Special Issue: "Digestive Inflammation and New Therapeutical Targets".

Inflammatory diseases commonly associated with humans are chronic inflammatory gastrointestinal diseases (CIGDs) [...].

Acinar-to-Ductal Metaplasia (ADM): On the Road to Pancreatic Intraepithelial Neoplasia (PanIN) and Pancreatic Cancer.

Adult pancreatic acinar cells show high plasticity allowing them to change in their differentiation commitment. Pancreatic acinar-to-ductal metaplasia (ADM) is a cellular process in which the differentiated pancreatic acinar cells transform into duct-like cells. This process can occur as a result of cellular injury or inflammation in the pancreas. While ADM is a reversible process allowing pancreatic acinar regeneration, persistent inflammation or injury can lead to the development of pancreatic intraepithelial neoplasia (PanIN), which is a common precancerous lesion that precedes pancreatic ductal adenocarcinoma (PDAC). Several factors can contribute to the development of ADM and PanIN, including environmental factors such as obesity, chronic inflammation and genetic mutations. ADM is driven by extrinsic and intrinsic signaling. Here, we review the current knowledge on the cellular and molecular biology of ADM. Understanding the cellular and molecular mechanisms underlying ADM is critical for the development of new therapeutic strategies for pancreatitis and PDAC. Identifying the intermediate states and key molecules that regulate ADM initiation, maintenance and progression may help the development of novel preventive strategies for PDAC.

Insights into the etiology and physiopathology of MODY5/HNF1B pancreatic phenotype with a mouse model of the human disease.

Maturity-onset diabetes of the young type 5 (MODY5) is due to heterozygous mutations or deletion of HNF1B. No mouse models are currently available to recapitulate the human MODY5 disease. Here, we investigate the pancreatic phenotype of a unique MODY5 mouse model generated by heterozygous insertion of a human HNF1B splicing mutation at the intron-2 splice donor site in the mouse genome. This Hnf1bsp2/+ model generated with targeted mutation of Hnf1b mimicking the c.544+1G>T (T) mutation identified in humans, results in alternative transcripts and a 38% decrease of native Hnf1b transcript levels. As a clinical feature of MODY5 patients, the hypomorphic mouse model Hnf1bsp2/+ displays glucose intolerance. Whereas Hnf1bsp2/+ isolated islets showed no altered insulin secretion, we found a 65% decrease in pancreatic insulin content associated with a 30% decrease in total large islet volume and a 20% decrease in total ß-cell volume. These defects were associated with a 30% decrease in expression of the pro-endocrine gene Neurog3 that we previously identified as a direct target of Hnf1b, showing a developmental etiology. As another clinical feature of MODY5 patients, the Hnf1bsp2/+ pancreases display exocrine dysfunction with hypoplasia. We observed chronic pancreatitis with loss of acinar cells, acinar-to-ductal metaplasia, and lipomatosis, with upregulation of signaling pathways and impaired acinar cell regeneration. This was associated with ductal cell deficiency characterized by shortened primary cilia. Importantly, the Hnf1bsp2/+ mouse model reproduces the pancreatic features of the human MODY5/HNF1B disease, providing a unique in vivo tool for molecular studies of the endocrine and exocrine defects and to advance basic and translational research. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Pancreatic Ductal Deletion of Hnf1b Disrupts Exocrine Homeostasis, Leads to Pancreatitis, and Facilitates Tumorigenesis.

BACKGROUND & AIMS: The exocrine pancreas consists of acinar cells that produce digestive enzymes transported to the intestine through a branched ductal epithelium. Chronic pancreatitis is characterized by progressive inflammation, fibrosis, and loss of acinar tissue. These changes of the exocrine tissue are risk factors for pancreatic cancer. The cause of chronic pancreatitis cannot be identified in one quarter of patients. Here, we investigated how duct dysfunction could contribute to pancreatitis development. METHODS: The transcription factor Hnf1b, first expressed in pancreatic progenitors, is strictly restricted to ductal cells from late embryogenesis. We previously showed that Hnf1b is crucial for pancreas morphogenesis but its postnatal role still remains unelucidated. To investigate the role of pancreatic ducts in exocrine homeostasis, we inactivated the Hnf1b gene in vivo in mouse ductal cells. RESULTS: We uncovered that postnatal Hnf1b inactivation in pancreatic ducts leads to chronic pancreatitis in adults. Hnf1bΔduct mutants show dilatation of ducts, loss of acinar cells, acinar-to-ductal metaplasia, and lipomatosis. We deciphered the early events involved, with down-regulation of cystic disease-associated genes, loss of primary cilia, up-regulation of signaling pathways, especially the Yap pathway, which is involved in acinar-to-ductal metaplasia. Remarkably, Hnf1bΔduct mutants developed pancreatic intraepithelial neoplasia and promote pancreatic intraepithelial neoplasia progression in concert with KRAS. We further showed that adult Hnf1b inactivation in pancreatic ducts is associated with impaired regeneration after injury, with persistent metaplasia and initiation of neoplasia. CONCLUSIONS: Loss of Hnf1b in ductal cells leads to chronic pancreatitis and neoplasia. This study shows that Hnf1b deficiency may contribute to diseases of the exocrine pancreas and gains further insight into the etiology of pancreatitis and tumorigenesis.

Adaptive ß-Cell Neogenesis in the Adult Mouse in Response to Glucocorticoid-Induced Insulin Resistance.

Both type 1 and type 2 diabetes are characterized by deficient insulin secretion and decreased ß-cell mass. Thus, regenerative strategies to increase ß-cell mass need to be developed. To characterize mechanisms of ß-cell plasticity, we studied a model of severe insulin resistance in the adult mouse and defined how ß-cells adapt. Chronic corticosterone (CORT) treatment was given to adult mice and led to rapid insulin resistance and adaptive increased insulin secretion. Adaptive and massive increase of ß-cell mass was observed during treatment up to 8 weeks. ß-Cell mass increase was partially reversible upon treatment cessation and reinduced upon subsequent treatment. ß-Cell neogenesis was suggested by an increased number of islets, mainly close to ducts, and increased Sox9 and Ngn3 mRNA levels in islets, but lineage-tracing experiments revealed that neoformed ß-cells did not derive from Sox9- or Ngn3-expressing cells. CORT treatment after ß-cell depletion partially restored ß-cells. Finally, ß-cell neogenesis was shown to be indirectly stimulated by CORT because serum from CORT-treated mice increased ß-cell differentiation in in vitro cultures of pancreatic buds. Altogether, the results present a novel model of ß-cell neogenesis in the adult mouse and identify the presence of neogenic factors in the serum of CORT-treated mice.

Metabolic Profiling of Multiorgan Samples: Evaluation of MODY5/RCAD Mutant Mice.

In the present study, we performed a metabolomics analysis to evaluate a MODY5/RCAD mouse mutant line as a potential model for HNF1B-associated diseases. Gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) of gut, kidney, liver, muscle, pancreas, and plasma samples uncovered the tissue specific metabolite distribution. Orthogonal projections to latent structures discriminant analysis (OPLS-DA) was used to identify the differences between MODY5/RCAD and wild-type mice in each of the tissues. The differences included, for example, increased levels of amino acids in the kidneys and reduced levels of fatty acids in the muscles of the MODY5/RCAD mice. Interestingly, campesterol was found in higher concentrations in the MODY5/RCAD mice, with a four-fold and three-fold increase in kidneys and pancreas, respectively. As expected, the MODY5/RCAD mice displayed signs of impaired renal function in addition to disturbed liver lipid metabolism, with increased lipid and fatty acid accumulation in the liver. From a metabolomics perspective, the MODY5/RCAD model was proven to display a metabolic pattern similar to what would be suspected in HNF1B-associated diseases. These findings were in line with the presumed outcome of the mutation based on the different anatomy and function of the tissues as well as the effect of the mutation on development.

Class I histone deacetylase inhibition improves pancreatitis outcome by limiting leukocyte recruitment and acinar-to-ductal metaplasia.

BACKGROUND AND PURPOSE: Pancreatitis is a common inflammation of the pancreas with rising incidence in many countries. Despite improvements in diagnostic techniques, the disease is associated with high risk of severe morbidity and mortality and there is an urgent need for new therapeutic interventions. In this study, we evaluated whether histone deacetylases (HDACs), key epigenetic regulators of gene transcription, are involved in the development of the disease. EXPERIMENTAL APPROACH: We analysed HDAC regulation during cerulein-induced acute, chronic and autoimmune pancreatitis using different transgenic mouse models. The functional relevance of class I HDACs was tested with the selective inhibitor MS-275 in vivo upon pancreatitis induction and in vitro in activated macrophages and primary acinar cell explants. KEY RESULTS: HDAC expression and activity were up-regulated in a time-dependent manner following induction of pancreatitis, with the highest abundance observed for class I HDACs. Class I HDAC inhibition did not prevent the initial acinar cell damage. However, it effectively reduced the infiltration of inflammatory cells, including macrophages and T cells, in both acute and chronic phases of the disease, and directly disrupted macrophage activation. In addition, MS-275 treatment reduced DNA damage in acinar cells and limited acinar de-differentiation into acinar-to-ductal metaplasia in a cell-autonomous manner by impeding the EGF receptor signalling axis. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that class I HDACs are critically involved in the development of acute and chronic forms of pancreatitis and suggest that blockade of class I HDAC isoforms is a promising target to improve the outcome of the disease.

Implication of epigenetics in pancreas development and disease.

Pancreas development is controlled by a complex interaction of signaling pathways and transcription factor networks that determine pancreatic specification and differentiation of exocrine and endocrine cells. Epigenetics adds a new layer of gene regulation. DNA methylation, histone modifications and non-coding RNAs recently appeared as important epigenetic factors regulating pancreas development. In this review, we report recent findings obtained by analyses in model organisms as well as genome-wide approaches that demonstrate the role of these epigenetic regulators in the control of exocrine and endocrine cell differentiation, identity, function, proliferation and regeneration. We also highlight how altered epigenetic processes contribute to pancreatic disorders: diabetes and pancreatic cancer. Uncovering these epigenetic events can help to better understand these diseases, provide novel therapeutical targets for their treatment, and improve cell-based therapies for diabetes.

Multi-Organ Contribution to the Metabolic Plasma Profile Using Hierarchical Modelling.

Hierarchical modelling was applied in order to identify the organs that contribute to the levels of metabolites in plasma. Plasma and organ samples from gut, kidney, liver, muscle and pancreas were obtained from mice. The samples were analysed using gas chromatography time-of-flight mass spectrometry (GC TOF-MS) at the Swedish Metabolomics centre, Umeå University, Sweden. The multivariate analysis was performed by means of principal component analysis (PCA) and orthogonal projections to latent structures (OPLS). The main goal of this study was to investigate how each organ contributes to the metabolic plasma profile. This was performed using hierarchical modelling. Each organ was found to have a unique metabolic profile. The hierarchical modelling showed that the gut, kidney and liver demonstrated the greatest contribution to the metabolic pattern of plasma. For example, we found that metabolites were absorbed in the gut and transported to the plasma. The kidneys excrete branched chain amino acids (BCAAs) and fatty acids are transported in the plasma to the muscles and liver. Lactic acid was also found to be transported from the pancreas to plasma. The results indicated that hierarchical modelling can be utilized to identify the organ contribution of unknown metabolites to the metabolic profile of plasma.

Hnf1b controls pancreas morphogenesis and the generation of Ngn3+ endocrine progenitors.

Heterozygous mutations in the human HNF1B gene are associated with maturity-onset diabetes of the young type 5 (MODY5) and pancreas hypoplasia. In mouse, Hnf1b heterozygous mutants do not exhibit any phenotype, whereas the homozygous deletion in the entire epiblast leads to pancreas agenesis associated with abnormal gut regionalization. Here, we examine the specific role of Hnf1b during pancreas development, using constitutive and inducible conditional inactivation approaches at key developmental stages. Hnf1b early deletion leads to a reduced pool of pancreatic multipotent progenitor cells (MPCs) due to decreased proliferation and increased apoptosis. Lack of Hnf1b either during the first or the secondary transitions is associated with cystic ducts. Ductal cells exhibit aberrant polarity and decreased expression of several cystic disease genes, some of which we identified as novel Hnf1b targets. Notably, we show that Glis3, a transcription factor involved in duct morphogenesis and endocrine cell development, is downstream Hnf1b. In addition, a loss and abnormal differentiation of acinar cells are observed. Strikingly, inactivation of Hnf1b at different time points results in the absence of Ngn3(+) endocrine precursors throughout embryogenesis. We further show that Hnf1b occupies novel Ngn3 putative regulatory sequences in vivo. Thus, Hnf1b plays a crucial role in the regulatory networks that control pancreatic MPC expansion, acinar cell identity, duct morphogenesis and generation of endocrine precursors. Our results uncover an unappreciated requirement of Hnf1b in endocrine cell specification and suggest a mechanistic explanation of diabetes onset in individuals with MODY5.

Epigenetic regulation of pancreatic islets.

Epigenetic mechanisms, including DNA methylation, histone modifications, and noncoding RNA expression, contribute to regulate islet cell development and function. Indeed, epigenetic mechanisms were recently shown to be involved in the control of endocrine cell fate decision, islet differentiation, ß-cell identity, proliferation, and mature function. Epigenetic mechanisms can also contribute to the pathogenesis of complex diseases. Emerging knowledge regarding epigenetic mechanisms suggest that they may be involved in ß-cell dysfunction and pathogenesis of diabetes. Epigenetic mechanisms could predispose to the diabetic phenotype such as decline of ß-cell proliferation ability and ß-cell failure, and account for complications associated with diabetes. Better understanding of epigenetic landscapes of islet differentiation and function may be useful to improve ß-cell differentiation protocols and discover novel therapeutic targets for prevention and treatment of diabetes.

Obesity-induced overexpression of miR-802 impairs glucose metabolism through silencing of Hnf1b.

Insulin resistance represents a hallmark during the development of type 2 diabetes mellitus and in the pathogenesis of obesity-associated disturbances of glucose and lipid metabolism. MicroRNA (miRNA)-dependent post-transcriptional gene silencing has been recognized recently to control gene expression in disease development and progression, including that of insulin-resistant type 2 diabetes. The deregulation of miRNAs miR-143 (ref. 4), miR-181 (ref. 5), and miR-103 and miR-107 (ref. 6) alters hepatic insulin sensitivity. Here we report that the expression of miR-802 is increased in the liver of two obese mouse models and obese human subjects. Inducible transgenic overexpression of miR-802 in mice causes impaired glucose tolerance and attenuates insulin sensitivity, whereas reduction of miR-802 expression improves glucose tolerance and insulin action. We identify Hnf1b (also known as Tcf2) as a target of miR-802-dependent silencing, and show that short hairpin RNA (shRNA)-mediated reduction of Hnf1b in liver causes glucose intolerance, impairs insulin signalling and promotes hepatic gluconeogenesis. In turn, hepatic overexpression of Hnf1b improves insulin sensitivity in Lepr(db/db) mice. Thus, this study defines a critical role for deregulated expression of miR-802 in the development of obesity-associated impairment of glucose metabolism through targeting of Hnf1b, and assigns Hnf1b an unexpected role in the control of hepatic insulin sensitivity.

Comparison of murine embryonic pancreatic development in vitro and in vivo.

OBJECTIVES: The purpose of the present study was to compare the development of murine embryonic pancreas in vitro and in vivo. METHODS: Murine embryonic pancreas at 12.5 days of gestation was dissected and cultured at the air-medium interface. At 1, 3, and 7 days of culture, the characteristics of cultured murine pancreas were assayed and compared with that of pancreas in vivo. RESULTS: The percentage of pancreatic duodenal homeobox-1 (PDX-1) and neurogenin 3 (Ngn3)-positive cells in pancreas cultured for 1 and 3 days was higher than that of pancreas at 13.5 and 15.5 days of gestation. Importantly, in comparison with embryonic pancreas in vivo, more insulin and glucagon-producing cells were developed in cultured pancreas. Furthermore, insulin was released in a regulated manner in response to glucose. The expressional kinetics of pancreatic markers of cultured pancreas was coincident with that of pancreas in vivo. CONCLUSIONS: The development of the murine pancreas cultured at the air-medium interface mimicked that of pancreas in vivo. Our simple culture system might offer the potential of a source of mature ß cells.

Specific control of pancreatic endocrine ß- and δ-cell mass by class IIa histone deacetylases HDAC4, HDAC5, and HDAC9.

OBJECTIVE: Class IIa histone deacetylases (HDACs) belong to a large family of enzymes involved in protein deacetylation and play a role in regulating gene expression and cell differentiation. Previously, we showed that HDAC inhibitors modify the timing and determination of pancreatic cell fate. The aim of this study was to determine the role of class IIa HDACs in pancreas development. RESEARCH DESIGN AND METHODS: We took a genetic approach and analyzed the pancreatic phenotype of mice lacking HDAC4, -5, and -9. We also developed a novel method of lentiviral infection of pancreatic explants and performed gain-of-function experiments. RESULTS: We show that class IIa HDAC4, -5, and -9 have an unexpected restricted expression in the endocrine ß- and δ-cells of the pancreas. Analyses of the pancreas of class IIa HDAC mutant mice revealed an increased pool of insulin-producing ß-cells in Hdac5(-/-) and Hdac9(-/-) mice and an increased pool of somatostatin-producing δ-cells in Hdac4(-/-) and Hdac5(-/-) mice. Conversely, HDAC4 and HDAC5 overexpression showed a decreased pool of insulin-producing ß-cells and somatostatin-producing δ-cells. Finally, treatment of pancreatic explants with the selective class IIa HDAC inhibitor MC1568 enhances expression of Pax4, a key factor required for proper ß-and δ-cell differentiation and amplifies endocrine ß- and δ-cells. CONCLUSIONS: We conclude that HDAC4, -5, and -9 are key regulators to control the pancreatic ß/δ-cell lineage. These results highlight the epigenetic mechanisms underlying the regulation of endocrine cell development and suggest new strategies for ß-cell differentiation-based therapies.

Directing cell differentiation with small-molecule histone deacetylase inhibitors: the example of promoting pancreatic endocrine cells.

Genes in the mammalian genome contain information necessary to build an organism during development. Epigenetic processes add a further degree of complexity. These mechanisms of temporal and spatial control of gene activity during the development of complex organisms modulate gene expression patterns without modifying the DNA sequence. Post-translational modifications of histones such as acetylation bestow the ability to modify genomic signals. Determining whether epigenetic changes are responsible for particular phenotypes is thus crucial to understand organ development. Here we review the role of histone deacetylase enzymes (HDACs) in guiding lineage commitment and driving cell differentiation, as well as their pharmacological manipulation using small-molecule HDAC inhibitors in various differentiation programs. In particular, we focus on the pancreas as we recently discovered that deacetylase inhibition favors generation of endocrine pancreatic cells. We also discuss the potential application of HDAC inhibitors for disease treatment, with particular emphasis on diabetes therapy.

Histone deacetylase inhibitors modify pancreatic cell fate determination and amplify endocrine progenitors.

During pancreas development, transcription factors play critical roles in exocrine and endocrine differentiation. Transcriptional regulation in eukaryotes occurs within chromatin and is influenced by posttranslational histone modifications (e.g., acetylation) involving histone deacetylases (HDACs). Here, we show that HDAC expression and activity are developmentally regulated in the embryonic rat pancreas. We discovered that pancreatic treatment with different HDAC inhibitors (HDACi) modified the timing and determination of pancreatic cell fate. HDACi modified the exocrine lineage via abolition and enhancement of acinar and ductal differentiation, respectively. Importantly, HDACi treatment promoted the NGN3 proendocrine lineage, leading to an increased pool of endocrine progenitors and modified endocrine subtype lineage choices. Interestingly, treatments with trichostatin A and sodium butyrate, two inhibitors of both class I and class II HDACs, enhanced the pool of beta cells. These results highlight the roles of HDACs at key points in exocrine and endocrine differentiation. They show the powerful use of HDACi to switch pancreatic cell determination and amplify specific cellular subtypes, with potential applications in cell replacement therapies in diabetes.

Crucial role of vHNF1 in vertebrate hepatic specification.

Mouse liver induction occurs via the acquisition of ventral endoderm competence to respond to inductive signals from adjacent mesoderm, followed by hepatic specification. Little is known about the regulatory circuit involved in these processes. Through the analysis of vHnf1 (Hnf1b)-deficient embryos, generated by tetraploid embryo complementation, we demonstrate that lack of vHNF1 leads to defective hepatic bud formation and abnormal gut regionalization. Thickening of the ventral hepatic endoderm and expression of known hepatic genes do not occur. At earlier stages, hepatic specification of vHnf1-/- ventral endoderm is disrupted. More importantly, mutant ventral endoderm cultured in vitro loses its responsiveness to inductive FGF signals and fails to induce the hepatic-specification genes albumin and transthyretin. Analysis of liver induction in zebrafish indicates a conserved role of vHNF1 in vertebrates. Our results reveal the crucial role of vHNF1 at the earliest steps of liver induction: the acquisition of endoderm competence and the hepatic specification.

Severe pancreas hypoplasia and multicystic renal dysplasia in two human fetuses carrying novel HNF1beta/MODY5 mutations.

Heterozygous mutations in the HNF1beta/vHNF1/TCF2 gene cause maturity-onset diabetes of the young (MODY5), associated with severe renal disease and abnormal genital tract. Here, we characterize two fetuses, a 27-week male and a 31.5-week female, carrying novel mutations in exons 2 and 7 of HNF1beta, respectively. Although these mutations were predicted to have different functional consequences, both fetuses displayed highly similar phenotypes. They presented one of the most severe phenotypes described in HNF1beta carriers: bilateral enlarged polycystic kidneys, severe pancreas hypoplasia and abnormal genital tract. Consistent with this, we detected high levels of HNF1beta transcripts in 8-week human embryos in the mesonephros and metanephric kidney and in the epithelium of pancreas. Renal histology and immunohistochemistry analyses of mutant fetuses revealed cysts derived from all nephron segments with multilayered epithelia and dysplastic regions, accompanied by a marked increase in the expression of beta-catenin and E-cadherin. A significant proportion of cysts still expressed the cystic renal disease proteins, polycystin-1, polycystin-2, fibrocystin and uromodulin, implying that cyst formation may result from a deregulation of cell-cell adhesion and/or the Wnt/beta-catenin signaling pathway. Both fetuses exhibited a severe pancreatic hypoplasia with underdeveloped and disorganized acini, together with an absence of ventral pancreatic-derived tissue. beta-catenin and E-cadherin were strongly downregulated in the exocrine and endocrine compartments, and the islets lacked the transporter essential for glucose-sensing GLUT2, indicating a beta-cell maturation defect. This study provides evidence of differential gene-dosage requirements for HNF1beta in normal human kidney and pancreas differentiation and increases our understanding of the etiology of MODY5 disorder.

A vHNF1/TCF2-HNF6 cascade regulates the transcription factor network that controls generation of pancreatic precursor cells.

Generation of pancreatic precursor cells in the endoderm is controlled by a network of transcription factors. Hepatocyte nuclear factor-6 (HNF6) is a key player in this network, because it controls the initiation of the expression of pancreatic and duodenal homeobox 1 (Pdx1), the earliest marker of pancreatic precursor cells. To further characterize this network, we have investigated how the expression of HNF6 is controlled in mouse endoderm, by using in vitro and in vivo protein-DNA interaction techniques combined with endoderm electroporation, transgenesis, and gene inactivation in embryos. We delineated Hnf6 regulatory regions that confer expression of a reporter gene in the embryonic endoderm but not in extraembryonic visceral endoderm. HNF6 expression in the embryonic endoderm was found to depend on an intronic enhancer. This enhancer contains functional binding sites for the tissue-specific factors of the forkhead box A and HNF1 families. Among the latter, variant HNF1 (vHNF1)/TCF2, which is expressed before HNF6 in the endoderm, was found to be critical for HNF6 expression. Therefore, the sequential activation of vHNF1, HNF6, and Pdx1 in the endoderm appears to control the generation of pancreatic precursors. This cascade may be used to benchmark in vitro differentiation of pancreatic precursor cells from embryonic stem cells, for cell therapy of diabetes.

The transmembrane semaphorin Sema6A controls cerebellar granule cell migration.

The transmembrane semaphorin protein Sema6A is broadly expressed in the developing nervous system. Sema6A repels several classes of developing axons in vitro and contributes to thalamocortical axon guidance in vivo. Here we show that during cerebellum development, Sema6A is selectively expressed by postmitotic granule cells during their tangential migration in the deep external granule cell layer, but not during their radial migration. In Sema6A-deficient mice, many granule cells remain ectopic in the molecular layer where they differentiate and are contacted by mossy fibers. The analysis of ectopic granule cell morphology in Sema6a-/- mice, and of granule cell migration and neurite outgrowth in cerebellar explants, suggests that Sema6A controls the initiation of granule cell radial migration, probably through a modulation of nuclear and/or soma translocation. Finally, the analysis of mouse chimeras suggests that this function of Sema6A is primarily non-cell-autonomous.