HREM workshop at the Division of Anatomy, Medical University of Vienna, 23-24 May 2019
HREM was the main imaging method used by the DMDD programme to enable phenotyping of embryos from genetically engineered mouse lines. Workshop tutors will include former members of the DMDD programme who developed and perfected the technique.
HREM can also be used to generate digital 3D volume data for a wide range of biological samples, analyse embryonic and adult tissue samples of various vertebrates and invertebrates, as well as samples from plants.
This international workshop will cover sample preparation, data generation and data analysis, as well as challenges, limitations and potential pitfalls.
The workshop is limited to 10 attendees, and will combine lectures, expert demonstrations and hands-on sessions. Attendees will be supervised working with HREM systems.
For the full workshop programme, enquiries, or to register, please contact Stefan Geyer at the Medical University of Vienna (firstname.lastname@example.org).
DMDD is publicising this workshop to those who may be interested in developing their knowledge of the HREM technique and understanding its potential, but the workshop is not a part of the DMDD programme.
It is widely known that a functional placenta is vital for normal embryonic development, but how much it may contribute to embryonic lethality has never before been systematically studied. Our research, published in Nature, demonstrates for the first time a remarkable co-association between embryonic lethality and placental defects.
A healthy placenta is vital to sustain normal pregnancy, ensuring proper supply of nutrients and oxygen to the baby. Abnormalities in the placenta can therefore have serious repercussions on fetal development, even causing miscarriage. Despite this, remarkably little is known about the identity of genes essential for a normal, functioning placenta and even less about the extent to which placental abnormalities contribute to defects that can arise as the fetus develops.
EXTENT AND IMPACT
We screened more than 100 mouse mutant lines in which affected embryos die before or immediately at birth. Almost 70% showed serious abnormalities in the placenta; in extreme cases this resulted in a placenta incapable of supporting embryo development beyond an early stage (Figure 1), in others, abnormalities in the developing embryo were accompanied by abnormalities in the placenta.
FIGURE 1 – mouse mid-gestation embryos and placentas shown at the same magnification
LEFT: a normal, wild-type (WT) genotype. RIGHT: Nubpl mutation (MUT) shows a growth-retarded and developmentally delayed embryo that will not survive until birth.
The placentas are stained for a marker of the exchange surface (MCT4, in green) across which nutrients are transported from the mother to the embryo. Note the complete absence of this cell type from the MUT placenta. Red staining is for a cell surface protein (CDH1) demarcating the cells underneath the MCT4-positive layer (arrows), which are greatly reduced in number in the MUT placenta.
EMBRYO AND PLACENTA DEFECTS ARE LINKED
Not only do these results identify a large number of genes essential for normal development of the placenta; in addition they show an intriguing link between placental defects and abnormalities affecting the brain , heart and vascular system of the embryo itself. The research, led by Dr Myriam Hemberger and her colleagues at the Babraham Institute demonstrates how common placental abnormalities are when embryos develop abnormally.
RESCUING EMBRYONIC LETHALITY
The team examined in detail three different genes that cause embryonic lethality, and showed that for two of them the loss of the gene affected proper differentiation of placental cell types. For one of these genes they were also able to show that embryo death was a direct result of gene loss in the placenta, by providing the mutant embryo with a genetically normal placenta, which prevented embryo death.
Although the DMDD study uses mice, the results are likely to be just as relevant for studying human pregnancy and the role the placenta may play in pregnancy complications and the origins of birth defects in newborn babies.
All image and phenotype data gathered by the DMDD programme is freely available to the scientific community at dmdd.org.uk. The research described in this blog post was funded by the Wellcome Trust with support from the Francis Crick Institute.
Our latest data release includes HREM image data for an additional 5 lines, and HREM phenotyping data for 4 lines. Five additional early lethal lines have also been identified, as well as placental phenotype data for more than 100 mutant lines, with associated placenta morphology and yolk sac images.
Throughout the DMDD project we continue to add data for existing lines, and in this release we have added P14 viability for mutant lines, Theiler stage (where assessed), and the voxel size of each HREM image stack.
Initial analysis of the new HREM phenotyping data shows two lines newly associated with heart defects.
Oaz1 ASSOCIATED WITH DORV
Oaz1 is a gene regulating levels of polyamines within the cell and is widely distributed in cells and tissues of the body. Our data now shows that removal of this gene causes a serious abnormality in heart development in which the vessel normally carrying blood from the left ventricle of the heart (the aorta) is in fact attached to the right ventricle (a defect known as “double outlet right ventricle” or DORV). As with many mutant lines, the embryos also show extensive swelling of the body (“edema”).
Cc2d2a ASSOCIATED WITH VSD AND OSTIUM PRIMUM DEFECT
Cc2d2a encodes a protein that plays a critical role in formation of cell cilia and mutations in this gene are associated with diseases such as Meckel syndrome type 6, which results in a broad range of symptoms such as polydactyly, cleft palate and kidney malformations. Our data reveals that removal of the Cc2d2a gene also has profound effects on heart development. Not only do the embryo hearts fail to complete separation of the left and right ventricular chambers (a “ventricular septal defect”), they also fail to form a proper wall between the left and right atrial chambers (an “ostium primum defect”). In addition, they have lost a swath of tissue at the junction between the atria and ventricles (the “vestibular spine”) that is essential for completing chamber separation.
Many of the genes studied by DMDD do not currently appear to be associated with any disease, however careful analysis of the phenotypes from lines such as these could contribute to the identification of new disease models, and our data is freely available at dmdd.org.uk in order to encourage this. For more information please email email@example.com.
At our BSCB/BSDB exhibitor stand, we created a little web testing station, complete with ‘booth’ – the computer and user are hidden behind our poster board in this photo. Volunteers were asked to spend 10 minutes testing our website, and in return were entered into a prize draw for a £50 Amazon voucher – won by Dr Seyed Beati, from the University of Dundee.
Volunteers were asked to complete some short tasks on our website, which were recorded using Silverback, a usability testing tool. Silverback not only records the user’s actions on screen, highlighting where they click. It also records a small image of the user, and although it doesn’t do eye-tracking it does allow us to see if users are looking all over the screen, or frowning or looking puzzled. Silverback does all of this unobtrusively, so that users are not distracted by their own image in a video; the only indicator that they are being recorded is the camera light. The movies generated can then be reviewed to see how easy or difficult specific tasks are for users.
This is the first time we’ve done user testing with a group of people, and although it was time-consuming, the information we gathered made it worth the effort, and made us look at the website through users’ eyes. We’ve now reviewed the movies, made notes of what we observed and categorised these observations.
Many of them are small things that are relatively trivial to fix. For example, we thought our website feedback link (Usersnap) on the right of the screen would be easily spotted as nothing else is around it. But it turns out that’s not the case – it seems that because no other content is near it, no-one actually looks there! So, it will be moved to the top right of the page, where most users naturally look for help.
Some of the more challenging fixes relate to the usability of the Stack Viewer, our online tool for exploring the HREM data. We made a lot of observations about how the controls are used on this page, and the problems with the current interface. One thing that we noticed was that the buttons to change orthogonal views are separate from the thumbnail images in the sidebar that show the view switching, so we’ll be bringing these together. In fact we’ll be bringing all the controls into a single ‘control panel’, rather than some on the side and some in the view pane.
The other parts of the website that presented challenges to users were Search and the ‘line page’, which shows all of the data for a particular gene. We have new search functionality already under development, and will now add in some additional changes to make this work better for users.
The line page is one of the busiest pages on the site, and we knew before the testing that it needed some improvements. The test results show us that we need to provide more context on the page to show users what’s what, and to make the actions that can be taken more prominent and easily understood (otherwise known as affordance).
Across the site we’ll also be adding more tooltips, to provide contextual help and information next to where the user is looking, as well as breadcrumb trails to help orient users.
We wouldn’t be able to make these improvements without your input, so we’d like to say a huge thanks to everyone who gave ten minutes of their time!