Ascl1 Balances Neuronal versus Ependymal Fate in the Spinal Cord Central Canal

Generation of neuronal types at the right time, location, and number is essential for building a functional nervous system. Significant progress has been reached in understanding the mechanisms that govern neuronal diversity. Cerebrospinal fluid-contacting neurons (CSF-cNs), an intriguing spinal cord central canal population, are produced during advanced developmental stages, simultaneous with glial and ependymal cells. It is unknown how CSF-cNs are specified after the neurogenesis-to-gliogenesis switch. Here, we identify delayed Ascl1 expression in mouse spinal progenitors during the gliogenic phase as key in CSF-cN differentiation. With fate mappings and time-controlled deletions, we demonstrate that CSF-cNs derive from Ascl1-expressing cells and that Ascl1 triggers late neurogenesis in the amniote spinal cord. Ascl1 abrogation transforms prospective CSF-cN progenitors into ependymocytes. These results demonstrate that late spinal progenitors have the potential to produce neurons and that Ascl1 initiates CSF-cN differentiation, controlling the precise neuronal and nonneuronal composition of the spinal central canal.Fil: Di Bella, Daniela Jesica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Instituto Leloir; ArgentinaFil: Carcagno, Abel Luis. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bartolomeu, M. Lucía. Fundación Instituto Leloir; ArgentinaFil: Pardi, Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Löhr, Heiko. University of Cologne; AlemaniaFil: Siegel, Nicole. Fundación Instituto Leloir; ArgentinaFil: Hammerschmidt, Matthias. University of Cologne; AlemaniaFil: Marin Burgin, Antonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Lanuza, Guillermo Marcos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Instituto Leloir; Argentin

United European Gastroenterology evidence-based guidelines for the diagnosis and therapy of chronic pancreatitis (HaPanEU)

Background:There have been substantial improvements in the management of chronic pancreatitis, leading to the publication of several national guidelines during recent years. In collaboration with United European Gastroenterology, the working group on Harmonizing diagnosis and treatment of chronic pancreatitis across Europe' (HaPanEU) developed these European guidelines using an evidence-based approach. Methods: Twelve multidisciplinary review groups performed systematic literature reviews to answer 101 predefined clinical questions. Recommendations were graded using the Grading of Recommendations Assessment, Development and Evaluation system and the answers were assessed by the entire group in a Delphi process online. The review groups presented their recommendations during the 2015 annual meeting of United European Gastroenterology. At this one-day, interactive conference, relevant remarks were voiced and overall agreement on each recommendation was quantified using plenary voting (Test and Evaluation Directorate). After a final round of adjustments based on these comments, a draft version was sent out to external reviewers. Results: The 101 recommendations covered 12 topics related to the clinical management of chronic pancreatitis: aetiology (working party (WP)1), diagnosis of chronic pancreatitis with imaging (WP2 and WP3), diagnosis of pancreatic exocrine insufficiency (WP4), surgery in chronic pancreatitis (WP5), medical therapy (WP6), endoscopic therapy (WP7), treatment of pancreatic pseudocysts (WP8), pancreatic pain (WP9), nutrition and malnutrition (WP10), diabetes mellitus (WP11) and the natural course of the disease and quality of life (WP12). Using the Grading of Recommendations Assessment, Development and Evaluation system, 70 of the 101 (70%) recommendations were rated as strong' and plenary voting revealed strong agreement' for 99 (98%) recommendations. Conclusions:The 2016 HaPanEU/United European Gastroenterology guidelines provide evidence-based recommendations concerning key aspects of the medical and surgical management of chronic pancreatitis based on current available evidence. These recommendations should serve as a reference standard for existing management of the disease and as a guide for future clinical research

Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis

This document is the Accepted Manuscript version of the following article: Riessland et al., 'Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis', The American Journal of Human Genetics, Vol. 100 (2): 297-315, first published online 26 January 2017. The final, published version is available online at doi: http://dx.doi.org/10.1016/j.ajhg.2017.01.005 © 2017 American Society of Human Genetics.Homozygous SMN1 loss causes spinal muscular atrophy (SMA), the most common lethal genetic childhood motor neuron disease. SMN1 encodes SMN, a ubiquitous housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. SMA-affected individuals harbor low SMN expression from one to six SMN2 copies, which is insufficient to functionally compensate for SMN1 loss. However, rarely individuals with homozygous absence of SMN1 and only three to four SMN2 copies are fully asymptomatic, suggesting protection through genetic modifier(s). Previously, we identified plastin 3 (PLS3) overexpression as an SMA protective modifier in humans and showed that SMN deficit impairs endocytosis, which is rescued by elevated PLS3 levels. Here, we identify reduction of the neuronal calcium sensor Neurocalcin delta (NCALD) as a protective SMA modifier in five asymptomatic SMN1-deleted individuals carrying only four SMN2 copies. We demonstrate that NCALD is a Ca(2+)-dependent negative regulator of endocytosis, as NCALD knockdown improves endocytosis in SMA models and ameliorates pharmacologically induced endocytosis defects in zebrafish. Importantly, NCALD knockdown effectively ameliorates SMA-associated pathological defects across species, including worm, zebrafish, and mouse. In conclusion, our study identifies a previously unknown protective SMA modifier in humans, demonstrates modifier impact in three different SMA animal models, and suggests a potential combinatorial therapeutic strategy to efficiently treat SMA. Since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in SMA and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies.Peer reviewedFinal Accepted Versio

<i>m1061</i> mutants show increased <i>th</i> expression and increase in number of hypothalamic DA neurons from 3 dpf onwards.

<p>Analysis of <i>th</i> gene expression in <i>cnot8^m1061</i> mutants and WT siblings as indicated in header. (A–F) <i>th</i> transcript levels are not altered in <i>m1061/m1061</i> mutants at 1 and 2 dpf. Embryos were genotyped by PCR. (G–N) <i>m1061</i> mutants at 3 dpf display stronger WISH signal indicating increased <i>th</i> mRNA levels in DA groups 1 to 7 (arrowhead) and NA sympathetic cells (asterisk). (K, L) NA locus coeruleus (LC) and DA pretectal cells (Pre) do not display altered <i>th</i> expression levels in <i>m1061</i> mutant embryos. (M, N) The number of DA retinal amacrine cells (rAC) is reduced in <i>m1061</i> mutant embryos. Additionally <i>m1061</i> mutants display a lens defect (arrowheads). Genotypes were inferred by <i>th</i> WISH analysis. (O, P) anti-TH immunohistochemistry of <i>m1061</i> mutant embryo and WT sibling at 4 dpf document DA neurons in the ventral diencephalon. Data confirm a higher cell number of DC7 DA neurons in the caudal hypothalamus of <i>m1061</i> mutants. Dorsal view z-projections of partial confocal stacks representing the ventral diencephalon are shown. (A, B, E, F, G, H, M, N) lateral views, anterior at left; (C, D, G, H, K.L, O, P) dorsal views, anterior at left. Scale bars 100 µm in A for A, B; in C for C–F; in G for G–N; in P for O, P. Abbreviations: DC - early diencephalic DA group (DC2, DC4), 1 - ventral thalamic DA group, 2,4,5,6 posterior tubercular and hypothalamic Orthopedia-dependent DA groups, 3 - medial hypothalamic DA group, 7 - caudal hypothalamic DA group, VT - ventral thalamus, PT/H - posterior tuberculum/hypothalamus, PO - preoptic area, AAC - arch associated catecholaminergic neurons/carotid body, sym - sympathetic NA neurons, Pr - pretectum, TC - telencephalon, CH - caudal hypothalamus, LC locus coeruleus, MO - medulla oblongata, rAC - retinal amacrine cells. (Q) Quantification of CA cell numbers in <i>m1061</i> embryos at 4 dpf. Cell counts of DC1-7 and LC TH-expressing cells. Bars show the average number of CA neurons in five <i>m1061</i> mutants and five WT sibling embryos. Error bars indicate standard deviation. Significance was evaluated using Mann-Whitney test (see text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113829#pone.0113829.s002" target="_blank">Table S1</a>).</p

<i>sim1a</i>, <i>otpa and nkx2.1a</i> expression in <i>cnot8^m1061</i> embryos and WT siblings.

<p>Analysis of <i>otpa</i> (A–D), <i>sim1a</i> (E, F) and <i>nkx2.1a</i> (G–J) gene expression in <i>cnot8^m1061</i> mutant embryos and wild-type siblings, stages as indicated. (A–F, I, J) lateral views. (G, H) dorsal views. Embryos were genotyped by PCR. Scale bar 100 µm.</p

<i>cnot8^m1061</i> mutant embryos are not generally delayed in development.

<p>Gene expression analysis of <i>emx1</i>, <i>fgf8</i> and <i>krox20/egr2b</i> in <i>cnot8^m1061</i> mutants and wild-type siblings at 1, 2 and 3 dpf. (A–F) <i>emx1</i>, (G–L) <i>fgf8</i> and (M–R) <i>krox20/egr2b</i>. Embryos were genotyped by PCR. All pictures show lateral views. Scale bars 100 µm.</p

<i>fgf3</i> gene expression is increased in <i>cnot8^m1061</i> mutants.

<p>Analysis of <i>fgf3</i> gene expression in <i>cnot8^m1061</i> mutants and WT siblings 2 dpf (A, B), and 3 dpf (C, D). All pictures show lateral views. Embryos were genotyped by PCR. Scale bar 100 µm.</p

<i>erm</i> and <i>pea3</i> expression in <i>cnot8^m1061</i> mutants and WT siblings.

<p>(A, B) <i>erm</i> expression in WT siblings and <i>cnot8^m1061</i> mutants at 3 dpf. (C–H) <i>pea3</i> expression in WT siblings and <i>cnot8^m1061</i> mutants at (C, D) 3 dpf and (E–H) 2 dpf. (E–G) <i>cnot8^m1061</i> mutants show higher <i>pea3</i> mRNA levels in cells surrounding the lens in comparison to WT siblings (arrowheads). (A–D, G, H) lateral views. (E, F) dorsal views, anterior at left. Embryos were genotyped by PCR. Scale bars 100 µm in B for (A, B), in D for (C, D) and in H for (E–H).</p

<i>oxtl</i>, <i>tphd2</i> and <i>crh</i> gene expression in <i>cnot8^m1061</i> embryos at 3 dpf.

<p>(A, B) <i>oxtl</i>, (C, D) <i>tphd2</i> and (E–H) <i>crh</i> WISH expression analysis in wild-type siblings and <i>cnot8^m1061</i> mutants. (A–F) lateral views. (G, H) dorsal views. Boxes indicate areas of cell counts. Sibling WT embryos developed on average 34.6 <i>crh</i> neurons and <i>cnot8^m1061</i> mutants developed on average 59.4 <i>crh</i> neurons. Significance was evaluated by Mann-Whitney test (p = 0.008). Embryos were genotyped by PCR. Scale bar 100 µm.</p