale bars, 50m.

A 3.340.21-fold increase within the ductal lining or epithelium and a 2.470.13-fold increase in the numbers of IdU + cells within the endocrine compartment were observed after 10 days of IdU treatment (Figure 19G). Of note, no difference in IdU + cell counts were observed in the acinar tissue comparing both groups.

In order to determine whether proliferating cells went through one or more cell division in Sst-Cre::Pax4-OE pancreata, control and transgenic mice were sequentially treated with CldU and IdU for 10 days each (Figure 20A). Despite the fact that the number of proliferative cells was, as shown before, increased in transgenic islets, no Idu + /CIdU + cells were detected in Sst-Cre::Pax4-OE nor control islets (Figure 20B-D). Concomitant with the plateau in islet hypertrophy observed after 5-months of age, comparative quantifications of endocrine and non-endocrine cell proliferation rates in 5-and 8-month old mutants did not reveal any differences (Figure 20E-F). Thus, these data suggest that the observed augmentations in islet size and number involve increased cell proliferation essentially originating in the ductal lining/epithelium, these proliferating cells eventually sprouting from the ductal compartment to reach the islets of Langerhans. Aiming to further ascertain a putative reenactment of endocrine developmental processes in Sst-Cre::Pax4-OE animals, we focused our analyses on the epithelialto-mesenchymal transition (EMT), a key developmental process by which cells located within an epithelial layer acquire the ability to spread and migrate to a distant site to form new structures [START_REF] Kang | Epithelial-Mesenchymal Transitions : Twist in Development and[END_REF]. EMT is orchestrated by a cascade of events, among which the loss of an epithelial cell identity (as outlined by the loss of E-cadherin), and the acquisition of a mesenchymal cell phenotype and associated markers (such as Vimentin and N-cadherin - [START_REF] Chiang | Single-cell transcript analysis of pancreas development[END_REF].

We therefore assessed the expression of Vimentin in 2-month old Sst-Cre::Pax4-OE pancreata versus control counterparts. Surprisingly, our analyses revealed a massive increase in the numbers of Vimentin + cells in the ductal lining and close to adjacent islets of transgenic pancreata (Figure 21D-E

VII. Adult somatostatin + cells convert into insulinproducing cells upon Pax4 misexpression following streptozotocin treatment

Adult Sst-Cre::Pax4-OE -cell plasticity was investigated in the context of chemically-induced -cell ablation. The glycemia of these mice was monitored throughout the experiment, prior to sacrifice at different time points (Figure 24A). A single dose of streptozotocin (STZ -115mg/kg) was administered to 2.5-month old transgenic mice and matching controls, the goal being to induce the destruction of a vast majority of -cells without complete ablation to avoid predictable death (Figure 24B,E). As expected after specific -cell loss, control and Sst-Cre::Pax4-OE mice developed hyperglycemia within 1 week post-injection (Figure 24H). 3 months postinjection, all control mice showed a glycemia >600mg/dl and a high rate of lethality (41%), while in contrast transgenic mice displayed an average glycemia of 350mg/dl (Figure 24H), suggesting a partial -cell mass restoration and an extended lifespan. months following STZ-mediated -cell destruction. In Sst-Cre::Pax4-OE transgenic mice, a progressive recovery of the -cell mass is observed. The monitoring of the glycemia revealed an expected peak in glycemic levels in both control and transgenic mice (H). 2.5 months following -cell ablation, Sst-Cre::Pax4-OE mice displayed lowered glycemic levels, while control animals remained hyperglycemic. Quantitative analyses indicate an approximate doubling in the -cell area in transgenic islets 3 months after STZ-administration (I). Lineage tracing experiments of somatostatin-expressing cells performed on pancreas sections of STZ-treated mice where -gal + /insulin + cells were detected in transgenic islets (inlets in J-K).

For the purpose of clarity, in selected photographs, islets are outlined with dashed white lines. Scale bars, 50m. All values are depicted as mean SEM of n>3 independent animals. Statistics were determined using Mann-Whitney test and multiple t-tests **** p<0.0001, * p<0.05.

To further assess these regeneration processes, the -cell mass was quantified in control and transgenic pancreata 3-month post-STZ administration. Although decreased compared to their non-injected counterparts, STZ-treated Sst-Cre::Pax4-OE islets exhibited 163.40% insulin-expressing cells compared to 81.77% in treated-control islets (Figure 24I). This difference represents an average 2-fold increase in islet -like cell content (Figure 24I). Immunohistochemical analyses and lineage tracing experiments were performed on STZ-injected control and transgenic pancreata to characterize the mechanisms underlying this partial -cell mass renewal. We thereby detected insulin + /-galactosidase + cells in Sst-Cre::Pax4-OE islets, 3 months following -cell mass ablation (Figure 24J-K). However, the results presented here suggest that adult somatostatin-expressing cells, upon Pax4 misexpression, are plastic and able to convert into insulin-producing cells also in the situation of a -cell loss. Although encouraging, one would have expected a better recovery upon STZ-mediated -cell destruction. Therefore, we initiated additional experiments using younger animals in order to take advantage of their improved cell neogenesis capability.

Here, we report that Pax4 misexpression in -cells leads to their conversion into insulin-producing -cells, further inducing an increase in islet number and islet hypertrophy provoked by an insulin + cell hyperplasia. Interestingly, the latter is associated with an improved glucose tolerance. Our data show that these effects essentially appear before 5-months of age and do not alter the relative endocrine cell composition as the respective endocrine cell proportions are maintained. Importantly, these processes involve the recruitment of putative endocrine progenitor cells located within the ductal lining or epithelium, such cells reactivating the Neurog3-controlled differentiation program prior to their delamination by EMT reawakening. Moreover, we demonstrate here that adult -cells are plastic and capable of converting into cells following chemically-induced diabetes upon the sole misexpression of Pax4.

I. Pax4 misexpression in -cells induces their conversion into -cells

Our results demonstrate that the misexpression of Pax4 in -cells is sufficient to induce their conversion into functional -like cells, further resulting in a massive insulin-producing cell hyperplasia. Indeed, we show that 2.8% of insulin + cells arise from -cells in 2-month old transgenics, such amount increasing to 4.7% in 5-month old Sst-Cre::Pax4-OE animals, suggesting continuous processes of conversion of cells into insulin + cells. According to previous studies, near-total -cell ablation in mice induces pancreatic -cells to convert into insulin-producing cells, such conversion only occurring in juveniles, before 2 months of age [START_REF] Chera | Diabetes recovery by age-dependent conversion of pancreatic δ-cells into insulin producers[END_REF].

Here, we confirm these results but also demonstrate that somatostatin-expressing cells retain the ability to be converted into -like cells until at least 5 months of age. In Moreover, it has been shown that during mouse pancreas morphogenesis, EMT processes [START_REF] Gouzi | Neurogenin3 initiates stepwise delamination of differentiating endocrine cells during pancreas development[END_REF]. Interestingly, our data show a massive increase in the expression of mesenchymal markers within the ductal lining of Sst-Cre::Pax4-OE transgenics, further supporting the notion of an endocrine cell neogenesis from the ductal epithelium.

Finally, the proliferation of ductal cells and the re-expression of Neurog3 were previously noted in regeneration processes [START_REF] Criscimanna | Duct cells contribute to regeneration of endocrine and acinar cells following pancreatic damage in adult mice[END_REF]. While ductal cells exhibit low proliferation rates in normal conditions [START_REF] Kopp | Stem cells versus plasticity in liver and pancreas regeneration[END_REF]

I. Somatostatin: a signal inducing -cell regeneration?

We hypothesized that the Pax4-mediated conversion of somatostatinexpressing cells induces a disruption of islet homeostasis, further leading to the recruitment of Neurog3-reexpressing ductal precursor cells adopting an endocrine cell identity to counteract the loss of -cells. However, further investigations are required.

Thus, the treatment of Sst-Cre::Pax4-OE mice with a somatostatin agonist or antagonist would allow for a better comprehension of such mechanisms. More specifically, the supplementation of Sst-Cre::Pax4-OE transgenics with a somatostatin agonist is expected to prevent Neurog3 re-expression and therefore like cell hyperplasia. In this context, one could wonder whether somatostatin antagonist administration to wild-type mice would induce such regeneration.

However, somatostatin exerts a wide-range of effects and its inhibition could lead to undesirable side effects. In addition, the precise expression of the different SST receptors within endocrine cells remains controversial. Therefore, inhibition of one or several of these receptors could have an effect on other islet cells, provoking uncontrolled side-effects. Several studies focusing on the effects of somatostatin receptor deficiency demonstrated changes in insulin secretion and alterations in islet architecture depending on the isoform deleted. SSTR2 loss leads to increased glucagon secretion while no difference in insulin levels was observed [START_REF] Strowski | Somatostatin inhibits insulin and glucagon secretion via two receptors subtypes: an in vitro study of pancreatic islets from somatostatin receptor 2 knockout mice[END_REF]. Ablation of SSTR1 or SSTR5 mostly results in glucose intolerance with decreased numbers of somatostatin-expressing cells being observed in SSTR1 deficient mice [START_REF] Wang | Alterations in glucose homeostasis in SSTR1 gene-ablated mice[END_REF]X. P. Wang et al., 2005). Remarkably, double mutant mice for SSTR1 and 5 show improved glucose tolerance, increased insulin secretion and islet cell hyperplasia [START_REF] Wang | Double-gene ablation of SSTR1 and SSTR5 results in hyperinsulinemia and improved glucose tolerance in mice[END_REF]. Although intriguing, understanding such results is challenging due to possible compensation in the expression of the different receptor subtypes. Moreover, the ability for somatostatin receptor to heterodimerize adds further difficulties in the interpretation [START_REF] Pfeiffer | Homo-and heterodimerization of somatostatin receptor subtypes. Inactivation of sst3 receptor function by heterodimerization with sst2A[END_REF][START_REF] Wang | Alterations in glucose homeostasis in SSTR1 gene-ablated mice[END_REF]X. P. Wang et al., 2005).

In addition, the production of SST-28 was found increased in type 1 diabetic patients and a -cell hyperplasia was observed (Xaio Ping Wang et al., 2005).

Understanding the role of SST and its receptors in the context of type 1 diabetes would therefore be of great interest. Importantly, we strongly believe that islet homeostasis is not only preserved by the hormones secreted by the different endocrine cell types, but also by cellular interactions, integrity and cohesion.

II. Model proposed

The aim of this study was to investigate the plasticity of -cells, aiming to eventually determine whether the sole misexpression of Pax4 in -cells could induce their conversion into insulin-producing cells in vivo. To address this question, we generated Sst-Cre::Pax4-OE double transgenic animals allowing the misexpression of Pax4 specifically in -cells. In this model, we demonstrated the conversion of adult somatostatin-expressing cells into insulin + cells upon the sole misexpression of Pax4.

Our data suggest that such conversion induces a disruption of islet homeostasis, leading to a reawakening of Neurog3-controlled endocrine differentiation program and associated developmental processes, including EMT (Figure 25). Importantly, we observed a progressive islet neogenesis and islet hypertrophy together with an increase in endocrine cell numbers, these augmentations plateauing after 5 months of age.

  INTRODUCTION ..................................................................................... 1 I. Diabetes Mellitus ............................................................................................

Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 7 :Figure 8 :Figure 9 :Figure 10 :Figure 11 : 44 Figure 12 :Figure 14 :Figure 15 :Figure 16 :Figure 17 :Figure 18 :Figure 19 :Figure 20 :Figure 24 :Figure 25 :

 12345678910114412141516171819202425 Figure 1: The two main types of diabetes -Type 1 and Type 2. ................................ 4

Figure 1 :

 1 Figure 1: The two main types of diabetes -Type 1 and Type 2. In healthy subjects,

Figure 2 :

 2 Figure 2: Human pancreas anatomy and histology (adapted from Encyclopaedia Britannica, 2010).

Figure 3 :

 3 Figure 3: Microarchitecture of human and mouse islets. Architecture of the human islet with non--cells scattered within -cell mass (A). Mouse islets display a mantle of non--cells at the periphery and a core typically constituted of -cells (B).

Figure 4 :

 4 Figure 4: Endocrine cell proportions of human and mouse islets. Endocrine proportions of human islets showing a majority of -and -cells (A). Differences in proportions in mouse islets are observed although -and -cells remain the main endocrine cells (B).

Figure 5 :

 5 Figure 5: Insulin release from pancreatic -cells (adapted from[START_REF] Ashcroft | K ATP channels and islet hormone secretion : new insights and controversies[END_REF]. Insulin secretion is inhibited at low glucose concentrations. At high glucose levels, glucose enters the -cell through its receptor GLUT2, increasing intracellular ATP concentration further leading to the closure of KATP channels, the depolarization of the plasma membrane and the opening of the Ca 2+ channels. As a result, the intracellular concentration of Ca2 + increases causing the exocytosis of insulin granules.

Figure 6 :

 6 Figure 6: Gut tube patterning and endoderm-derived organs (adapted from[START_REF] Zorn | Vertebrate endoderm development and organ formation[END_REF]. Schematic representation of the gut tube of a mouse embryo at E10.5. The gut tube is divided into Foregut, Midgut and Hindgut segments through anterior-posterior and dorsal-ventral axes and will give rise to digestive and respiratory tracts and associated organs.

Figure 7 :

 7 Figure 7: Ventral and dorsal pancreas specification (adapted from Mastracci and Sussel, 2012). Most signals received by the dorsal pancreas originate from the notochord (A), while the ventral pancreas is subjected to septum transversum mesenchyme and cardiac mesoderm signals (B).

Figure 8 :

 8 Figure 8: Primary and secondary transitions(from Benitez et al., 2012). Mouse pancreas development is characterized by two overlapping transitions from E9.5 to birth. MPCs proliferation induces the thickening of the foregut endoderm therefore leading to pancreatic bud emergence(A) and microlumen formation (B). The onset of the secondary transition is marked by the segregation of two domains known as tip/trunk compartmentalization with Ptf1a and Nkx6.1 as key regulators (C). The bipotent "trunk" cells express Nkx6.1 and will give rise to endocrine and ductal cells while the multipotent "tip" domain contains MPCs capable of differentiating into acinar, ductal and endocrine cells, quickly restricted to acinar progenitors (D).

Figure 9 :

 9 Figure 9: Simplified model of pancreatic cell fate determination (adapted from[START_REF] Shih | Pancreas Organogenesis: From Lineage Determination to Morphogenesis[END_REF]. Multipotent pancreatic progenitors (MPCs) located in the prepancreatic epithelium express a set of TFs, key regulators of pancreas development (including Pdx1, Hnf1, Ptf1a, Mnx1 and Sox9). At the onset of the secondary transition, the tip/trunk segregation appears dependent on the Notch signaling pathway. The specification of the acinar, ductal and endocrine cell lineages is governed by additional TFs, acting in synergy and described in the main text.

  activation and repression of Neurog3 expression have been suggested to be dependent on Notch signaling. A recent study demonstrated that high Notch activity induces the expressions of Hes1 and Sox9, leading to Neurog3 inhibition and the subsequent blockade of endocrine differentiation. However, intermediate Notch activity leads to the expression of Sox9 alone and the derepression of Neurog3

  c. -cellssomatostatin Somatostatin-producing -cell specification during pancreas development is not well understood, mostly due to a lack of identification of -cell-specific transcription factors. As previously mentioned, total depletion of Pax4 expression leads to an absence -cells (Sosa-Pineda et al., 1997). Conversely, Ganon et al. observed increased numbers of -cells after specific the deletion of Pdx1 in -cells, such supplementary somatostatin-expressing cells not arising from the conversion of

Figure 10 :

 10 Figure 10: Cell sources used in the replacement of -cells (adapted from[START_REF] Bouwens | The use of stem cells for pancreatic regeneration in diabetes mellitus[END_REF]. Able of long-term self-renewal, pluripotent (1) or multipotent stem cells (2) are used for directed differentiation into insulin-secreting cells. Multiple strategies using endogenous pancreatic cells for -cell replacement involve acinar (3), duct (4) or endocrine cell conversion (5) or -cell self-replication (6).

  requires embryos at the blastocyst stage to create ESC lines, therefore leading to ethical concerns with the use of human embryonic cells[START_REF] De Wert | Human embryonic stem cells: Research, ethics and policy[END_REF][START_REF] Mclaren | Ethical and social considerations of stem cell research[END_REF]. Another barrier is that transplanted exogenous ESCs can trigger host immune rejection, thus requiring lifelong immunosuppressive treatments. Induced pluripotent stem cells (iPSCs) represent a second type of PSCs, exhibiting similar properties to ESCs. They are obtained from the reprogramming of somatic cells into pluripotent cells able to self-renew. By expressing 4 specific transcription factors, Oct4, Sox2, Klf4 and c-Myc, fibroblasts were reprogrammed into pluripotent stem cells

  induce their proliferation. An alternative approach used by Cano et al. demonstrated the regeneration of the -cell mass through inactivation of c-Myc expression specifically in insulin-expressing cells. The authors showed that this regeneration occurred via -cell replication[START_REF] Cano | Regulated beta-cell regeneration in the adult mouse pancreas[END_REF]. Inducing -cell self-replication is therefore of interest but unsuitable in advanced stages of T1DM where very few insulin cells remain following autoimmune destruction.Another potential source of -cells was revealed with the discovery of -cell plasticity. Several experimental models contributed to the demonstration of the ability of -cells to convert into insulin-producing cells. Using transgenic mice expressing the diphtheria toxin receptor (DTR) specifically in insulin-cells, Thorel et al. showed that near total -cell loss forced the transdifferentiation of adult -cells into insulinproducing cells able to restore normoglycemia[START_REF] Thorel | Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta-cell loss[END_REF]. Chung et al. also described -to--conversion following the combination of PDL and alloxan-mediated -cells destruction[START_REF] Chung | Pancreatic β-Cell neogenesis by direct conversion from mature α-Cells[END_REF]. Other approaches involving genetic manipulations demonstrated the ability of -cells to transdifferentiate into -like cells upon ectopic expression of Pdx1 in Neurog3-positive cells[START_REF] Yang | Context-specific α -to-β -cell reprogramming by forced Pdx1 expression service re programming by forced Pdx1 expression[END_REF] or through epigenetic modifications[START_REF] Bramswig | Epigenomic plasticity enables human pancreatic alpha to beta cell reprogramming[END_REF]. Moreover, -to--like cell conversion was demonstrated upon Pax4 misexpression/Arx loss-of-function (further detailed below).V. Pax4-mediated -cell regenerationAs discussed in section III, Pax4 is a key transcription factor in specifying and maintaining -cell identity. It was shown that the ectopic expression of Pax4 in adult or embryonic -cells induces their conversion into -like cells[START_REF] Al-Hasani | Adult duct-lining cells can reprogram into β-like cells able to counter repeated cycles of toxin-induced diabetes[END_REF][START_REF] Collombat | The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into α and subsequently β Cells[END_REF]. Conversely, the specific loss of Arx in glucagon-expressing cells similarly triggers their conversion into functional insulin-producing cells[START_REF] Courtney | The Inactivation of Arx in Pancreatic α-Cells Triggers Their Neogenesis and Conversion into Functional β-Like Cells[END_REF].Equally important was the finding that the -to--like cell conversion observed in both models, induces the re-expression of Neurog3 in ductal cells and their differentiation into endocrine cells through the reawakening of the epithelial-tomesenchymal transition (EMT). The newly formed -cells being yet again converted into -like cells upon -cell-specific Pax4 misexpression/Arx loss-of-function, such a cycle of conversion and neogenesis results in -like cell hyperplasia over time (Figure11). Importantly, such β-like cells are functional and can revert the consequences of chemically-induced diabetes.

Figure 11 :

 11 Figure 11: Pax4 misexpression in adult or embryonic -cells induce their neogenesis and conversion into -like cells. Following Pax4 misexpression in α-cells (1), these can be converted into β-like cells (2,3). This leads to a shortage in glucagon (4) responsible for the mobilization of ductal precursor cells, these re-expressing the pro-endocrine gene Neurog3 (5), prior to undergo EMT (6) and adopt an endocrine cell identity (7). Such a continuous cycle of conversion/regeneration results in insulin + cell hyperplasia (8).

Figure 12 :

 12 Figure 12: Transgenic mouse line allowing Pax4 misexpression in somatostatinexpressing cells

  PCR results were analyzed by using the QIAxcel Advanced System(QIAGEN) and the QIAxcel DNA Screening Kit (929004, Qiagen). The presence of the GFP gene was assessed by observation of our mice under blue light while wearing goggles equipped with green filters. -2'-deoxyuridine, BrdU, is a synthetic analogue of thymidine which is incorporated into DNA during the S phase (part of the cell cycle when the DNA is replicated). 5-iodo-2'-deoxyuridine (IdU) and 5-chloro-2'-deoxyuridine (Cldu), are modified forms of BrdU. To assess cellular proliferation, animals were treated with IdU, CldU, or BrdU (MP Biomedicals -Figure13) in drinking water for different durations prior to examination (1 mg/mL). The solution was changed every two days and protected from light. IdU-positive cells were detected using a mouse anti-BrdU primary antibody while CldU-positive cells were detected using a rat anti-BrdU antibody.

Figure 13 :

 13 Figure 13: Cldu and Idu labelling. The first cell division is labeled with CldU (here in green) and the second cell division with IdU (here in red). Therefore, sequential cell division results in co-labeled cells with both CldU and IdU (green/red).

For

  intraperitoneal glucose tolerance tests (ipGTT) and intraperitoneal insulin tolerance tests (ipITT), animals were fasted for 16-18 hours and injected intraperitoneally with 2g/kg of bodyweight of D-(+)-glucose (G7201, Sigma) or 0.75U/kg of insulin (Novo Nordisk A/S). Blood glucose levels were measured at the indicated time points post-injection with a ONETOUCH glucometer (Life Scan, Inc., CA).

  PCR reactions were carried out with the QuantiTect SYBR Green RT-PCR kit (Roche) and Qiagen-validated primer probes on a LightCycler 480 ® instrument (Roche Life Science). The program used for the RT-PCR was the following (Table3), the fluorescence being monitored during each amplification PCR cycle.housekeeping gene GAPDH (validated primers from Qiagen) was used as an internal control to normalize transcript expression.

V

  Mice were euthanized by cervical dislocation and the pancreatic tissue was isolated and placed in cold PBS. Tissue was fixed for 30 min at 4°C in Antigenfix (paraformaldehyde solution pH 7,2-7,4; Microm Microtech France), washed 5 x 20 min in cold PBS and incubated 1 hour in 0.86% saline. Following dehydration through a series of increasing ethanol dilutions (50%, 70%, 80%, 90%, and 100%), the pancreas was treated with isopropanol and toluene, a hydrophobic agent, to remove alcohol from the tissue. Finally, the organ was incubated in clean paraffin baths (1 hour, 3 hours and overnight) and embedded in paraffin in metal molds on a cold plate. Paraffin blocks were stored at 4°C prior to sectioning. 8m sections were cut using a rotary microtome (Leica) and placed on polarized glass slides (Fisher Scientific). Paraffin sections were allowed to dry 45 min at 42°C into a heating block and kept at 37°C overnight. Slides were subsequently stored at 4°C until staining.

Finally

  , slides were washed 3 x 5 min in PBS, dried, and mounted with coverslips and Vectashield HardSet Mounting Medium (Vector) with 4', 6-diamidino-2-phenylindole (DAPI) as a nuclear counterstain. Slides were kept at 4°C prior to observation.

  Figure 14A). Our analyses of the pancreata from the resulting Sst-Cre::ROSA26--

Figure 14 :

 14 Figure 14: Transgenic mouse line allowing Pax4 misexpression in somatostatinexpressing cells. Control Sst-Cre::ROSA26--gal double transgenic mice were obtained by crossing Sst-Cre animals with the ROSA26--gal line (in which the Rosa26 promoter is upstream of a Neomycin resistance cassette and a STOP codon flanked by LoxP sites and followed by the -galactosidase reporter) (A). Sst-Cre mice were also crossed with Pax4-OE animals (in which a CAG promoter is upstream of the GFP gene and a STOP codon flanked by LoxP sites and followed by the Pax4 and the -galactosidase cDNA sequences) (B). In

Figure 15 :

 15 Figure 15: Pax4 misexpression in -cells leads to islet hypertrophy and -cell hyperplasia. Representative pictures of insulin (red) immunohistochemical analyses on pancreas sections of 2-(B), 5-(C) and 8-(D) month old transgenics and controls (A) showing a dramatic increase in islet number and size. Quantitative analyses of at least 3 animals from each group revealed a ~1.5x increase in islet size (F) and 2x in islet number (E)

  -B), a preferential localization at a pole of the islets, close to adjacent ducts, was evidenced for these non--cells in 2-month old Sst-Cre::Pax4-OE islets (Figure 16C-D).

Figure 16 :

 16 Figure 16: Abnormal positioning of non--cells within the islets of Sst-Cre::Pax4-OE transgenic mice. Representative photographs of somatostatin-, glucagon-and insulinexpressing cells within control islets (A-B) or 2-month old transgenic islets (C-D). While somatostatin-and glucagon-positive cells were found distributed in the mantle of control islet (A-B), a preferential localization close to adjacent ducts of such cells was observed in Sst-Cre::Pax4-OE islets (C-D). For the purpose of clarity, pancreatic ducts are outlined in dashed orange lines. Scale bars, 50m.

  investigate the fate/destiny of Pax4 misexpressing -cells in Sst-Cre::Pax4-OE pancreata, further immunohistochemical analyses were undertaken. Interestingly, a fraction of somatostatin-positive cells were found to ectopically express insulin in 2month old Sst-Cre::Pax4-OE islets (Figures 17B, 18A-B), such bi-hormonal cells being expectedly absent in control pancreata (Figure 17A). As these results suggested a putative conversion of somatostatin + cells into insulin + cells, we investigated the consequences of Pax4 misexpression in -cells using lineage tracing. The expression of the -galactosidase tracer was therefore monitored comparing Sst-Cre::ROSA26--gal double transgenics with Sst-Cre::Pax4-OE animals.

Figure 17 :

 17 Figure 17: Conversion of adult -cells into insulin-producing cells upon Pax4 misexpression. Immunohistochemical analyses on pancreas sections of 2-month old mice revealing cells co-expressing somatostatin and insulin in transgenics (inlet and arrows in B), such cells being absent in control islets (A). Representative images of lineage tracing experiments of somatostatin-expressing cells performed on pancreas sections of 2-(D) and 5-(E) month old transgenics and controls (C). While -galactosidase + cells did not label insulin + cells in control islets (C), immunohistochemical analyses showed several insulin + cells marked by the -galactosidase tracer in Sst-Cre::Pax4-OE pancreata (inlets and arrows in D-E) indicative of a conversion of -cells into insulin + cells. Further examinations revealed that 2.80.25% of insulin + cells expressed -galactosidase in 2-month old transgenics, such

  core and the mantle of 2-month old Sst-Cre::Pax4-OE pancreata (Figure17D, 18C-D). Interestingly, similar lineage tracing experiments performed on 5-month old transgenics revealed an increased number of insulin + cells labeled with galactosidase (Figure17E, 18E-F). These results were further confirmed using quantitative analyses. Indeed, while no insulin + cells expressed -galactosidase in control islets, 2.80.25% and 4.70.27% of insulin + cells expressed -galactosidase in 2-and 5-month old transgenic islets respectively (Figure17F). Importantly, a proportion of 90.87% of supplementary -like cells appeared -galactosidase + in 2month old Sst-Cre::Pax4-OE mice, this proportion being maintained in older transgenics. Together, these findings demonstrate the conversion of somatostatinexpressing cells into insulin + cells upon the sole misexpression of Pax4. Remarkably, the proportion of insulin + cells from a -cell origin faithfully follows the observed islet size increase. This suggests continuous processes of conversion of -cells into insulin + cells.

Figure 18 :

 18 Figure 18: Additional examples illustrating the conversion of -cells to insulinproducing cells upon Pax4 misexpression. Immunohistochemical analyses on pancreas sections of 2-month old mice revealing cells co-expressing somatostatin and insulin-cells in transgenics (inlets in A, B), such cells being absent in control islets. Representative images of lineage tracing experiments of somatostatin-expressing cells performed on pancreas sections of 2-(C-D) and 5-(E-F) month old transgenics, suggesting a -to-insulin + cell conversion. Scale bars, 50m.

Figure 19 :

 19 Figure 19: Increased cell proliferation within the ductal epithelium of Sst-Cre::Pax4-OE pancreata. Cell proliferation was assessed evaluating IdU incorporation (detected with a BrdU antibody) in 2 month-old transgenics and controls after short-(3 days) or long-(10 days) term IdU pulse. Representative images of immunohistochemical analyses on pancreas sections of control (A-C) and Sst-Cre::Pax4-OE (D-F) mice. An increase in proliferation was observed in the ductal lining of 2-month old transgenics after a short-term IdU pulse (A, D).

Figure 20 :

 20 Figure 20: Increased proliferation within the ductal epithelium of Sst-Cre::Pax4-OE pancreata occurring before 2 month of age. CldU and IdU were administered consecutively for 10 days with a washout period of 24 hours (A). Representative pictures of immunohistochemical analyses on pancreas sections of 2-month old transgenics (C-D) and matched controls (B) showing no IdU/CldU double positive cells in both groups. Quantification of endocrine and non-endocrine BrdU + cells in 5-(E) and 8-(F) month old mutants did not reveal any differences compared to controls. For the purpose of clarity, islets are outlined with dashed white lines. All values are depicted as mean SEM of n>3 independent animals. Statistics were performed with Mann-Whitney test. Scale bars, 50m

Figure 21 :

 21 Figure 21: Re-expression of Neurog3 in duct-associated cells upon Pax4 misexpression in -cells and reawakening of epithelial-to-mesenchymal transition. Representative photographs of immunohistochemical analyses on pancreas sections of 2month old transgenics (B, E, G, I) and matched controls (A, D, F, H). Neurog3 expression was assessed in 2-month old controls (A) and transgenics (B). Neurog3 was found reexpressed in the ductal lining (B inlet yellow arrow) and epithelium (B white arrow) of transgenic mice while being absent in controls (A). Accordingly, in Sst-Cre::Pax4-OE

  , H-I). Of note, the examination of 10 days BrdU-treated Sst-Cre::Pax4-OE animals also unraveled a number of duct-lining BrdU + cells expressing Vimentin (Figure 21F-G), such short-term lineage tracing suggesting that proliferating ductal cells undergo EMT prior to acquire an endocrine cell identity. These results indicate that Pax4 misexpression in -cells eventually results in a reawakening of the Neurog3-controlled endocrine differentiation program and associated developmental processes, including EMT.

Figure 22 :

 22 Figure 22: Sst-Cre::Pax4-OE endogenous and supplementary insulin + cells display a cell phenotype. Representative photographs of immunohistochemical analyses on pancreas sections of controls (A-B, E-F, I-J, M-N, Q-R, U-V) and Sst-Cre::Pax4-OE (C-D, G-H, K-L, O-P, S-T, W-X). All insulin + cells uniformly expressed the bona fide -cell markers PC1/3 (A-D), Glut-2 (E-H), Nkx6.1 (I-L), Pdx1 (M-P) and the pan-endocrine markers NeuroD1 (Q-T) and Pax6 (U-X). Scale bars, 50m.

Figure 23 :

 23 Figure 23: Improved -cell function in transgenic Sst-Cre::Pax4-OE mice. 2 month-old transgenic and controls were challenged with glucose. Transgenic mice displayed an improved glucose clearance with a lower peak in glycemia and a faster recovery to euglycemia (A). The relative area under the curve (AUC) confirmed this improved glucose tolerance in 2 month-old Sst-Cre::Pax4-OE animals. Upon insulin tolerance tests (ITT), glycemia levels of 2 month-old transgenic and matched controls were monitored. Transgenic mice exhibited unaltered glycemia levels, indicative of a standard insulin sensitivity (B). AUC were indeed similar in both groups. The relative AUC are expressed as a percentage relative to the average AUC of the controls. All values are depicted as mean SEM of n>3 independent animals. Statistics were determined using Mann-Whitney test ** p<0.01, * p<0.05, ns p>0.05.

  This is further supported by the detection of insulin-expressing cells in islets of control and transgenic mice, at different time after STZ-mediated -cell ablation. Indeed, while after 1 week of STZ administration, control and Sst-Cre::Pax4-OE animals displayed a massive loss of insulin-expressing cells (Figure24B, E), aprogressive restoration of such cells was observed in transgenic mice (Figure24F, G), such partial recovery being absent in matching controls (Figure24C, D).

Figure 24 :

 24 Figure 24: Pax4 misexpression in -cells can induce functional -like cell neogenesis upon chemically-induced -cell ablation. 2.5 month-old mice were treated with streptozotocin and sacrificed at different time points (A). Immunohistochemical analyses of insulin-expressing cells in control (B, D) or transgenic (E, G) islets 1 week, 1 month and 3

  addition, we provide evidence that -cell plasticity does not require -cell ablation but the sole misexpression of Pax4. Finally, the absence of further augmentations in 8months old Sst-Cre::Pax4-OE animals further suggest that -cells might lose their plasticity and ability to convert into insulin + cells after 5 months of age, possibly due to aging processes.While these results are of interest, the mechanisms by which Pax4 induces such -to--like cell conversion remain to be elucidated. Accordingly, the identification of Pax4 targets would provide a better understanding of the mechanisms involved. Interestingly, Hhex could represent a reasonable target as it is required for -cell specification. Surprisingly, the Hhex locus, in both humans and mice, appears to contain binding sites for Pax4. As demonstrated in this work, Pax4 misexpression in -cells induce their reprogramming toward a -like cell identity. One hypothesis could be a role for Pax4 in Hhex inhibition to promote the acquisition of a -like cell phenotype. Hhex expression was investigated in Sst-Cre::Pax4-OE transgenics but none of available Hhex antibodies worked in our hands. Another legitimate alternative would be Mnx1 as its inactivation in endocrine cells induces a -to--cell conversion[START_REF] Pan | Inactivating the permanent neonatal diabetes gene Mnx1 switches insulin-producing β-cells to a δlike fate and reveals a facultative proliferative capacity in aged β-cells[END_REF]. Interestingly, the Mnx1 locus was also found to contain binding sites for Pax4. One could therefore assume that Pax4 could be an indirect or a direct activator of Mnx1 expression, its ectopic expression in -cells leading to their reorientation toward a -cell phenotype. In addition,Chera et al. suggested that adult -cells can convert into -like cells upon FoxO1 inhibition. In all three instances, it would be of interest to investigate the involvement of these putative targets in the processes underlying the reprogramming of -cells into -like cells.II. Recruitment of Neurog3 expressing ductal cells and EMT reawakeningIn this study, we demonstrated that a proportion of 9% of supplementary -like cells appeared to arise from -cells in 2-month old Sst-Cre::Pax4-OE mice, this proportion being maintained in older transgenics. Therefore, the observed conversion rate of -cells misexpressing Pax4 cannot explain such increase in islet count and size, suggestive of the involvement of additional processes.As previously mentioned, Neurog3 is a pro-endocrine gene only expressed in endocrine progenitors during development, and is responsible for the generation of the main endocrine cell types. Here, we demonstrate the re-expression of Neurog3 within or close to the ductal lining epithelium upon Pax4 misexpression in somatostatin-expressing cells. Emphasizing that Neurog3 is responsible for the specification of all the endocrine cell lineages following specific ratios, the generation of endocrine cell types from the adult ductal epithelium would respect the standard endocrine proportions (80% of -cells/10% of -cells/10% of -cells). Our analyses of islets from animals misexpressing Pax4 in -cells support this hypothesis with a clear favoring toward -like cells neogenesis. Accordingly, our results demonstrate an increase in -cell contents perfectly matching the progressive islet hypertrophy.

  , our analyses reveal a significant augmentation in cell proliferation within the ductal epithelium, a number of these proliferating cells eventually acquiring a mesenchymal phenotype. Together, these results clearly suggest that Pax4 misexpression in -cells induces their conversion into -like cells and a subsequent cycle of endocrine cell neogenesis through recapitulation of developmental processes. Our analyses also demonstrate that Pax4 misexpression in -cells induces their conversion into -like cells following chemically-induced -cell ablation, resulting in a partial -cell mass restoration. Although not exhibiting euglycemia as expected, Sst-Cre::Pax4-OE transgenics displayed an average glycemia of 350mg/dl and an extended lifespan. Therefore, as -cells plasticity seems to decline with age, it would be of interest to induce -cell ablation before 2 months of age. Accordingly, additional experiments putting to the test -cells plasticity at 1 month of age are currently ongoing. Moreover, to definitively ascertain the requirement of Neurog3 for the endocrine cell neogenesis observed in Sst-Cre::Pax4-OE transgenics, it would be of interest to use an inducible knock down of Neurog3 within the ductal epithelium.

Figure 25 :

 25 Figure 25: Neurog3-mediated EMT reawakening upon Pax4 misexpression in -cells.Following Pax4 misexpression in -cells (1), these can be converted into β-like cells (2,3). This leads to the disruption of islet homeostasis responsible for the mobilization of ductal precursor cells (4), these re-expressing the pro-endocrine gene Neurog3 (5). Neurog3endocrine progenitor cells give rise to insulin + , glucagon + and somatostatin + cells following normal endocrine endogenous proportions prior to undergo EMT (6). Such a continuous cycle of conversion/regeneration results in insulin + cell hyperplasia and islet hypertrophy (7).

TABLE OF CONTENT

 OF

TABLE INDEX Table 1 :

 INDEX1 Blood glucose levels for non-diabetic, prediabetic and diabetic patients .....

Table 2 :

 2 Primer sequences used for genotyping .......................................................

Table 3 :

 3 RT PCR program used ................................................................................

Table 4 :

 4 Primary antibodies used for IHC ..................................................................

Table 5 :

 5 Secondary antibodies used for IHC .............................................................

Table 1 : Blood glucose levels in non-diabetic and diabetic patients

 1

(adapted from the glocal diabetes community website http://www.diabetes.co.uk/).

Table 2 : Primer sequences used for genotyping

 2

Table 3 : RT PCR program used

 3

Table 4 : Primary antibodies used for IHC

 4

Table 5 : Secondary antibodies used for IHC

 5

ACKNOWLEDGEMENTS

Soyons reconnaissants aux personnes qui nous donnent du bonheur ;

elles sont les charmants jardiniers par qui nos âmes sont fleuries.

Marcel Proust

VIII. Data analyses and statistics

All values are depicted as mean ± SEM of at least data from 3 animals. Data were analyzed using GraphPrism 6 software. Normality was tested using D'Agostino-Pearson omnibus normality test and appropriate statistical tests were performed.

Results are considered significant if p < 0.0001 (****), p < 0.001 (***), p < 0.01 (**), and p < 0.05 (*).

VI. Supplementary insulin-expressing cells in Sst

DISCUSSION

CONCLUSION / PERSPECTIVES