Michal Reichman-Fried

Transcription

1 Zebrafish Development and Genetics course 2013 Cell labeling and migration Imaging primordial germ cell migration Instructors: Erez Raz Michal Reichman-Fried Introduction: On this day we will label germ cells with fluorescent proteins and follow the migration of this cell population using time-lapse microscopy. To visualize germ cells, purified mrna encoding fluorescent-protein fusions will be injected (by Michal) at the 1-cell stage. To alter germ cell migration, we will co-inject either specific morpholinos or various mrna encoding mutated regulatory proteins. Injection of mrna containing a universal 3 untranslated region (UTR) e.g., that of the globin gene at the 1-cell stage results in uniform expression of the protein encoded by the mrna (in our case a fluorescent protein). Directing the expression of the protein preferentially to germ cells, is achieved by fusing a specific 3 UTR, such as that of the nanos1 (nos1) gene (a germ cell marker critical for their normal development [1]) to the open reading frame of interest. The model system of primordial germ cell (PGC) migration provides a unique system to study directed cell migration in a three-dimensional in vivo environment [2]. Our lab previously demonstrated that directed germ cell migration and their ability to reach the target relies on the chemokine Cxcl12 and its receptor CXCR4b [3]. While migrating, germ cells produce blebs in a manner that is dependent of myosin contractions [4]. We have also shown that Rho GTPases Rac and RhoA are activated at the front of the migrating germ cells to produce an effective leading edge [5]. Disruption of Rac1 or RhoA signaling adversely affects actin dynamics and consequently, proper germ cell migration. The aim of this experiment is to image germ cells and monitor their normal and abnormal (upon manipulations) modes of migration within the developing embryo. Germ cells will be labeled using EGFP-lifeact-nos3 UTR that enables visualization of actin dynamics during migration. The somatic environment within which germ cells migrate will be labeled in red by injection of mcherry-f - 1

2 Zebrafish Development and Genetics course 2013 Cell labeling and migration globin3 UTR mrna. Normal and abnormal migratory behavior of germ cells will be imaged at a high magnification (63x reagents 1, 2, 3 and 4), while long-term migration, either directional or non-directional, will be imaged at a low magnification (10x reagents 5 and 6). The following reagents will be used: 1. Lifeact EGFP nanos 3 UTR RNA - marks actin in the germ cells [6] and mcherryf globin 3 UTR RNA labels the membranes of all cells. 2. C rock_dn- nanos 3 UTR directs the expression of a dominant negative form of the ROCK protein to the germ cells. 3. rhoa-v14 nanos 3 UTR RNA directs the expression of an activated version of the RhoA protein to the germ cells. 4. N17 rac1-dn- nanos 3 UTR RNA directs the expression of a dominant negative version of the Rac protein to the germ cells. 5. cxcr4b morpholino inhibits the translation of the chemokine receptor CXCR4b. 6. control morpholino serves as control for 5. RhoA and ROCK regulate acto-myosin contraction, which is essential for proper germ cell migration [4, 5]. Using the activated and dominant-negative forms of these proteins will reveal the effect of myosin deregulation on PGC migration and actin dynamics. Rac is essential for formation of maintenance of the leading edge in migrating germ cells. Expression of the dominant-negative form of Rac thus leads to loss of polarity and abnormal blebbing. Cxcr4 morpholino reduces the level of the chemokine receptor resulting in non-directed PGC migration. Following the Pit Talk you will be provided with ~20hpf embryos for imaging, that were injected with samples 4, 5 and 6 on the previous day (by Michal) to look at germ cells that arrived at the target. Subsequently, embryos of ~6hpf injected with any 2 of the samples below will be provided for imaging at a high magnification (63x). Movies of migrating cells will be recorded upon imaging at 5 or 10-second intervals for min. 2

3 Zebrafish Development and Genetics course 2013 Cell labeling and migration Migrating cells will be tracked for path and speed at 10x imaging at a 1 min interval for at least 20 min. Samples include reagents: 1. 1 & & & & & 6 RhoA and ROCK regulate acto-myosin contraction, which is essential for proper germ cell migration [4, 5]. Using the activated and dominant-negative forms of these proteins, the effect of myosin deregulation on PGC migration and actin dynamics will be revealed. Cxcr4 morpholino reduces the level of the chemokine receptor resulting in non-directed PGC migration. Image processing 1. We use ImageJ or Metamorph software to analyze raw data 2. ImageJ can be down loaded at 3. A PDF with instruction for basic use of ImageJ is part of the course package References: 1. Koprunner, M., et al., A zebrafish nanos-related gene is essential for the development of primordial germ cells. Genes Dev, (21): p Raz, E. and Reichman-Fried, M. Attraction rules: germ cell migration in zebrafish. Curr Opin Genet Dev., (4): p Doitsidou, M., et al., Guidance of primordial germ cell migration by the chemokine SDF-1. Cell, (5): p Blaser, H., Reichman-Fried., et al, Migration of zebrafish primordial germ cells: a role for myosin contraction and cytoplasmic flow. Dev Cell, (5): p Kardash, E., et al., A role for Rho GTPases and cell-cell adhesion in single-cell motility in vivo. Nat Cell Biol, (1): p ; sup pp Riedl, J., et al., Lifeact: a versatile marker to visualize F-actin. Nat Methods, (7): p

4 Zebrafish Development and Genetics course 2013 Cell labeling and migration Schedule Friday August 9th 13:00-14:00- Pit Talk: Time lapse imaging of germ cell migration: Erez Raz 14:00-15:30- microscopy; imaging ~20 hpf embryos 15:30-18:00 - time lapse microscopy of migrating germ cells in 6 hpf embryos. 20:00-22:00 data processing and preparation of presentations. Saturday August 10th 9:00 11:00 Wrap-up; student presentations. 4

5 Image processing using ImageJ basics Erez Raz and Michal Reichman-Fried MBL 2011

6 - Home Page: Installation - Instructions for installation: - for making a quick time and processing, use the ImageJ.app change memory allocation - memory: edit>options>memory>1024 restart the application many command paths can be found when searched under help using keywords!

7 Uploading data into ImageJ - To open an image file: simply drag the tif file or stack into ImageJ menu -Always keep a copy of the raw data, since the software modifies the file and has limited Undo options - Save files (.tiff) in between manipulations!!! (because you might want to go back to the original ones)

8 preparing an image for presentation 1. Select a Region Of Interest (ROI): draw it using the cursor as you like, or: Edit>selection>specify 2. press t or Analyze>Tools>ROI manager - a window of ROI manager will appear 3. To copy the ROI from one file to the other, select the target stack, double click the Roi identification number in the ROI Manager panel, the ROI will appear at the same location on the target stack or Edit>Selection>Restore Selection on the second image. 4. For cropping the region of interest and/or selecting a range of individual frames from the stack, use the Duplicate function - Image>Duplicate or Right mouse>duplicate

9 Background correction - To correct for uneven illumination in a normal epi-fluorescence: - ImageJ menu: Process>subtract background rolling ball radius: usually (try several values) - how to test yourself : draw a line along the image before and after subtraction, press k => you will get the intensity profile plot, the pattern of intensity within your region of interest should not change - For confocal (even illumination): Process>Math>Subtract (define value) you can determine the mean background value to be subtracted by drawing a ROI in the background area and press m. define a ROI from the background to be used as the value to be subtracted from every pixel (suitable for even illumination (e.g., confocal) where same value is subtracted (unlike the background subtraction in the case of uneven illuminaiton) to get the value press 'm' and 'mean' is

10 Saving images for presentation 1. intensity range set up : Image>Adjust>brightness/ contrast you can set up certain intensity range (important when showing differences in intensities) 2. color selection for individual stacks Image>Lookup tables. select a color. this step is not a pre-requisite for merging stacks. 3. To keep the color: Image>Type>RGB color (will lose the intensities values information, used only for presentation) 4. Merging channels Image>Color>Merge channels 5. Scale bars - First define the dimensions: Analyze>Set scale For example, typical values that should be derived from the camera specification are- 63x, bin 2: distance in pixels : 1 Known distance: 0,2 µm - Putting scale bar: Analyze>Tools>scale bar

11 correct for bleaching download bleach correction plugin (EMBL tools) use imagej 64 to use it. simple ration method is ok

12 Making a movie 1. making substack - to keep part of the stacks or selected frames only(optional): image> stacks >tools > make substack 2. Optional: Combining stacks (convert to RGB before!!!) image> stacks >tools > combine 3. writing text on images - activate the letter icon in the ImageJ command bar (and save by Edit>Draw) 4. time stamper - image> stacks > label 5. Save the stack with all the information as a tiff file 6. File > Save as quick time movie > Compression: animation Quality: maximum keep a short name find all the parameters for the time stamping

13 combining movies convert stack to RGB (Image>Type>RGB color) image > stack> tools > combine

14 Generating tracks and analyzing speed download the manual tracker plugin- ij/plugins/track/track.html and save it in the plugin folder. Restart ImageJ open the stack and Plugins> Manual Tracking A window opens and parameters should be provided; camera specifications are considered for speed. select add track > click the tracked object as you move from one frame to the next > end track. A data file is generated that can be opened as an Excel file to be analyzed. to open tifs into a stack in imagej file> import> image sequence

15 Creating and recording macros - To generate a sequence of commands and record it as a single path referred to as a macro : Plugins>Macros>Record Important is to remember that it always creates a certain path, so stick to a certain computer or have a memory stick

16 Useful links - Plugins: - Mailing list - contains searchable archive where you can find the information: