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The Role of Cdep in the Embryonic Morphogenesis of Drosophila Melanogaster

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The Role of Cdep in the Embryonic Morphogenesis of Drosophila melanogaster Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer.nat.) vorgelegt der Fakultät Mathematik und Naturwissenschaften der Technischen Universität Dresden von Anne Morbach Geboren am 05. August 1984 in Nordhausen, Deutschland Eingereicht am 30. Oktober 2015 Die Dissertation wurde in der Zeit von April 2012 bis Oktober 2015 im Max-Planck-Institut für molekulare Zellbiologie und Genetik angefertigt Gutachter: Prof. Dr. Elisabeth Knust Prof. Dr. Christian Dahmann Now, here, you see, it takes all the running you can do, to keep in the same place. Lewis Carroll, Through the Looking-Glass I Contents List of Figures V List of Tables VII Abstract IX List of Abbreviations XI 1 Introduction 1 1.1 Epithelial cell polarity . 1 1.1.1 Cellularization and formation of the primary epithelium . 1 1.1.1.1 Establishment of epithelial polarity and adhesion . 2 1.1.2 The epithelial polarity network . 3 1.1.3 Cell-cell adhesion . 5 1.1.3.1 Adherens junctions . 5 1.1.3.2 Septate junctions . 6 1.2 Epithelial movements in Drosophila embryonic morphogenesis . 6 1.2.1 Epithelial tube formation during Drosophila embryogenesis . 7 1.2.2 Coordinated migration of epithelial sheets during Dros. embryogenesis 7 1.2.2.1 FERM domain proteins in epithelial migration . 9 1.2.2.2 Cdep . 10 1.3 Mutagenesis with the CRISPR/Cas9 system . 12 2 Aim of My PhD Thesis Work 15 3 Preliminary Work 17 4 Results 19 4.1 A screen for novel regulators in Drosophila embryonic morphogenesis . 19 4.1.1 Deficiencies on the left arm of chromosome 3 cause defects in SG lumen morphology . 19 4.1.2 A locus in the overlap of two deficiencies on the right arm of chromosome 3 causes defects in Drosophila embryonic dorsal closure 20 4.2 Two uncharacterized genes regulate SG lumen diameter in Drosophila embryos 22 4.2.1 Mutations in two uncharacterized genes on chromosome 3 cause intermittent tube closure in the Drosophila SG . 22 4.2.2 CG17362/ CG9040/ CG17364 play a role in the maintenance of SG lumen width after lumen expansion . 24 4.2.3 CG17362 is exclusively expressed in the embryonic SG . 25 4.2.4 CG17362 and CG9040 do not contain known protein domains and are only conserved in Drosophilidae . 28 4.3 Loss of Cdep causes different defects in Drosophila embryonic morphogenesis 30 4.3.1 Insertions in the ORF of Cdep cause defects in DC and HI . 30 4.3.2 Embryos transheterozygous for PBac{5HPw+}CdepB122 and Mi{MIC} CdepMI00496 show defects in the LE during DC . 34 4.3.3 Insertions in the ORF of Cdep cause defects in tracheal and Malpighian tubule morphogenesis . 34 4.3.4 The CRISPR/Cas9 system was used to generate loss-of-function mutants of Cdep ............................. 35 4.3.5 LOF mutants of Cdep show a variety of phenotypes . 37 II Contents 4.3.6 LOF mutants of Cdep cause denticle belt defects and ventral holes in Drosophila larval cuticles . 38 4.3.7 Phenotypes in Cdep−/− mutant embryos are likely not caused by maternal defects . 44 4.3.8 LOF mutants of Cdep cause segments to fuse . 44 4.3.9 Defects in Cdep−/− mutants are not due to actin mislocalization . 51 4.3.10 Cdep genetically interacts with yurt . 51 4.3.11 Cdep genetically interacts with cora . 55 5 Discussion 57 5.1 The role of CG17362 and CG9040 in SG lumen stability . 57 5.1.1 Impairments in the expression of CG17362 and CG9040 could be the cause for the intermittent tube closure in the embryonic SGs . 57 5.1.2 CG17362 and CG9040 could be necessary for SG lumen dilation and stability of lumen diameter . 57 5.2 The role of Cdep in Drosophila embryonic morphogenesis . 59 5.2.1 Cdep is a regulator of the JNK pathway . 60 5.2.1.1 Mutations in TGF-β pathway components cause LE bunching and gaps in the tracheal dorsal trunk . 60 5.2.1.2 The JNK pathway, like Cdep, is instrumental in GBR, DC and HI . 61 5.2.1.3 Mouse Farp2 acts upstream of the JNK pathway . 61 5.2.2 Does Cdep act as a GEF? . 62 5.2.3 Cdep might regulate epithelial migration in parallel to Yrt and Cora 63 5.3 Conclusion and Outlook . 64 6 Materials and Methods 67 6.1 Cell strains, plasmids and DNA constructs . 67 6.2 Culture media . 68 6.2.1 Lysogeny Broth (Bertani, 1951) . 68 6.2.2 Super Optimal Catabolite repression (SOC) medium (Hanahan, 1983) 68 6.3 Molecular biology methods . 69 6.3.1 Amplifying DNA by standard PCR technology . 69 6.3.2 Molecular cloning . 70 6.3.2.1 Cloning with restriction endonucleases (Old and Primrose, 1980) . 70 6.3.2.2 Gibson Assembly (Gibson et al., 2009) . 70 6.3.3 Detecting DNA via agarose gel electrophoresis . 71 6.3.4 Purifying DNA from E.coli ....................... 71 6.3.5 Extracting DNA from Drosophila adults . 71 6.3.5.1 Extracting mRNA from Drosophila embryos and reverse transcription into cDNA . 72 6.3.6 Making mRNA probes for in situ hybridization . 72 6.3.7 Measuring DNA concentrations using the NanoDrop . 72 6.3.8 DNA sequencing . 72 6.3.9 Transformation . 73 6.3.10 Mutagenesis of Cdep with the CRISPR-Cas9 system . 73 6.3.10.1 CRISPR/Cas9 tools for mutagenesis in D.melanogaster . 73 Contents III 6.3.11 Strategies for CRISPR/Cas9 mutagensis . 74 6.3.11.1 vectors and oligomers used for mutagenesis of Cdep via CRISPR-Cas9 system . 75 pCFD3-dU6:3-CdepEx2gRNA . 75 CdepSTOP-ssODN . 76 pCFD4-CdepExc_2sgRNAs . 77 pDsRed-5’HA-3’HA . 78 6.3.11.2 Screening for successful mutagenesis events in flies modified with the CRISPR/Cas9 system . 79 Insertion of an in-frame stop codon into the Cdep ORF . 79 Replacing the entire Cdep locus with dsRed . 80 6.3.12 Extracting protein from Drosophila embryos . 83 6.3.13 Detecting and separating proteins by mass via SDS-PAGE . 83 6.3.14 Detection of specific polypeptides by Western blot analysis (Towbin et al., 1979) . 84 6.3.14.1 Transferring polypeptides to nitrocellulose membrane . 84 6.3.14.2 Detecting specific polypeptides via immonublotting . 85 6.4 Online tools for prediction of protein domains, interactions, sequence alignments, etc. 86 6.5 Cell culture growth and harvest . 86 6.5.1 Growing E.coli cells for vector amplification . 86 6.5.2 Cell harvest . 86 6.6 Work with Drosophila melanogaster ...................... 87 6.6.1 Fly stocks used: deficiencies, alleles, duplications and balancers . 88 6.6.2 Recombining alleles located on the same chromosome . 93 6.6.3 Collecting Drosophila melanogaster embryos . 93 6.6.4 Histochemistry . 94 6.6.4.1 Embryo dechorionation . 94 6.6.4.2 Embryo fixation for light microscopy of whole specimens . 94 Heat fixation of Drosophila embryos . 94 Formaldehyde fixation of Drosophila embryos . 94 6.6.4.3 Embryo fixation for light microscopy of semi-thin sections (Tepass and Hartenstein, 1994) . 95 6.6.4.4 Antibody staining of embryos for Immunofluorescence . 95 6.6.4.5 Phalloidin and DAPI staining of embryos . 96 6.6.4.6 Embryo mounting for analysis via fluorescence microscopy 96 6.6.4.7 Embryo mounting for live imaging . 97 6.6.4.8 Embedding of embryos for semi-thin sectioning . 97 6.6.4.9 Cuticle preparation from hatched and unhatched Drosophila larvae . 97 6.6.4.10 Preparation and staining of ovarian follicles from Drosophila females . 98 6.6.4.11 mRNA in situ hybridization of whole-mount Drosophila embryos . 99 Bibliography XV Acknowledgements XXIX Erklärung entsprechend §5.5 der Promotionsordnung XXXI V List of Figures 1.1 Schematic representation of Drosophila cellularization . 2 1.2 The cell polarity network in insects . 4 1.3 Cell adhesions in insects . 6 1.4 Schematic overview over morphogenesis of tubular organs in the Drosophila embryo....................................... 8 1.5 Schematic overview over germ band retraction, dorsal closure and head involution . 9 1.6 Schematic overview over the type II CRISPR/Cas9 system . 12 1.7 Schematic overview over the modified CRISPR/Cas9 system for use in eukaryotes . 13 1.8 Example for indels in Drosophila white gene caused by CRISPR/Cas9-induced mutagenesis . 13 1.9 Schematic overview over strategy to replace an endogenous gene with a marker gene via CRISPR/Cas9 . 14 4.1 Overview over deficiencies used to find salivary gland phenotypes. 20 4.2 Examples of salivary gland phenotypes caused by deficiencies . 21 4.3 Salivary gland phenotypes found in embryos homozygous for CG17364MB01315 and CG17362MB04765. .............................. 21 4.4 Dorsal closure phenotype in embryos transheterozygous for Df(3R)ED5066 and Df(3R)Exel6143. 23 4.5 Localization of Df(3R)ED5066 and Df(3R)Exel6143 in the Drosophila genome 23 4.6 Overlap of Df(3L)ED4284 with Df(3L)ED4287 and Df(3L)ED4515 with Df(3L)ED4502. 24 4.7 Localization of the genomic insertions CG17364MB01315 and CG17362MB04765. 25 4.8 In embryos transheterozygous for CG17364MB01315 and Df(3L)ED4515, the SG lumen collapses during stage 17 . 27 4.9 In situ staining of CG17362 mRNA in WT embryos. 27 4.10 Overlap between Df(3R)ED5066 and Df(3R)Exel6143 . 31 4.11 Overview over the Cdep locus with insertion sites of PBac{5HPw+}CdepB122 and Mi{MIC}CdepMI00496 ............................ 31 4.12 Dorsal closure phenotypes in embryos homozygous or transheterozygous for PBac{5HPw+}CdepB122 and Mi{MIC}CdepMI00496.
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  • The 2Q37-Deletion Syndrome: an Update of the Clinical Spectrum Including Overweight, Brachydactyly and Behavioural Features in 14 New Patients

    The 2Q37-Deletion Syndrome: an Update of the Clinical Spectrum Including Overweight, Brachydactyly and Behavioural Features in 14 New Patients

    European Journal of Human Genetics (2013) 21, 602–612 & 2013 Macmillan Publishers Limited All rights reserved 1018-4813/13 www.nature.com/ejhg ARTICLE The 2q37-deletion syndrome: an update of the clinical spectrum including overweight, brachydactyly and behavioural features in 14 new patients Camille Leroy1,2,3, Emilie Landais1,2,4, Sylvain Briault5, Albert David6, Olivier Tassy7, Nicolas Gruchy8, Bruno Delobel9, Marie-Jose´ Gre´goire10, Bruno Leheup3,11, Laurence Taine12, Didier Lacombe12, Marie-Ange Delrue12, Annick Toutain13, Agathe Paubel13, Francine Mugneret14, Christel Thauvin-Robinet3,15, Ste´phanie Arpin13, Cedric Le Caignec6, Philippe Jonveaux3,10, Myle`ne Beri10, Nathalie Leporrier8, Jacques Motte16, Caroline Fiquet17,18, Olivier Brichet16, Monique Mozelle-Nivoix1,3, Pascal Sabouraud16, Nathalie Golovkine19, Nathalie Bednarek20, Dominique Gaillard1,2,3 and Martine Doco-Fenzy*,1,2,3,18 The 2q37 locus is one of the most commonly deleted subtelomeric regions. Such a deletion has been identified in 4100 patients by telomeric fluorescence in situ hybridization (FISH) analysis and, less frequently, by array-based comparative genomic hybridization (array-CGH). A recognizable ‘2q37-deletion syndrome’ or Albright’s hereditary osteodystrophy-like syndrome has been previously described. To better map the deletion and further refine this deletional syndrome, we formed a collaboration with the Association of French Language Cytogeneticists to collect 14 new intellectually deficient patients with a distal or interstitial 2q37 deletion characterized by FISH and array-CGH. Patients exhibited facial dysmorphism (13/14) and brachydactyly (10/14), associated with behavioural problems, autism or autism spectrum disorders of varying severity and overweight or obesity. The deletions in these 14 new patients measured from 2.6 to 8.8 Mb.
  • Pathogenic Nssnps That Increase the Risks of Cancers Among the Orang

    Pathogenic Nssnps That Increase the Risks of Cancers Among the Orang

    www.nature.com/scientificreports OPEN Pathogenic nsSNPs that increase the risks of cancers among the Orang Asli and Malays Nurul Ain Khoruddin1,2, Mohd NurFakhruzzaman Noorizhab1,3, Lay Kek Teh1,3, Farida Zuraina Mohd Yusof1,2 & Mohd Zaki Salleh1,3* Single-nucleotide polymorphisms (SNPs) are the most common genetic variations for various complex human diseases, including cancers. Genome-wide association studies (GWAS) have identifed numerous SNPs that increase cancer risks, such as breast cancer, colorectal cancer, and leukemia. These SNPs were cataloged for scientifc use. However, GWAS are often conducted on certain populations in which the Orang Asli and Malays were not included. Therefore, we have developed a bioinformatic pipeline to mine the whole-genome sequence databases of the Orang Asli and Malays to determine the presence of pathogenic SNPs that might increase the risks of cancers among them. Five diferent in silico tools, SIFT, PROVEAN, Poly-Phen-2, Condel, and PANTHER, were used to predict and assess the functional impacts of the SNPs. Out of the 80 cancer-related nsSNPs from the GWAS dataset, 52 nsSNPs were found among the Orang Asli and Malays. They were further analyzed using the bioinformatic pipeline to identify the pathogenic variants. Three nsSNPs; rs1126809 (TYR), rs10936600 (LRRC34), and rs757978 (FARP2), were found as the most damaging cancer pathogenic variants. These mutations alter the protein interface and change the allosteric sites of the respective proteins. As TYR , LRRC34, and FARP2 genes play important roles in numerous cellular processes such as cell proliferation, diferentiation, growth, and cell survival; therefore, any impairment on the protein function could be involved in the development of cancer.