SUMMARYOne major unresolved question in the field of pancreas biology is whether ductal cells have the ability to generate insulinproducing b-cells. Conclusive examination of this question has been limited by the lack of appropriate tools to efficiently and specifically label ductal cells in vivo. We generated Sox9CreER T2 mice, which, during adulthood, allow for labeling of an average of 70% of pancreatic ductal cells, including terminal duct/centroacinar cells. Fate-mapping studies of the Sox9 + domain revealed endocrine and acinar cell neogenesis from Sox9 + cells throughout embryogenesis. Very small numbers of non-b endocrine cells continue to arise from Sox9 + cells in early postnatal life, but no endocrine or acinar cell neogenesis from Sox9 + cells occurs during adulthood. In the adult pancreas, pancreatic injury by partial duct ligation (PDL) has been suggested to induce b-cell regeneration from a transient Ngn3 + endocrine progenitor cell population. Here, we identify ductal cells as a cell of origin for PDL-induced Ngn3 + cells, but fail to observe b-cell neogenesis from duct-derived cells. Therefore, although PDL leads to activation of Ngn3 expression in ducts, PDL does not induce appropriate cues to allow for completion of the entire b-cell neogenesis program. In conclusion, although endocrine cells arise from the Sox9 + ductal domain throughout embryogenesis and the early postnatal period, Sox9 + ductal cells of the adult pancreas no longer give rise to endocrine cells under both normal conditions and in response to PDL.

Summary The molecular mechanisms that underlie cell lineage diversification of multipotent progenitors in the pancreas are virtually unknown. Here, we show that the early fate choice of pancreatic progenitors between the endocrine and acinar cell lineage is restricted by cross-repressive interactions between the transcription factors Nkx6.1/Nkx6.2 (Nkx6) and Ptf1a. Using genetic loss- and gain-of-function approaches, we demonstrate that Nkx6 factors and Ptf1a are required and sufficient to repress the alternative lineage program and to specify progenitors toward an endocrine or acinar fate, respectively. The Nkx6/Ptf1a switch only operates during a critical competence window when progenitors are still multipotent and can be uncoupled from cell differentiation. Thus, cross-antagonism between Nkx6 and Ptf1a in multipotent progenitors governs the equilibrium between endocrine and acinar cell neogenesis required for normal pancreas development.

SUMMARY Neurodegenerative diseases can occur so early as to affect neurodevelopment. From a cohort of over 2000 consanguineous families with childhood neurological disease, we identified a founder mutation in four independent pedigrees in cleavage and polyadenylation factor I subunit (CLP1). CLP1 is a multifunctional kinase implicated in tRNA, mRNA and siRNA maturation. Kinase activity of the CLP1 mutant protein was defective, and the tRNA endonuclease complex (TSEN) was destabilized, resulting in impaired pre-tRNA cleavage. Germline clp1 null zebrafish showed cerebellar neurodegeneration that was rescued by wild type but not mutant human CLP1 expression. Patient-derived induced neurons displayed both depletion of mature tRNAs and accumulation of unspliced pre-tRNAs. Transfection of partially processed tRNA fragments into patient cells exacerbated an oxidative stress-induced reduction in cell survival. Our data links tRNA maturation to neuronal development and neurodegeneration through defective CLP1 function in humans.

All pancreatic endocrine cell types arise from a common endocrine precursor cell population, yet the molecular mechanisms that establish and maintain the unique gene expression programs of each endocrine cell lineage have remained largely elusive. Such knowledge would improve our ability to correctly program or reprogram cells to adopt specific endocrine fates. Here, we show that the transcription factor Nkx6.1 is both necessary and sufficient to specify insulin-producing beta cells. Heritable expression of Nkx6.1 in endocrine precursors of mice is sufficient to respecify non-beta endocrine precursors towards the beta cell lineage, while endocrine precursor- or beta cell-specific inactivation of Nkx6.1 converts beta cells to alternative endocrine lineages. Remaining insulin+ cells in conditional Nkx6.1 mutants fail to express the beta cell transcription factors Pdx1 and MafA and ectopically express genes found in non-beta endocrine cells. By showing that Nkx6.1 binds to and represses the alpha cell determinant Arx, we identify Arx as a direct target of Nkx6.1. Moreover, we demonstrate that Nkx6.1 and the Arx activator Isl1 regulate Arx transcription antagonistically, thus establishing competition between Isl1 and Nkx6.1 as a critical mechanism for determining alpha versus beta cell identity. Our findings establish Nkx6.1 as a beta cell programming factor and demonstrate that repression of alternative lineage programs is a fundamental principle by which beta cells are specified and maintained. Given the lack of Nkx6.1 expression and aberrant activation of non-beta endocrine hormones in human embryonic stem cell (hESC)–derived insulin+ cells, our study has significant implications for developing cell replacement therapies.

Exome sequencing of 118 patients with neurodevelopmental disorders shows that this technique is useful for identifying new pathogenic mutations and for correcting diagnosis in ~10% of cases.

Pediatric-onset ataxias often present clinically with developmental delay and intellectual disability, with prominent cerebellar atrophy as a key neuroradiographic finding. Here we describe a novel clinically distinguishable recessive syndrome in 12 families with cerebellar atrophy together with ataxia, coarsened facial features and intellectual disability, due to truncating mutations in sorting nexin 14 (SNX14), encoding a ubiquitously expressed modular PX-domain-containing sorting factor. We found SNX14 localized to lysosomes, and associated with phosphatidyl-inositol (3,5)P2, a key component of late endosomes/lysosomes. Patient cells showed engorged lysosomes and slower autophagosome clearance rate upon starvation induction. Zebrafish morphants showed dramatic loss of cerebellar parenchyma, accumulated autophagosomes, and activation of apoptosis. Our results suggest a unique ataxia syndrome due to biallelic SNX14 mutations, leading to lysosome-autophagosome dysfunction.

DEVELOPMENT 2491 RESEARCH ARTICLE INTRODUCTIONRecent studies suggest that the most promising approach for the in vitro derivation of insulin-producing pancreatic beta-cells from stem cells is by recapitulating embryonic beta-cell differentiation (D'Amour et al., 2006). The in vitro generation of fully functional beta-cells for transplantation, however, will require further improvement of existing differentiation protocols. Such refinement will require detailed knowledge of the molecular mechanisms that underlie beta-cell differentiation. Lineage-tracing studies in mice have shown that all pancreatic lineages, which include the exocrine and endocrine compartment, arise from a common, proliferative progenitor cell population that is marked by the transcription factor Pdx1 (Gu et al., 2002). Expression of the transcription factor Ngn3 (also known as Neurog3 -Mouse Genome Informatics) further restricts progenitors to five distinct endocrine cell fates: alpha-, beta-, delta-, PP-or epsilon-cells, which produce the hormones glucagon, insulin, somatostatin, pancreatic polypeptide and ghrelin, respectively (Gu et al., 2002;Heller et al., 2005;Prado et al., 2004). Initially, scattered endocrine cells differentiate throughout the organ, but these cells aggregate into so-called islets of Langerhans at the end of gestation (Slack, 1995).In recent years, major progress has been made in our understanding of the molecular pathways that control endocrine differentiation (Jensen, 2004). Much of this knowledge stems from genetic gain-and loss-of-function experiments in mice. Such studies have shown that Ngn3 activity is essential for the differentiation of all endocrine cells and, conversely, that Ngn3 is sufficient to restrict Pdx1-positive (Pdx1 + ) progenitors to an endocrine fate (Apelqvist et al., 1999;Gradwohl et al., 2000;Schwitzgebel et al., 2000). Based on the observation that only select endocrine lineages are affected in null mutant mice for Nkx2.2 (Nkx2-2), NeuroD (Neurod1), Pax4, Arx, Hb9 (also known as Hlxb9 -Mouse Genome Informatics) or Nkx6.1 (Nkx6-1), these transcription factors have been proposed to be downstream effectors of Ngn3 (Collombat et al., 2003;Harrison et al., 1999;Li et al., 1999;Naya et al., 1997;Sander et al., 2000b;Schwitzgebel, 2001;Sosa-Pineda et al., 1997;Sussel et al., 1998;Wilson et al., 2003). Although the loss of Pax4, Arx and NeuroD expression in Ngn3 mutant mice (Collombat et al., 2003;Gradwohl et al., 2000) indeed suggests a function of these genes downstream of Ngn3 in endocrine differentiation, the evidence for Nkx6.1 being downstream of Ngn3 is less clear.Deficiency for Nkx6.1 results in a specific abrogation of beta-cell neogenesis during embryogenesis without affecting cell survival or the development of any other cell type in the pancreas (Sander et al., 2000b). In Nkx6.1 mutant mice, a marked reduction in beta-cell numbers is first apparent at embryonic day (E)14, the time-point at which the first mature beta-cells differentiate. The specific defect in the beta-cell lineage and th...

A number of mutations in genes that encode ubiquitously expressed RNA-binding proteins cause tissue specific disease. Many of these diseases are neurological in nature revealing critical roles for this class of proteins in the brain. We recently identified mutations in a gene that encodes a ubiquitously expressed polyadenosine RNA-binding protein, ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), that cause a nonsyndromic, autosomal recessive form of intellectual disability. This finding reveals the molecular basis for disease and provides evidence that ZC3H14 is essential for proper brain function. To investigate the role of ZC3H14 in the mammalian brain, we generated a mouse in which the first common exon of the ZC3H14 gene, exon 13 is removed (Zc3h14Δex13/Δex13) leading to a truncated ZC3H14 protein. We report here that, as in the patients, Zc3h14 is not essential in mice. Utilizing these Zc3h14Δex13/Δex13mice, we provide the first in vivo functional characterization of ZC3H14 as a regulator of RNA poly(A) tail length. The Zc3h14Δex13/Δex13 mice show enlarged lateral ventricles in the brain as well as impaired working memory. Proteomic analysis comparing the hippocampi of Zc3h14+/+ and Zc3h14Δex13/Δex13 mice reveals dysregulation of several pathways that are important for proper brain function and thus sheds light onto which pathways are most affected by the loss of ZC3H14. Among the proteins increased in the hippocampi of Zc3h14Δex13/Δex13 mice compared to control are key synaptic proteins including CaMK2a. This newly generated mouse serves as a tool to study the function of ZC3H14 in vivo.