HREM WORKSHOP

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.

THE WORKSHOP

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.

REGISTRATION

For the full workshop programme, enquiries, or to register, please contact Stefan Geyer at the Medical University of Vienna (stefan.geyer@meduniwien.ac.at).

FURTHER INFORMATION ON HREM

DISCLAIMER

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.

JOIN OUR HREM AND PHENOTYPING WORKSHOP

Generation and interpretation of HREM data from normal and mutant E14.5 mouse embryos in the DMDD programme

 

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20 – 22 October 2017

The Medical University of Vienna

 

Deciphering the Mechanisms of Developmental Disorders (DMDD) is a large-scale imaging and phenotyping programme for genetically modified mouse embryos. For embryos at E14.5, the key imaging technique is High Resolution Episcopic Microscopy (HREM), and the resulting images are used to comprehensively phenotype the embryos using a systematic approach.

With a combination of lectures, demonstrations and hands-on sessions, this three-day workshop will introduce HREM technology and discuss the value of the resulting images when used to score morphological phenotypes. The HREM procedure will be described, while sample preparation and data generation will be demonstrated.

As an introduction to phenotyping, the workshop will cover the normal anatomy of E14.5 mouse embryos and the morphology, topology and tissue architecture of their organs as presented in HREM data. A special focus will be given to developmental peculiarities and norm variations in anatomy. A protocol for scoring abnormalities will be demonstrated, after which hands-on sessions will allow participants to practice scoring both wild-type and mutant embryos whilst receiving feedback.


Registration

http://www.bioimaging-austria.at/web/pages/training/by-cmi-technology-units.php

Early registration is recommended to secure a place, as this workshop is limited to 8 attendees.

The registration fee of Euro 300 (payable by invoice) includes access to all workshop sessions, tea, coffee and lunch each day, and dinner on the first evening. Lunches are sponsored by Indigo Scientific.


Full programme

FRIDAY 20 OCTOBER

Session 1, The DMDD Programme

Background and workflow (lecture)

Data collection and the DMDD website (lecture and demonstration)


Session 2, High Resolution Episcopic Microscopy (HREM)

Workflow, specimen harvesting and preparation (lecture and demonstration)

Data generation and data quality (lecture, demonstration and hands-on)

Data management and analysis (lecture, demonstration and hands-on)

Limitations and artefacts (lecture and demonstration)

 

SATURDAY 21 OCTOBER

Session 3, Phenotyping using 3D models from HREM data

Producing and interpreting 3D models using HREM data (lecture and demonstration)

Staging 3D models of E14.5 embryos (lecture and demonstration)

Using 3D models to score external embryo phenotypes (lecture and hands-on)

Morphometry of 3D embryo models (lecture and hands-on)

 

Session 4, Phenotyping using 2D HREM section images

Annotation using the Mammalian Phenotype ontology (lecture and demonstration)

Phenotyping protocol (lecture, demonstration and hands-on)

Stage-dependent peculiarities (lecture, demonstration and hands-on)

 

SUNDAY 22 OCTOBER

Session 5, Phenotyping examples and pitfalls

Norm variations (lecture and demonstration)

Artifacts (lecture and demonstration)

Supervised phenotyping of genetically normal embryos (hands-on)

 

Session 6, Phenotyping mutant embryos

Supervised phenotyping of mutant embryos (hands-on)

 

Session 7, Feedback and questions


General information

Workshop timings

Daily from 09.30 – 12.30 and 13.30 – 17.30

Location

Division of Anatomy, The Medical University of Vienna, Waehringerstr. 13, A-1090 Vienna

Facilities

Hands-on sessions will take place in groups of two. Each pair will have access to both a high-end Mac and PC operating the required software.

Faculty

WJ Weninger, LH Reissig, B Maurer Gesek, J Rose, SH Geyer (Medical University of Vienna)

TJ Mohun (The Francis Crick Institute, London)

DMDD WEBSITE ENHANCEMENTS

Following user testing of the DMDD website at the BSCB/BSDB meeting earlier this year, we’ve been working behind the scenes on some enhancements to our site. The changes went live today, and here’s a rundown of what’s new.


HOMEPAGE

Perhaps the first thing you might notice is our updated homepage. If you’re planning to search the database for genes or phenotypes, a featured ‘SEARCH’ form on the homepage will make this quicker and easier.

 

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The enhanced DMDD homepage has a dedicated ‘SEARCH’ form, as well as additional information about the project.

We’ve introduced a ‘mega menu’ at the top of the page, so you can see more information about the different sections of the website and quickly navigate to the right place. The height of the homepage has also been decreased, so it’s easier to see all the content on laptop devices.

 

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A ‘mega menu’ provides additional information about each page to help with navigation.

 

Also on the homepage: get bite-sized bits of information about the DMDD programme from the new ‘ticker’ text, sign up to our email newsletter or refresh the page to see a variety of featured images from our dataset.


NEW CONTENT

DMDD recently released a temporal gene expression profile for wild type embryos – the first step towards providing molecular phenotypes for our embryonic lethal lines. We have now added a dedicated wild type gene expression profiling page, which provides some background information and a link to the dataset itself in Expression Atlas.

A new section titled ‘OUR RESEARCH’ includes content about the DMDD consortium, our analysis techniques and a list of our publications. Protocols for placental histology, transcriptional profiling and immunohistochemistry are now available, allowing users to repeat any results of interest, while the contribution by each of our consortium member institutions is detailed on the DMDD pipeline page.

We have added additional information about the mutant lines and how they were generated, updating the About the mutants page to include allele diagrams and information about how to request lines for future research.

Finally, a dedicated section about High Resolution Episcopic Microscopy (HREM) now provides detailed instructions for the HREM imaging technique. Over time this hub of information will grow to include videos and additional information on sample dissection for HREM imaging.

A new 'HREM methods' section is a hub on information about the HREM imaging technique.
A new ‘HREM methods’ section is a hub on information about the HREM imaging technique.

COMMUNICATIONS

Our website now has a dedicated news page highlighting data releases, additional content on the DMDD blog and other news related to the project. It’s also a place to find out where you can meet us at forthcoming events, and how to connect with us via social media.

We are also keen for you to tell others about our data. You can easily download flyers or other promotional materials about the DMDD programme from our Communications materials page.


STYLE

Finally, we’ve made some changes to the style of the site to increase its usability. The most immediately visible change is the switch from a serif font, which has small projecting features called ‘serifs’ at the end of strokes, to a sans serif font, which has no ‘serifs’ and better letter spacing to make the text more readable.

We’ve also changed the layout of each embryo page to make it immediately apparent what data is available for each embryo, without requiring multiple clicks.


YOUR FEEDBACK

Part of our user testing showed that users didn’t notice the feedback button on the right edge of pages, so we’ve moved this to the top right, where users look for it.

We’re excited to launch the enhanced version of our website and would like to know what you think. If you have any suggestions just click the ‘Feedback’ link at the top right of any page on our website, or post a comment below, to let us know your ideas. Make sure to leave your details if you’d like a response or are happy for us to contact you to find out more.

HREM IMAGING REVEALS DETAILS OF ABNORMAL EMBRYO DEVELOPMENT

High Resolution Episcopic Microscopy (HREM) is a technique that allows embryos to be imaged in unprecedented 3D detail. It’s the main imaging method used by the DMDD programme – we use it to identify a wide range of developmental abnormalities that result from mouse gene knockouts. The detail revealed by HREM models can be seen in this video of an embryo with a profound ventricular septal defect. At the time of imaging, the embryo was just over 1cm long.

 

 

But despite the many benefits of HREM it’s still a relatively uncommon technique to use, because a commercial system has only recently become available. In this post we’ll give a quick overview of how HREM works (and why it’s so useful).


MAXIMISING 3D IMAGE RESOLUTION

Many 3D imaging methods work by taking a series of images through the whole volume of a tissue sample, after cutting it into thin sections. The resulting ‘stack’ of section images can then be used to build a 3D model using software such as Osirix or Amira. The resolution of the final model depends on both the thickness of the sections and the accuracy with which the final images can be aligned. Sometimes this can involve using external markers to help align the images.

However, sections can often become distorted as they are cut and captured – even using external markers the final alignment can often be poor. HREM overcomes this problem by imaging the face of the block (so-called ‘episcopic imaging’) rather than the individual sections.

The samples (in our case, embryos) are embedded in a hard plastic resin, which contains fluorescent dyes. Tissue at the surface of the block can be visualised against the bright background of the fluorescent plastic, and this simple approach gives remarkable detail. The sections are not distorted, and the relative alignment of the images is well known. As a result the resolution of the 3D model is limited only by the thickness of the cut sections.

 

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HREM models at 3 µm resolution allow detailed 3D investigation of developing embryos.

 

Since the plastic resin is very hard, HREM sections can be cut as thin as 1 µm. In the DMDD programme we use sections between 1 and 3 µm (depending on the size of the embryo), and around 3000 images are recorded for an embryo at E14.5. At this resolution, features such as individual nerves and blood vessels can be identified, as shown in the image above.


WHY USE HREM?

HREM data has allowed the DMDD team to identify nearly 400 different phenotypes in knockout mouse embryos. These range from large-scale organ malformations to the abnormal development of nerves and blood vessels. In the image below, a mutant embryo was found to have an absent hypoglossal nerve. Since this nerve controls tongue movement, the phenotype typically results in neonatal death when the pup is unable to suckle.

 

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This view from the DMDD database shows a mutant embryo (right) from the knockout line D930028MRik, and its wild-type litter-mate (left). The mutant embryo has an absent hypoglossal nerve on both the right and left sides of the head. The same nerves are clearly visible in the wild-type embryo.

 

The DMDD programme has used HREM to image whole mouse embryos. But the technique has also been used to image embryos, organs and tissues from other animals, as well as samples from plants. It provides a way to resolve details that may not be visible in other 3D models.

If you would like more information or advice about how to image a tissue sample using HREM, please email contact@dmdd.org.uk.

DMDD HREM image and phenotype data can be found at dmdd.org.uk.

NEW DMDD YOUTUBE CHANNEL

This week sees the launch of the DMDD YouTube channel. We’ve added videos that explore different aspects of our embryonic-lethal mouse data, providing a new way to visualise phenotypes that we’ve observed in our knockout lines.

For example, this video compares HREM data from a Psph mutant embryo with its wild-type litter-mate.

 

 

Detailed phenotyping of the mutant revealed a double outlet right ventricle, absent tongue, increased rib number and abnormal hindbrain morphology. The data can be explored in more detail on the DMDD website.

In the video below we erode into a Brd2 mutant embryo to reveal a profound ventricular septal defect.

 

 

We are just getting started, so more videos will be added soon including instructional videos on how to access and analyse our data online. Visit the DMDD YouTube channel now to see more.


GENERATE YOUR OWN 3D MODELS OF DMDD DATA

Our HREM embryo data can easily be visualised using 3D modelling software such as Osirix, allowing you to explore phenotypes in great detail. To request the offline data for 3D modelling, or if you have any questions, please email contact@dmdd.org.uk.

DATA RELEASE – AUGUST 2016

New embryonic-lethal knockout mouse lines are now available on the DMDD database.

If you haven’t previously taken a look at our data (or even if you have) now would be a good time to explore our website.  We’ve added new embryo phenotype data and HREM images for many knockout lines, taking our total dataset to more than 4 million images of 550 embryos. We also have placental histology images and phenotypes available for over 100 mutant lines.

This post explores some of the phenotypes observed in the new data, and highlights new lines that could be relevant for clinicians researching rare diseases and developmental disorders. But there isn’t enough space here to include every interesting feature of the data – the best thing to do is to explore it yourself.


EMBRYO PHENOTYPES

Our phenotypers at the Medical University of Vienna have observed many interesting phenotypes in the new data.

Embryos from the line Adamts3 display both subcutaneous edema and bifid ureter. A bifid ureter is the most common malformation of the urinary system, [1] in which there is a duplex kidney drain into separate ureters. This observation highlights the incredible resolution of HREM images, which allow detailed phenotypes to be scored for each embryo.

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Bifid ureter (left side) observed in an Adamts3 mutant embryo. The red arrows highlight a single ureter on the right side, but two branches on the left side.

 

Embryos from the line H13 suffered from severe abnormalities in heart morphology, and had an abnormal heart position within the body. The stomach situs was also inverted, as shown in the image below. Note that severely malformed embryos often have different tissue characteristics, which can result in reduced image clarity.

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Comparison between a H13 mutant embryo (left) and its wild-type litter-mate (right). The yellow arrows indicate situs invertus of the stomach.

 

Embryos from the line Brd2 exhibited a profound ventricular septal defect, as shown in the video below.

 


PLACENTAL PHENOTYPES

Our placental image and phenotype dataset is growing rapidly and now contains more than 100 lines.

H13 knockout placentas were smaller than their wild-type counterparts and showed reduced vascularisation in the placental labyrinth, the region of the placenta that allows nutrient and gas exchange between the mother and the developing embryo.

Click to view larger image.
A comparison of the placenta from a H13 mutant embryo and that of its wild-type litter-mate.

Vascularisation of the labyrinth is crucial to allow the embryo to receive the oxygen and nutrients needed for normal development. This is just one example, but many more placental phenotypes are available on our website.


LINKS TO CLINICAL STUDIES

Systematic knockout mouse screens can offer a wealth of information about the genetic basis of rare diseases. Many DMDD lines have human orthologues known to be associated with developmental disorders, and the nature of our study means that it would not be possible to derive equivalent systematic data from human patients.

New knockout lines of potential clinical interest include:

Brd2: the human ortholog of this gene is associated with epilepsy, generalised, with febrile seizures plus, type 5.

Cog6: in humans, COG6 is linked to Shaheen Syndrome and congenital disorder of glycosylation, type IiI.

Npat: the human ortholog is associated with Ataxia-telangiectasia, a rare inherited disorder affecting the nervous and immune systems.

Nsun2: in humans, NSUN2 is linked to mental retardation, autosomal recessive 5 and Dubowitz syndrome.


A FULL LIST OF NEW DATA

Embryo phenotype data added for: Adamts3, Brd2, Cog6, Cpt2, Dhx35, H13, Mir96, Npat, Nsun2, Pdzk1.

HREM embryo images added for: Atg16l1, Capza2, Cog6, Coro1c, Cyfip2, Dhx35, Gm5544, Nadk2, Nrbp1, Rab21, Rpgrip1l, Syt1.

Placenta image and phenotype data added for: 1110037F02Rik, Actn4, Atg16l1, Camsap3, Capza2, Cfap53, Coro1c, Crim1, Crls1, Cyfip2, Dmxl2, Gm5544, Gtpbp3, H13, Nsun2, Rab21, Rala, Rpgrip1l, Syt1, Trim45.


REFERENCES

[1] Obstructed bifid ureteric system causing unilateral hydronephrosis, A. Bhamani1 and M. Srivastava2, Rev Urol. 2013, 15(3) p.131–134, PMC3821993.

1 Department of General Medicine, The Ipswich Hospital, Ipswich, UK.
2 Department of Radiology, Barking, Havering and Redbridge NHS Trust, Romford, Essex, UK.