Patrick Collombat

The Ectopic Expression of Pax4 in the Mouse Pancreas Converts Progenitor Cells into α and Subsequently β Cells

We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell mass and curing diabetes in animals that have been chemically depleted of beta cells.

Embryonic endocrine pancreas and mature β cells acquire α and PP cell phenotypes upon Arx misexpression

Aristaless-related homeobox (Arx) was recently demonstrated to be involved in pancreatic alpha cell fate specification while simultaneously repressing the beta and delta cell lineages. To establish whether Arx is not only necessary, but also sufficient to instruct the alpha cell fate in endocrine progenitors, we used a gain-of-function approach to generate mice conditionally misexpressing this factor. Mice with forced Arx expression in the embryonic pancreas or in developing islet cells developed a dramatic hyperglycemia and eventually died. Further analysis demonstrated a drastic loss of beta and delta cells. Concurrently, a remarkable increase in the number of cells displaying alpha cell or, strikingly, pancreatic polypeptide (PP) cell features was observed. Notably, the ectopic expression of Arx induced in embryonic or adult beta cells led to a loss of the beta cell phenotype and a concomitant increase in a number of cells with alpha or PP cell characteristics. Combining quantitative real-time PCR and lineage-tracing experiments, we demonstrate that, in adult mice, the misexpression of Arx, rather than its overexpression, promotes a conversion of beta cells into glucagon- or PP-producing cells in vivo. These results provide important insights into the complex mechanisms underlying proper pancreatic endocrine cell allocation and cell identity acquisition.

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

The specification of the different mouse pancreatic endocrine subtypes is determined by the concerted activities of transcription factors. However, the molecular mechanisms regulating endocrine fate allocation remain unclear. In the present study, we uncover the molecular consequences of the simultaneous depletion of Arx and Pax4 activity during pancreas development. Our findings reveal a so far unrecognized essential role of the paired-box-encoding Pax4 gene. Specifically, in the combined absence of Arx and Pax4, an early-onset loss of mature α- and β-cells occurs in the endocrine pancreas, concomitantly with a virtually exclusive generation of somatostatin-producing cells. Furthermore, despite normal development of the PP-cells in the double-mutant embryos, an atypical expression of the pancreatic polypeptide (PP) hormone was observed in somatostatin-labelled cells after birth. Additional characterizations indicate that such an expression of PP was related to the onset of feeding, thereby unravelling an epigenetic control. Finally, our data provide evidence that both Arx and Pax4 act as transcriptional repressors that control the expression level of one another, thereby mediating proper endocrine fate allocation.

IA1 is NGN3-dependent and essential for differentiation of the endocrine pancreas

Neurogenin 3 (Ngn3) is key for endocrine cell specification in the embryonic pancreas and induction of a neuroendocrine cell differentiation program by misexpression in adult pancreatic duct cells. We identify the gene encoding IA1, a zinc‐finger transcription factor, as a direct target of Ngn3 and show that it forms a novel branch in the Ngn3‐dependent endocrinogenic transcription factor network. During embryonic development of the pancreas, IA1 and Ngn3 exhibit nearly identical spatio‐temporal expression patterns. However, embryos lacking Ngn3 fail to express IA1 in the pancreas. Upon ectopic expression in adult pancreatic duct cells Ngn3 binds to chromatin in the IA1 promoter region and activates transcription. Consistent with this direct effect, IA1 expression is normal in embryos mutant for NeuroD1, Arx, Pax4 and Pax6, regulators operating downstream of Ngn3. IA1 is an effector of Ngn3 function as inhibition of IA1 expression in embryonic pancreas decreases the formation of insulin‐ and glucagon‐positive cells by 40%, while its ectopic expression amplifies neuroendocrine cell differentiation by Ngn3 in adult duct cells. IA1 is therefore a novel Ngn3‐regulated factor required for normal differentiation of pancreatic endocrine cells.

Specifying pancreatic endocrine cell fates.

Cell replacement therapy could represent an attractive alternative to insulin injections for the treatment of diabetes. However, this approach requires a thorough understanding of the molecular switches controlling the specification of the different pancreatic cell-types in vivo. These are derived from an apparently identical pool of cells originating from the early gut endoderm, which are successively specified towards the pancreatic, endocrine, and hormone-expressing cell lineages. Numerous studies have outlined the crucial roles exerted by transcription factors in promoting the cell destiny, defining the cell identity and maintaining a particular cell fate. This review focuses on the mechanisms regulating the morphogenesis of the pancreas with particular emphasis on recent findings concerning the transcription factor hierarchy orchestrating endocrine cell fate allocation.

Inactivation of Arx, the murine ortholog of the X-linked lissencephaly with ambiguous genitalia gene, leads to severe disorganization of the ventral telencephalon with impaired neuronal migration and differentiation

ARX loss-of-function mutations cause X-linked lissencephaly with ambiguous genitalia (XLAG), a severe neurological condition that results in profound brain malformations, including microcephaly, absence of corpus callosum, and impairment of the basal ganglia. Despite such dramatic defects, their nature and origin remain largely unknown. Here, we used Arx mutant mice as a model to characterize the cellular and molecular mechanisms underlying the basal ganglia alterations. In these animals, the early differentiation of this tissue appeared normal, whereas subsequent differentiation was impaired, leading to the periventricular accumulation of immature neurons in both the lateral ganglionic eminence and medial ganglionic eminence (MGE). Both tangential migration toward the cortex and striatum and radial migration to the globus pallidus and striatum were greatly reduced in the mutants, causing a periventricular accumulation of NPY+ or calretinin+ neurons in the MGE. Arx mutant neurons retained their differentiation potential in vitro but exhibited deficits in morphology and migration ability. These findings imply that cell-autonomous defects in migration underlie the neuronal localization defects. Furthermore, Arx mutants lacked a large fraction of cholinergic neurons and displayed a strong impairment of thalamocortical projections, in which major axon fiber tracts failed to traverse the basal ganglia. Altogether, these results highlight the critical functions of Arx in promoting neural migration and regulating basal ganglia differentiation in mice, consistent with the phenotype of XLAG patients.

Arx Is a Direct Target of Dlx2 and Thereby Contributes to the Tangential Migration of GABAergic Interneurons

The Arx transcription factor is expressed in the developing ventral telencephalon and subsets of its derivatives. Mutation of human ARX ortholog causes neurological disorders including epilepsy, lissencephaly, and mental retardation. We have isolated the mouse Arx endogenous enhancer modules that control its tightly compartmentalized forebrain expression. Interestingly, they are scattered downstream of its coding region and partially included within the introns of the downstream PolA1 gene. These enhancers are ultraconserved noncoding sequences that are highly conserved throughout the vertebrate phylum. Functional characterization of the Arx GABAergic enhancer element revealed its strict dependence on the activity of Dlx transcription factors. Dlx overexpression induces ectopic expression of endogenous Arx and its isolated enhancer, whereas loss of Dlx expression results in reduced Arx expression, suggesting that Arx is a key mediator of Dlx function. To further elucidate the mechanisms involved, a combination of gain-of-function studies in mutant Arx or Dlx tissues was pursued. This analysis provided evidence that, although Arx is necessary for the Dlx-dependent promotion of interneuron migration, it is not required for the GABAergic cell fate commitment mediated by Dlx factors. Although Arx has additional functions independent of the Dlx pathway, we have established a direct genetic relationship that controls critical steps in the development of telencephalic GABAergic neurons. These findings contribute elucidating the genetic hierarchy that likely underlies the etiology of a variety of human neurodevelopmental disorders.

ghrelin Is a Novel Target of Pax4 in Endocrine Progenitors of the Pancreas and Duodenum

Pax4‐deficient mice have a severe gastrointestinal endocrine deficiency: they lack most pancreatic cells that produce insulin or somatostatin and various duodenal endocrine cell types. Remarkably, Pax4‐deficient mice also have an overabundance of ghrelin‐expressing cells in the pancreas and duodenum. Detailed analysis of the Pax4 nullizygous pancreas determined that the mutant islets are largely composed of a distinctive endocrine cell type that expresses ghrelin, glucagon, islet amyloid polypeptide (IAPP), and low levels of Pdx1. Lineage‐tracing analysis revealed that most of these unique endocrine cells directly arose from Pax4‐deficient progenitors. Previous in vitro work reported that Pax4 is a transcriptional repressor of islet amyloid polypeptide (IAPP) and glucagon. In this study, we expanded those results by showing that Pax4 is also a repressor of gherlin. Together, our data further support the notion that Pax4 activity is necessary to establish appropriate patterns of gene expression in endocrine progenitors of the digestive tract. Developmental Dynamics 237:51–61, 2008.

Reprogramming into pancreatic endocrine cells based on developmental cues.

Due to the increasing prevalence of type 1 diabetes and the complications arising from actual therapies, alternative treatments need to be established. In order to compensate the beta-cell deficiency associated with type 1 diabetes, current research focuses on new strategies to generate insulin-producing beta-cells for transplantation purpose, including the differentiation of stem or progenitor cells, as well as the transdifferentiation of dispensable mature cell types. However, to successfully force specific cells to adopt a functional beta-cell fate or phenotype, a better understanding of the molecular mechanisms underlying beta-cell genesis is required. The present short review summarizes the hitherto known functions and interplays of several key factors involved in the development of the different endocrine cell lineages during pancreas morphogenesis, as well as their potential to direct the generation of beta-cells. Furthermore, an emphasis is made on beta-cell regeneration and the determinants implicated.

The homeobox gene Arx is a novel positive regulator of embryonic myogenesis

Skeletal muscle fibers form in overlapping, but distinct phases that depend on the generation of temporally different lineages of myogenic cells. During primary myogenesis (E10.5–E12.5 in the mouse), embryonic myoblasts fuse homotypically to generate primary fibers, whereas during later development (E14.5–E17.5), fetal myoblasts differentiate into secondary fibers. How these myogenic waves are regulated remains largely unknown. Studies have been hampered by the lack of markers which would distinguish embryonic from fetal myoblast populations. We show here that the homeobox gene Arx is strongly expressed in differentiating embryonic muscle, downstream of myogenic basic helix–loop–helix (bHLH) genes. Its expression progressively decreases during development. When overexpressed in the C2C12 myogenic cell line, Arx enhances differentiation. Accordingly, it stimulates the transcriptional activity from the Myogenin promoter and from multimerized E-boxes when co-expressed with MyoD and Mef2C in CH310T1/2. Furthermore, Arx co-immunoprecipitates with Mef2C, suggesting that it participates in the transcriptional regulatory network acting in embryonic muscle. Finally, embryonic myoblasts isolated from Arx-deficient embryos show a delayed differentiation in vivo together with an enhanced clonogenic capacity in vitro. We propose here that Arx acts as a novel positive regulator of embryonic myogenesis by synergizing with Mef2C and MyoD and by establishing an activating loop with Myogenin.