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.

DMDD DATA TO REMAIN ACCESSIBLE TO ALL

 

 

As DMDD’s five-year Wellcome Trust grant draws to a close, the analysis of all 250 knockout mouse lines is almost complete. The remaining image and phenotype data will be added to our website over the coming weeks. We are excited to announce that we have secured additional funding from the Wellcome Trust, which we believe will allow us to maintain the website for a further two years. In the long term we are seeking to identify a suitable place to archive the data, so that it can remain accessible to all.


STAY UP TO DATE

Follow DMDD on Twitter to be notified which archive(s) the data is moved to, and also to find out about any future publications. We thank you for your support and hope you will continue to use the DMDD website in the future.

PLACENTAL DEFECTS ARE HIGHLY PREVALENT IN EMBRYONIC LETHALS

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.


REFERENCES

The Advance Online Publication on Nature, ‘Placentation defects are highly prevalent in embryonic lethal mouse mutants is available now .


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.

LATEST DATA RELEASE HIGHLIGHTS INCLUDING NEW HEART DEFECT ASSOCIATIONS

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”).

Left panel: a view of the heart seen from the right side and showing both the pulmonary trunk (red arrow) and the aorta (yellow arrow) drain from the right ventricle. Right panel: a cross section through the body at the level of the heart shows the extent of swelling (arrows) in tissue beneath the skin.

 


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.

Shows three views of the embryo heart. The lefthand panel shows the ventricular septal defect; the middle panel shows the osmium primum defect and the right panel shows the absence of vestibular spine tissue which normally enables the atrial and ventricular septal walls to attach to each other.

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 contact@dmdd.org.uk.


A FULL LIST OF NEW DATA IN THE LATEST RELEASE

JOIN OUR HREM AND PHENOTYPING WORKSHOP

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

 

Click to view larger image.

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)

WHAT DOES NORMAL HEART DEVELOPMENT REALLY LOOK LIKE?

Every developing heart is subtly different. Hearts and their blood vessels don’t always grow at the same rate, to exactly the same size or precise shape, and this can complicate things if we want to identify an abnormality. To be sure if any feature of a heart is abnormal, first we need to understand the range of differences that we might see in normal hearts as they grow.

Surprisingly, this is a much-understudied area – something that it has only recently been possible to determine using modern imaging techniques. A new article published in Journal of Anatomy [1] uses high resolution 3D imaging to study more than 200 genetically normal mouse embryos from the DMDD programme, identifying the typical range and occurrence of different variations in the heart’s development. The image below shows the hearts of two genetically normal mouse embryos that were determined to be at exactly the same stage of development. However, one feature of their hearts that is very different is the extent to which the ventricular septum has grown to separate what was initially a single cavity into the right and left ventricles. The heart on the left has only a very small gap left in the developing septum, while the heart on the right has a much larger gap. Without this sort of study we wouldn’t be able to tell whether the heart on the right is normal or whether it has a ventricular septal defect – the most common congenital heart defect in newborns.

 

Click to view larger image.
The hearts of two genetically normal mouse embryos at precisely the same developmental stage show significant differences in the development of the ventricular septum.

 

The new data will be a valuable reference when identifying phenotypes in the heart and vessels of mouse embryos around the 15th day of gestation.

[1] Morphology, topology and dimensions of the heart and arteries of genetically normal and mutant mouse embryos at stages S21-S23, S. H. Geyer et al., J. Anat (2017), doi: 10.1111/joa.12663

 

 

9.5 MILLION EMBRYO IMAGES NOW AVAILABLE

A new set of DMDD embryo and placenta data has been released today, taking our total dataset to 9.5 million images of around 1300 embryos. Phenotypes are available for embryos from 73 different knockout lines, and we have phenotyped the placentas from 124 lines. We have also added data on the sex of each embryo.

Visitors to our website can now compare HREM embryo images with the closest-matching, annotated histological section from the Kaufman Atlas of Mouse Development. This follows a major project by the eMouseAtlas team at the University of Edinburgh to digitise the Kaufman Atlas at high resolution. The annotated Kaufman sections can be viewed alongside DMDD embryo images to help users who are unfamiliar with the detailed morphological features of a mouse embryo as it develops.

All DMDD data can be freely accessed at dmdd.org.uk, or you can continue reading for highlights from the latest lines to be made publicly available.


SEVERE BRAIN PHENOTYPES

Phenotyping of Hmgxb3 knockout embryos revealed severe brain defects, with half of the embryos displaying exencephaly. Embryos from this line also had a range of phenotypes including edema, abnormalities of the optic cup, and defects of the venous system including an abnormal ductus venosus valve and blood in the lymph vessels.

 

Click to view larger image.
An Hmgxb3 homozygous knockout embryo displays exencephaly.

 


 

POTENTIAL MODELS OF HUMAN DISEASE

A number of genes studied by DMDD have already been associated with human diseases. For example, Prmt7 mutations have been associated with Short Stature Brachydactyly Obesity Global Developmental Delay Syndrome, an autosomal recessive disease characterised by developmental delay, learning disabilities, mild mental retardation, delayed speech, and skeletal abnormalities. Strikingly, in the Prmt7 knockout embryos studied, the most common phenotypes included neuroma of the motoric part of the trigeminal nerve (a tumour within the skull, affecting the nerve controlling the jaw movements needed for speaking and chewing) and abnormalities of the hypoglossal nerve (which controls movement of the tongue) and the ribs.

Image data has been added for both Cc2d2a and Xpnpep1 knockouts. Mutations of the Cc2d2a gene are known to cause Meckel and Joubert syndromes, while Xpnpep1 has been associated with billiary atresia.

Many of the genes studied by DMDD do not currently appear to be associated with any disease, for example Hmgxb3 or Cbx6. There is potential that 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 in order to encourage this.


A DETAILED DESCRIPTION OF NORMAL MOUSE EMBRYO DEVELOPMENT

The Atlas of Mouse Development by Professor Matthew Kaufman describes normal mouse embryo anatomy using a series of hundreds of annotated histological sections. Even today, twenty three years after its publication, it is still considered to be the gold standard for describing mouse embryo development. As part of a project to update the book in 2012, the original sections were digitised by the Edinburgh Mouse Atlas Group and made freely available on their eHistology resource.

The images have now been integrated into the DMDD database, and users can directly compare any HREM embryo image with the closest-matching annotated Kaufman section.

 

Click to view larger image.
Each HREM embryo image can now be viewed alongside the closest-matching section from the Kaufman Atlas of Mouse Development.

 

This new feature is intended to help users who are not fully confident of the details of mouse developmental anatomy. It means that mutant mouse data can now be explored alongside a fully-annotated wild-type reference point.


A FULL LIST OF NEW DATA

Embryo phenotype data added for: Hmgxb3 and Prmt7

Embryo image data added for: Cbx6, Cc2d2aHmgxb3, Prmt7 and Xpnpep1

Placenta images and phenotypes added for: Mir96

To explore the data, visit dmdd.org.uk or for more information please email contact@dmdd.org.uk.

MEET US AT ISDB 2017

 

The International Society for Developmental Biologists 2017 meeting is taking place in Singapore from 18 – 22 June, and we’re currently putting the finishing touches to our exhibit. On stand 11, Emily and Chris will be able to show you detailed phenotype data for embryonic lethal knockout mouse lines, together with high resolution images of the embryos, like this Actn4 knockout embryo with abnormal lens epithelium morphology. You’ll also be able to try out our database at dmdd.org.uk where you can search and analyse all DMDD data free of charge.

 

 

While you’re there you can pick up a free DMDD mug, along with promotional flyers for our database.

 

DMDD promotional mugs
Sign up to our mailing list at the meeting to get a free DMDD mug.

 

We’re excited to be sharing the stand with our colleagues from eMouseAtlas, so you can also find out more about their digital atlas of mouse development. We look forward to meeting you!

FIND DMDD PHENOTYPE DATA IN THE MGI DATABASE

DMDD embryo phenotype data is now available in the Mouse Genome Informatics (MGI) database, complimenting the existing morphological phenotype data that is held there. To date we have contributed detailed phenotypes for 63 knockout lines, and will continue to provide additional data as it becomes available.

Each allele overview page shows the high level phenotypes that have been identified.

Click to view larger image.
An overview of the Sh3pxd2a tm1b(EUCOMM)Wtsi allele and the related high-level phenotypes identified by both DMDD and the IMPC.

 

Each high-level phenotype can then be expanded to show all relevant annotated terms. The image below shows all phenotypes related to the respiratory system for Sh3pxd2a tm1b(EUCOMM)Wtsi knockout embryos.

 

Respiratory system phenotypes scored for Sh3pxd2a tm1b(EUCOMM)Wtsi embryos.

 

For any DMDD phenotype, the original annotated embryo image and the full embryo image stack can be found in the DMDD database.

NEW COMPLEXITIES IN RELATIONSHIP BETWEEN GENE MUTATION AND EMBRYO DEVELOPMENT

A large-scale study of DMDD data [1] has shown that inactivating the same gene in mouse embryos that are virtually genetically identical can result in a wide range and severity of physical abnormalities. This suggests that the relationship between gene mutation and embryo development is more complex than previously thought.

The article ‘Highly variable penetrance of abnormal phenotypes in embryonic lethal knockout mice‘ was published in Wellcome Open Research.


REVEALING THE DETAILED EFFECTS OF GENE MUTATION

The study considered 220 mouse embryos, each with one of 42 different genes inactivated. These genes are part of a set known as ‘embryonic lethal’, because they are so crucial to development that an embryo missing any one of them can’t survive to birth. Studying these genes can help us understand how embryos develop, why some miscarry and why some mutations can lead to abnormalities.

Each embryo was scanned in minute detail, meaning that even the smallest differences in features could be identified – right down to the level of whether the structures of individual nerves, muscles and small blood vessels were abnormal. It was also possibe to see whether having the same, single missing gene affected all embryos in exactly the same way.

Clinicians commonly find that people with the same genetic disease can show different symptoms or be affected with differing severity. In part this is likely to be due to the fact that we all differ in our precise genetic makeup. However, the results of this study in mice shows that even when individuals have virtually identical genomes, the same mutation can lead to a variety of different outcomes amongst affected embryos.

 

Click to view larger image.
A comparison of two embryos that are both missing the embryonic lethal gene Coro1c. The embryo on the right has abnormal viscerocranium (facial skeleton) morphology, while the embryo on the left does not.

 

A total of 398 different abnormalities (known as abnormal phenotypes) were observed across the 220 embryos. Surprisingly, almost none of the phenotypes were found in every embryo with the same missing gene. A phenotype that does not occur for every embryo missing a particular gene is said to have ‘incomplete penetrance’ and this phenomenon was seen regardless of how profound the abnormality was. Incompete penetrance was observed for phenotypes ranging from severe heart malformations to relatively minor defects such as the abnormal positioning of nerves.

Dr Tim Mohun, who led the study at DMDD said:

“This is a striking result, coming as it does from such a large study in which embryos have been analysed in unprecedented detail. It shows us that even with an apparently simple and well-defined mutation, the precise outcome can be both complex and variable. We have a lot to learn about the roles of these lethal genes in embryonic development to understand why this happens.”

The result was put in context by Dr Andrew Chisholm, Head of Cellular and Developmental Sciences at the Wellcome Trust, who added that “the fundamental processes driving how we develop have been conserved through evolution, which makes studying animal models enormously helpful in increasing our understanding of why some babies develop birth defects. This study throws new light on what we thought was a fairly straightforward relationship between what’s coded in our genes and how we develop. Researchers need to appreciate this added layer of complexity, as well as endeavouring to unpick the intricate processes of genetic control at play.”

All image and phenotype data gathered by the DMDD programme is freely available to the scientific community at dmdd.org.uk. Dr David Adams, Group Leader at the Wellcome Trust Sanger Institute who contributed to the work, said “this study highlights the power of genetic analyses in mice and provides an unprecedented resource of data to inform clinical genetic studies in humans.”

The research described in this blog post was funded by the Wellcome Trust with support from the Francis Crick Institute.


REFERENCES

[1] Wilson R, Geyer SH, Reissig L et al. Highly variable penetrance of abnormal phenotypes in embryonic lethal knockout mice, Wellcome Open Res 2016, 1:1 (doi: 10.12688/wellcomeopenres.9899.2)