Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
USING THE JATLAS VIEWER AND USING THE GENE EXPRESSION DATABASE What is the JAtlas viewer? This is a tool developed by the MRC Human Genetics Unit here in Edinburgh to display a 3-dimensional embryo and show virtual sections through it How do I get the digital embryos? These can be obtained from the Embryology Learning tools page in EEVeC https://www.eevec.vet.ed.ac.uk/index.asp . Select Teaching tools and resources/Embryology/JAtlas. No log-in is required Choose the one you wish to study (the following describes the 60h chick embryo) and down-load it as a zipped file into your workspace. Unzipping the file reveals a folder containing the digital embryo as a file with the extension .wlz and an additional Anatomy folder. How do I open the chick 60h embryo in JAtlas viewer? From the same page on EEVeC you can launch the JAtlas viewer (it takes a little while). Then use File/open from the menu to navigate to and load the digital embryo .wlz file that you have unzipped into your workspace. You should have two open windows. The left is the viewing tool for rotating the 3-D embryo or its domains. The right is the anatomy ‘tree’. For the 60h chick embryo, you should also have a surface representation of it in the 3-D rotation window These surface representations do take up memory and it is best to turn it off. Drag across the screen to show how you can rotate the object and then use the Show 3D menu to unclick both the bounding box and the 3D surface How do I view the virtual sections through the chick 60h embryo? 1. The menu option ‘view section’ allows you to choose between three cutting planes Choose the the zx plane (90,90,90) of the 60h chick embryo. A new section viewer window opens. Click on the section slider near the bottom of this window (note that you are at section 0). Repetitive clicking on the right arrow key on your keyboard will move you through the sections in a positive direction (don't click too fast as it can cause the software to hang up). As you click through, the bottom right number will change to show the section number. You can always return quickly to a particular section by typing the section number into the bottom right box followed by return. This is a quick way, for example, of returning to section 0 in the middle of the stack. Try it. Exercise A Can you find this section? You can move back through the sections using the left arrow on the keyboard. When you go below zero, the slide numbers show as negative. As you do this, observe the changing appearance of the Central Nervous System, in particular the spinal cord. The embryo is an imperfect shape so that different regions of the spinal cord appear on different sections. Exercise B Observe the optic stalk, optic vesicle and lens appearing in the region of the forebrain in the section range -45 to -85 2. We have ‘painted’ selected ‘domains’ of the embryo for you and these appear in the Anatomy tree to the right of the 3-D rotation window. These domains are loaded by single clicking on the little horizontal arm of ‘embryo’ and then similarly with CNS. Double clicking on the final CNS colours the central nervous system on the section and loads a surface representation of that domain. You can change the colour of the domain from the default value using the Anatomy key (click on colour box) Exercise C Rotate the embryo in the 3D window and observe the structure of the brain, optic vesicle, otic vesicle and some cranial nerves. Then turn painted volume off by using delete in the Anatomy Key. 3. Choose view section xy (0,0,0) and yz (90,0,90). This opens two new sections windows at right angles to the original xz (90,90,90) plane. Choose Show/ 'intersection of views in this original section window If you go to the xy window (0,0,0) and click on the section slider, you can move through the sections as you have done before. By default, you will start at section zero and the position of this section relative to zx (90,90,90) is shown as an intersection line in the zx window with a colour corresponding to the colour in the top right box of the xy window (yellow in examples above). As you click through the sections in the xy window, the line in the zx window will move to show the section postion. At any stage, you can choose an anatomy component and display it as a painted volume to help you understand the relationship between the sections and the object. But best to delete the volume when you are moving through sections Exercise D As you move through the negative sections in the xy (0,0,0) window, you will get good views of the olfactory pits, the optic stalk and vesicle and the lens. You will also see the relatively early stages of caudal development compared to the rostral components. This is typical of the chick. Development proceeds much faster rostrally than caudally. As you go through the positive sections, you will see the pharynx, the truncus arteriosus and ventricle (and the point where these two become continuous), the otic vesicle, the lung buds (sections 75-95) and regions of the gut tube. Right at the dorsal end of the sections, you will see the segmentation of the somites Exercise E There are two other chick embryo data files (50h and 40h) that could be investigated but perhaps the best thing to do is to look at their corresponding movies at EEVeC/Teaching tools and resources/Embryology/Movies Chick 40h fluorescent and painted movies Chick 50h fluorescent and painted movies How do I view the sections through the 21 day pig embryo? The digital chick and pig embryos are fundamentally different in the ways in which they were obtained. The chick embryos are obtained by a process of optical tomography. They are relatively low resolution because of this method of collection but the virtual sections through them produce equivalent quality in any plane. The pig embryos are reconstructions from an actual series of sections. When viewed in the plane of these original sections, they are of high quality and good resolution. However, when viewed in different planes, the quality is much poorer because of the imperfections and distortions in both the sections themselves and their alignment. Choose the datafile named 'pig 21 day transverse' and down-load it as a zipped file into your workspace. Unzipping the file reveals a folder containing the digital embryo as a file with the extension .wlz and an additional Anatomy folder. Open the .wlz file in the JAtlas viewer. The 3D view that loads into the 3D window is not very informative but it does show the process of reconstruction from the transverse sections that has been carried out. Turn off the 3D view and bounding box. 1. Choose View section xy (0,0,0). This is the original plane of sectioning and shows high quality detail. Exercise F The default section 0 shows details of ventricle, liver, mesonephros, forelimb bud, neural tube, gut tube, lung bud and dorsal aorta. Try identifying these structures. As before, you can check whether your identification is correct by choosing a particular domain on the anatomy tree and observing both its 3D reconstruction and its painted area in the section. Remember to delete the volumes from the anatomy tree once you have finished and before you move through the sections 2. Click on the pointer in the section slider and move through the negative numbered sections Exercise G As you move, you will see the limb bud connection being made (around section -12) and the second limb bud appearing around section -28. Observe the splitting of the dorsal aorta around section -62. The olfactory epithelium lining the olfactory pits becomes evident around section -65 to -95. An optic vesicle comes into view around section -144. Aortic arches are difficult to see in these sections but if you look at the series -153 to -160 you will see an aortic arch connection forming between the dorsal aorta and the heart. It might be best to turn the arterial volume on (Anatomy tree) in order to see that. The joining of the laryngo-tracheal groove to the pharynx can be seen around section -202 to -212. Type in 0 to the bottom right box to return to the middle of the stack. Moving through the positive sections, the ventricle disappears as the liver and mesonephros become very prominent. Try now to follow the gut tube. The gut tube expands into the stomach around section 50. The herniated gut tube can be seen between sections 150 to 180. Exercise H There is also a sagittal data file that could be investigated but perhaps the best thing to do is to look at their corresponding movies at Pig 21 day sagittal painted Pig 21 day transverse painted Pig 21 day transverse unpainted What is the gene expression database? The UK MRC Human Genetics Unit in Edinburgh has developed a digital database (the Edinburgh Mouse Atlas Project) as a resource for spatially mapped data such as in situ gene expression. http://genex.hgu.mrc.ac.uk/ 1. Choose Emage gene expression database, then choose web search EMAGE/browse 2. Choose Search gene/protein. Enter Pax6 (this opens a complete listing of the study of Pax6 expression at different stages and with different methods) 3. Scroll to EMAGE 361 and select by clicking 4. This is material we have looked at in the lecture. You can view the image, view the movie which shows the context of the section studied in the 3D embryo, view the probe information and the method of visualisation A second portion of the database contains tools for viewing sections through the reconstructed embryo. If you return to: http://genex.hgu.mrc.ac.uk/ and choose 3D embryo anatomy atlas 1. Choose the Theiler stage 15 embryo (you may need to adjust the level of the split top/bottom screens by dragging at the interface) 2. Clicking on the view of the whole embryo give a section at that level Sagittal, transverse or frontal sections can be chosen. This is very similar to JAtlas 3. In the bottom window there are movies to play. Try the section movies which animate a series of continuous sections in one chosen plane Examples of detection of gene expression in Drosophila embryos Anterior pole -Gal reporter B C Horse radish peroxidase conjugated to second antibody In-situ hybridisation Questions: 1) These figures represent the pattern of expression of genes involved in Drosophila body plan development. Different techniques have been used to show the expression of these genes. Which are the gene types represented in the panels? 2) Which defects in body plan development do you expect if the gene in ‘B’ is inactivated?