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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 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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