Gene expression profiling during gliogenesis in the Drosophila embryo. Angela Becker

Transcription

1 Gene expression profiling during gliogenesis in the Drosophila embryo Angela Becker

2 Introduction to the modellorganism Drosophila melanogaster the embryonic development structure and development of the central nervous system (CNS) neural stem cells and the regulation of cell fate choice

3 The embryonic CNS of Drosophila C

4 Three types of neural precursor cells in the CNS Neuroblast Neuro-Glioblast Glioblast

5 Regulation of cell fate choice NB Neuronal genes Neuronal fate NGB Neuron GB gcm repo pointed????? Activation of glial genes????? tramtrack Repression of neuronal genes Glia glial cells missing

6 Design and performance of a dual microarray approach

7 Aim: Identification of gcm-dependent target genes Genome-wide search based on microarray-experiments

8 Microarray Experiments wildtype situation mutant situation Cy3 labelled cdna RNA Preparation of total RNA (targets) Reverse transcription of mrna Labelling with fluorescent dyes RNA Cy5 labelled cdna

9 wildtype situation mutant situation Hybridization on microarrays Scanning the slide expressed in wildtype expressed in both expressed in both expressed in mutant

10 FlyArray, Heidelberg Genome annotation, gene prediction, primer calculation, PCR, and spotting were done by the FlyArray consortium Heidelberg (Groups of J. Hoheisel, R. Paro, F. Sauer) High density array with spots: double spotted ORFs plus 2502 controls,

11 Aim: Identification of gcm-dependent target genes Genome-wide search based on microarray-experiments two approaches : ectopic expression of gcm with the UAS/Gal4 System (gain-of-function/gof)

12 The UAS/GAL4 System Brandt and Perrimon, 1993

13 Ektopic expression of gcm transforms potential neurons into glial cells Stadium (früh) wt Mz1060:gcm Repo/BP102 anterior

14 Aim: Identification of gcm-dependent target genes Genome-wide search based on microarrayexperiments two approaches : ectopic expression of gcm with the UAS/Gal4 System (gain-of-function/gof) loss-of-function of gcm(lof)

15 Gcm loss-of-function embryos lack nearly all glial cells Stadium 12 (früh) wt gcm - Repo/BP102 anterior

16 Microarray time course approach Mz 1060 Gal4 x UAS-gcm (gain-of-function, GOF) Stage 9 Stage 10 Stage 11 Stage 12 Stage 13 Stage 14 Stage 15 Stage 16 wt Stage 9 Stage 10 Stage 11 Stage 12 Stage 13 Stage 14 Stage 15 Stage 16 gcm N7-4 (loss-of-function, LOF) Stage 10 Stage 11 Stage 12 Stage 13 Stage 14 Stage 15 Stage 16 Repo/axonal marker

17 Checklist for quality controls and replicates before microarray evaluation Check developmental and genetic background (Antibody-staining) before RNA-Isolation Check degradation of Total-RNA (RNA-Gel) include dye-swaps (Cy3/Cy5) at least 4 replicates for each stage (double-spotted arrays)

18 Evaluation and result of the microarray approach

19 Quality control and determination of the methodical and genetic background determination of significantly wt vs. wt Gal4 vs. UAS differentially regulated genes Ratio Ratio Ratio equitation of non-equal genetic backgrounds Ratio by a pooled Ratio 1.4 wildtype 7 control 669 (GOF only) Ratio 1.5 Ratio Ratio Ratio reliable and more sensitive evaluation of data Ratio Ratio

20 Use of microarray-software Genepix as spotfinder : extraction of raw data MChips-Software: background subtraction normalization determination of correlation coefficient: comparison of replicates on the array filtering by intensity: at least one value above threshold filtering by ratio 1.5: at least one value above threshold filtering by reproducibility correspondance analysis (CA)

21 CA-Plot graphical illustration to see comparability of your replicated arrays and the difference between your data (wt:mutant)

22 Problem: Still too many candidate genes GOF No. of diff. reg. genes (total) Upregultated genes (>1.5) Downregulated genes (<-1.5) St. 9 St. 10 St. 11 St. 12 St. 13 St. 14 St. 15 St. 16 Total No Mean overlap LOF St. 9 St. 10 St. 11 St. 12 St. 13 St. 14 St. 15 St. 16 Total No. No. of diff. reg. genes (total) Upregultated genes (>1.5) Downregulated genes (<-1.5) Mean overlap

23 The advantage of time course experiments: Profiling differentially regulated genes above ratio potential candidate genes GOF Profiling GOF 790! 1080" LOF - expression profile in time course - strength of expression - comparison between GOF and LOF - profile of potential glial gene 131! 18!" 149" LOF

24 Manual Profiling of candidate genes Gene St. 9 St. 10 St. 11 St. 12 St. 13 St. 14 St. 15 St. 16 Sortieren aller 1,5fach differentiell regulierten Gene CG ZNS ubiquitär entsprechend ihrem Expressionsprofil ISH result, Literature CG ZNS, Glia + Neurone, BDGP CG ZNS ab 14 schwach (LG, EG) CG ML, ZNS Glia? CG ZNS, Glia? Ptp10D Entfernen von Genen ohne Änderungen ZNS, der Glia differentiellen + Neurone csw ZNS ubiquitär CG8983 Regulation entlang der Zeitachse ML CG tendon cells CG BDGP, schwach in LG gcm CG Auswahl potentieller gcm-zielgene aufgrund ZNS ubiquitär, desevtl EG UbcD2 Verlaufsprofils, der Reproduzierbarkeit und ZNS ubiquitär, der Stärke nicht PNS der smid ZNS ubiquitär differentiellen Regulation Cam ZNS ubiquitär, PNS einzelne Zellen CG ZNS, -ubiquitär CG ZNS, Glia + Neurone CG CBG RpII ubiquitär, im ZNS in einzelnen Zellen (G+N) CG3227 Gruppierung von positiv getesteten Genen BDGP, und LG, EG, Auswahl PG neuer glu ZNS ubiquitär, später LG, EG, +, Egger Uba2 Kandidaten mit vergleichbaren Expressionsprofilen ZNS ubiquitär BcDNA:LD ZNS Glia + Neurone, Egger CG ZNS Neurone + Glia? Map BDGP, ZNS ubiquitär, evtl. EG, LG CG ZNS ubiquitär CG ZNS Glia + Neurone CG LG, EG, SPG?, CG?, Neurone aus mehr als 2900 differentiell regulierten Genen 1.25 wurden 400 Kandidaten gefiltert und getestet pnut ZNS ubiquitär CG tendon cells CG PNS, ZNS, SPG CG viele Glia!, BDGP CG BDGP, LG, SPG, CBG, PG, - EG, CG Freeman, BDGP, CBG CG exklusiv PG, CBG,ML, ZNS EG:22E PG, SPG, CBG, CG, EG

25 Profiling of temporal regulation gain-of-function loss-of-function 2,5-1 Ratio 2 1,5 Ratio -1,5-2 -2,5-3 early regulated genes , Stage Stage 2,5-1 Ratio 2 1,5 Ratio -1,5-2 -2,5-3 mid regulated genes Stage -3, Stage 2,5-1 Ratio 2 1,5 Ratio -1,5-2 late regulated genes Stage -2, Stage

26 Array-Profile versus in vivo 3-1 2,5-1,5 Ratio 2 Ratio -2 1,5-2, gcm mutant Stage ISH, stage ectopic gcm StageISH, stage 14 GOF CG6218 LOF Stage 11 Stage 12 Stage 13 Stage 14 Stage 15 Stage 16 wt ISH

27 Are the filtered candidate genes expressed in glial cells? HFA15994 HFA16115 HFA03125 HFA02311 HFA12894 wt ISH, stage 14 wt FISH #-Repo, stage 14, anterior

28 Potential candidate genes tested novel gcm targets known gcm targets non-neuronal expression unconfirmed results exclusively expressed in glia 12 expressed in glia and neurons 26 ubiquitously in CNS and PNS 10 expressed in hematopoetic lineage 8 expressed in muscle tendon cells

29 Further ways to explore the data filtering for GO-Annotation filtering for pathways Transkriptionsfaktoren 64; 25% 18; 7% 27; 10% Signaltransduktion 4-fold increase compared to genome Posttransl. Protein- Modifikationen Zellzyklus,Proliferation,Mitose 27; 10% Nukleinsäure Metabolismus 21; 8% 34; 13% 9; 3% 11; 4% 27; 10% 11; 4% 16; 6% strukt. Moleküle, Cytoskelett,Adhäsion Transporter,Antiporter,Ionen- Kanäle Exozytose,Endozytose Metabolismus,Enzyme andere unbekannte Funktion