7 MOUSE PHENOTYPE RESOURCES

If you are interested in mouse phenotypes, you’ll have noticed that there are a wealth of resources available. Here’s our round-up of some of the best databases out there. Did we miss your favourite? Let us know by contacting us on Twitter @dmdduk.


Mouse Genome Informatics

 

 

 

 

 

 

Almost everyone will be familiar with this one, but no list of mouse resources would be complete without the MGI database. It covers gene characterisation, nomenclature, phenotypes, gene expression and tumour biology amongst many other datasets.

Use this resource: for a broad picture of mouse genetics. www.informatics.jax.org


Facebase

 

 

 

 

Around half of all birth defects involve the face, but in many cases the reason they occur remains unknown. The Facebase resource aims to tackle this problem with their database of head, skull and craniofacial data. The first five-year phase concentrated on the middle of the human face and the genetics of disorders such as cleft lip and palate. The second phase (which is currently underway) will expand Facebase to include other regions of the face, as well as developing new online search and analysis tools for the data.

Use this resource: if you’re specifically interested in craniofacial phenotypes. www.facebase.org


Monarch

 

 

 

 

The Monarch resource allows cross-species comparison of phenotype data without the user having detailed knowledge of each species’ genetics, development, anatomy, or the terminology used to describe it. The database contains phenotype data for many species including human, mouse, zebrafish and flies, which has been gathered from other dedicated phenotyping projects. The tools developed by Monarch allow users to explore phenotypic similarity between species and are intended to facilitate the identification of animal models of human disease.

Use this resource: to compare mouse phenotype data with phenotypes from many other species. www.monarchinitiative.org


Deciphering the Mechanisms of Developmental Disorders

DMDD LOGO

 

The DMDD database contains high-resolution images and detailed whole-embryo phenotype data for embryonic lethal knockout mouse lines. The High Resolution Episcopic Microscopy technique used for imaging allows phenotypes to be identified down to the level of abnormal positioning or morphology of individual nerves and blood vessels. Parallel screens identify placental phenotypes and carry out whole-embryo gene expression profiling, with all data freely available online. Around 80 lines have been phenotyped to date, with new data added regularly.

Use this resource: for whole-embryo images and phenotype datasets – primary screen data at an unprecedented level of detail. dmdd.org.uk


International Mouse Phenotyping Consortium

 

 

 

 

The IMPC has the ambitious goal of phenotyping knockout mice for 20,000 known and predicted mouse genes. For adult mice, the project provides primary screen data for all the major organ systems, and for many embryonic lethal lines there is also embryo data available. With nearly 6000 lines already analysed, there’s an enormous amount of data to explore.

Use this resource: to access phenotype data for a huge number of knockout mouse lines. www.mousephenotype.org


Origins of Bone and Cartilage Disease

 

 

 

OBCD is a collaboration working to identify the genetic causes of bone and cartilage disease – an important goal when you consider that around half of adults are affected by a bone or cartilage disorder. OBCD aims to phenotype mice from 1750 different knockout lines, and they have made a heatmap of their data freely available online. With nearly 500 lines phenotyped so far, there’s already a huge amount of data and much more to come.

Use this resource: if you’re specifically interested in phenotypes related to the bones and joints. www.boneandcartilage.com


eMouseAtlas

 

 

 

 

Last but not least, if you’re interested in mouse phenotypes you will probably also need information about normal mouse development. The eMouseAtlas resource provides 3D computer models of the developing mouse, covering everything from gross anatomy to detailed structure. It’s a useful point of comparison for phenotypes that have been observed in mutant mouse strains. As a nice project they have also re-digitised the original histological sections from Kaufman’s definitive book ‘The Atlas of Mouse Development‘, making the images available online in high resolution for the first time, together with their original annotations.

Use this resource: for a detailed description of normal mouse embryo morphology at any stage of development. www.emouseatlas.org

 

Tweet us if we missed your favourite database @dmdduk.

Cover image by Rama (Own work) [CC BY-SA 2.0 fr], via Wikimedia Commons.

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.

CAN WE IDENTIFY MORE GENES WITH LINKS TO MISCARRIAGE?

Around 1 in 4 pregnancies ends in miscarriage, but in many cases a definite cause cannot be found. It’s an all-too-common situation that is heart breaking for parents, and incredibly frustrating for the clinicians involved.

Miscarriage can happen for many reasons, including infection and hormonal imbalances. But around half of all miscarriages that occur before 12 weeks of pregnancy are thought to be caused by a gene mutation or chromosomal abnormality that prevents the baby from developing as it should. One approach to understanding, and potentially preventing, pregnancy loss is to identify gene mutations that have an adverse effect on embryo development. This is an area in which mouse embryo screening programmes such as DMDD and the IMPC can make an important contribution.


EMBRYONIC LETHAL GENES AND MISCARRIAGE

Recurrent miscarriage, the loss of 3 or more consecutive pregnancies, affects around 1% of couples who are trying to conceive. The condition has already been linked to mutations in several genes, including F2, F5 and ANXA5, which are all involved in blood clotting. This suggests that there may be other genes linked to miscarriage that have not yet been discovered.

The DMDD programme studies the effect of inactivating single genes in mouse embryos. For each inactivated gene, we record any abnormalities in the embryo’s development – from brain and heart defects down to tiny problems at the level of individual nerves and blood vessels. Our study is limited to a set of genes called ‘embryonic lethal’. By definition, inactivating any one of these genes causes developmental abnormalities so serious that the embryo is not able to survive past birth. These genes have clear relevance to miscarriage research, and the data we are gathering could be key to understanding more about the genetic causes of pregnancy loss.

 

Click to view larger image.
Detailed imaging of embryos allows us to identify abnormalities down to the level of individual nerves and blood vessels.

CANDIDATE GENES FOR VERY EARLY PREGNANCY LOSS

Around a third of the genes studied by DMDD, if inactivated, cause mouse embryos to die in the very early stages of development. We call these genes ‘early lethal’, and if a mouse embryo is missing any one of them it cannot survive to 9.5 days of gestation. In the mouse, 9.5 days is mid-gestation, but this stage of development is actually comparable with week 4 for a human embryo.

To date we have found more than 60 genes that are lethal in the first 9.5 days of gestation. This data could be a starting point for identifying genes whose mutations might be responsible for miscarriage in the first few weeks of pregnancy.


MISCARRIAGE LATER IN PREGNANCY

DMDD also studies genes that cause mouse embryos to die around 14.5 days of gestation, which is roughly equivalent to week 8 of a human pregnancy. By 14.5 days’ gestation, mouse embryos have grown to around 1cm in length and are big enough for us to look in detail for abnormalities in their development. We see a wide range of problems, but very common abnormalities include abnormalities of the hypoglossal nerve, which controls tongue movement, and a range of different heart defects.

Many of the embryonic lethal genes we have studied at 14.5 days’ gestation have not yet been associated with human disease or miscarriage. The data is available to explore at dmdd.org.uk, and these genes may be interesting candidates for those researching the genetic basis of miscarriage.

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)

JOIN US AT THE BSCB/BSDB/GENETICS SOCIETY MEETING


The joint BSCB/BSDB/Genetics Society Spring Meeting is taking place at the University of Warwick from 2 – 5 April, and DMDD will be there to exhibit our data. Come and visit our stand to find out more about phenotype data for embryonic lethal knockout mice.

Emily, Dorota, Emma and Jenna from the DMDD team will be on hand to give you a demo of the data and answer any questions you might have. You can even take away a free mug if you sign up to our email newsletter.

 

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

 

On Wednesday 5 April, Myriam Hemberger from the Babraham Institute will speak about her team’s work to phenotype placentas from DMDD embryonic lethal lines. Plus if you’re interested in our recent publications, you can visit one of the three posters we’re presenting:

Poster 66: Staging mouse embryos harvested on embryonic day 14 (E14.5). (Original article in Journal of Anatomy).

Poster 77: Highly variable penetrance of abnormal phenotypes in embryonic lethal knockout mice. (Original article in Wellcome Open Research).

Poster 83: Interpreting neonatal lethal phenotypes in mouse mutants.

 

We’re looking forward to meeting you!

HOW MICE ARE KEY TO UNDERSTANDING GENETIC HEART CONDITIONS

Nearly 1 in every 100 babies is born with a heart defect. These are the most common type of birth defect and can range from simple, symptomless cases to life-threatening conditions that require treatment within hours of birth.

Environmental influences such as excessive alcohol consumption or exposure to other toxins are known to cause heart defects. But many are also the result of faulty genes that can be passed on from one generation to the next. Identifying the genes involved is key to understanding when a baby might be at risk, and could also help us develop new ways to treat or prevent these heart defects. But with more than 20,000 genes in the human genome there are a mind-boggling number of possibilities.

A screen of mouse embryos by the DMDD programme (Deciphering the Mechanisms of Developmental Disorders) has identified a huge number of genes related to heart defects and other developmental abnormalities, and is now a potential goldmine of information on the genetic basis of heart conditions. Part of an open data initiative by the Wellcome Trust, the project has made all of its data available online, with a goal to spark further research into heart defects and rare disease.

 

Click to view larger image
Photographs of isolated mouse embryo hearts. On the right is a ‘normal’ heart — this mouse was not missing any genes. On the left, due to a missing gene, the heart has a defect called ‘Double Outlet Right Ventricle’. Here, the heart’s two major arteries (the pulmonary artery and the aorta) both connect to the right ventricle. In a normal heart the aorta connects to the left ventricle.

INACTIVATING GENES TO UNDERSTAND RARE DISEASE

The DMDD team studies mouse embryos that have been bred to have a single one of their 20,000 genes inactivated – a process that’s known as knocking out a gene. As each embryo grows, any abnormalities in the way it develops are likely to be due to the missing gene and this provides powerful information about the sort of birth defects that the gene could be linked to. Although we might look very different, mice and humans are thought to share around 98% of our genes, so the effects of a missing gene on a developing mouse can tell us a lot about what we might expect if the same faulty gene is found in humans.

We concentrate specifically on genes that when knocked out cause a mouse embryo to die before birth.  On the face of it, studying these genes might not seem so important to people living with rare genetic diseases – these people have already survived past birth. But genes like these are a rich source of information about human genetic diseases.

Many rare disease patients have mutations that act as genetic dimmer switches, increasing or decreasing a gene’s activity rather than completely turning it off. A particular gene may only be partially turned on (called a hypomorph mutation) or it may be turned on more than normal (known as a hypermorph mutation). If we are able to understand the effect of fully turning off a gene, we can then begin to infer what might happen to patients who have a hypomorph or hypermorph mutation.

 

Like genetic dimmer switches, gene mutations can increase or decrease the activity of a gene, or knock it out completely.

 

By the end of the project, DMDD will have studied the effects of 250 different gene knockouts – it’s a huge opportunity to learn more about genetic causes of rare diseases in humans. And the biggest surprise in the results so far is the overwhelming prevalence of defects in the developing embryo hearts.


IDENTIFYING HEART DEFECTS

So far the team have analysed more than 200 embryos, each with one of 42 different genes knocked out. But unexpectedly, more than 80% of the gene deletions resulted in heart defects. Using an imaging technique called High Resolution Episcopic Microscopy the embryos were reconstructed in incredible 3D detail and studied down to the level of individual nerves and blood vessels. In the image below, the developing embryo heart is less than 2 mm across – smaller than the thickness of a matchstick – yet even the tiniest abnormality can be picked up.

“Even though we know that heart defects are common, we were really surprised that they were caused by more than 80% of our gene deletions. The data is a potential goldmine of information about the genetic basis of many different types of heart condition.” Dr Tim Mohun, DMDD.

 

Click to view larger image.
A 3D model of a mouse embryo heart using data from High Resolution Episcopic Microscopy.

 

The most common defects were problems with the walls that separate the right and left chambers of the heart. But there were also many defects in the heart valves, the outflow vessels (which carry blood out of the heart to the body or the lungs) and in the structure of the heart itself.

Several of the gene knockouts result in developmental defects that mimic known human genetic disorders. For example knocking out the genes Psph or Psat1 causes a range of developmental defects that appear similar to Neu-Laxova syndrome, a serious condition that leads to miscarriage or neonatal death. Tim Mohun commented “we know that many rare genetic diseases cause problems with the heart as it develops. Having so much new data about heart defects is exciting, because there is the potential for us to learn more about rare disease.”


THE PLACENTA: A NEW WAY TO UNDERSTAND THE HEART?

In the first study of its kind the placentas from a large collection of knockout embryos have also been analysed, and the results show an unexpected link between the placenta and the heart. Around a third of gene knockouts that cause placental abnormalities also cause a heart defect in the developing embryo. A more detailed statistical study of the data (publication in progress) has shown that this is a genuine link. Myriam Hemberger of the Babraham Institute, who performed the work as part of the DMDD programme, said “it could be that the restricted nutrient supply or blood circulation defects caused by an abnormal placenta adversely affect heart development. It does suggest there is more we could learn about some rare heart conditions by studying the placenta.

 [The results] suggest there is more we could learn about some rare heart conditions by studying the placenta. Myriam Hemberger, DMDD.


Initial analysis of the DMDD embryo and placenta data has shown it to be a rich resource for those studying rare disease and developmental disorders. But, unexpectedly, it may shed particular light onto the genetic basis of heart disease.

All data from the DMDD programme is freely available at dmdd.org.uk. For further information please email contact@dmdd.org.uk.

 

MEET THE AUTHOR – STEFAN GEYER

This week sees the publication of a new technique to precisely find the developmental stage of a mouse embryo, simply by looking at its fingers. We talked to the lead author, Stefan Geyer, to find out more about the technique, and the impact it could have on the field.

Stefan Geyer


What’s the main question that you have been trying to answer during your research career?

I have been fascinated by developmental biology since I was a student, and during my diploma thesis I became really interested in imaging. I was using High Resolution Episcopic Microscopy (HREM) and it was such a powerful tool that we could do 3D analysis and measurements of tiny structures like blood vessels and arteries in mouse embryos. I’m especially interested in the pharyngeal arch arteries – transient structures that undergo complex re-modelling during development. Abnormal re-modelling of these arteries accounts for a range of congenital cardiovascular abnormalities, so it’s really important that they develop as normal.

I’m also trying to expand the potential applications of HREM imaging. It was originally developed to study mouse, chick and zebrafish embryos but I believe that it has incredibe potential to be used with adult tissue samples, or even to study non-biological materials.

 

You published a paper today in Journal of Anatomy – it describes a new way to very precisely find the developmental stage of a mouse embryo. Why is this such an important result?

In the DMDD programme we screen a lot of mouse embryos – both those that have had a single gene knocked out and wild-type controls (with no gene knocked out). We study their morphology in great detail and look for developmental abnormalities, as a way to gain understanding about the function of the different genes that have been knocked out. Very early on in the project we realised that even though we were looking at embryos all harvested at E14.5 (14.5 days gestation) there was a dramatic variation in their appearance. And crucially this applied to the wild-type controls, as well as the knockouts. Some looked as you might expect for an embryo harvested at E13.5, others looked older. Even embryos from the same litter didn’t all look the same.

Obviously this is a big problem if you want to detect malformations in mouse embryos. If the embryos that are supposed to be ‘normal’ are so diverse in appearance, how can you begin to tell what is ‘abnormal’? Our new staging technique splits ‘normal’ at E14.5 into 6 developmental stages. Once we have identified the precise developmental stage of a knockout embryo using these new descriptions, we can compare it to what is normal for that stage and truly know whether it has developed as it should.

The appearance of the forelimb at stages S21 and S23.
The forelimb has the appearance of a paddle at stage S21 (left) but by stage S23 (right) it has developed into a hand with separate fingers.

 

Can you describe your technique in a few sentences?

It’s actually quite simple. We defined the 6 new developmental stages based on the amount of webbing between the embryo’s fingers. Between E14 and E15 an embryo’s forelimbs develop from paddles to hands with individual fingers. We defined the stages S21, S22-, S22, S22+, S23- and S23 according to how much of this webbing remained.

I think the simplicity is one of the main advantages of this technique. We use it on 3D models produced from HREM data but it could also be used on data derived from a variety of other imaging techniques.

 

In the past, people have staged mouse embryos using the Theiler stages (a set of 26 stages covering the whole of embryonic development). Why do we need more detailed staging than this?

Because there are structures and organs that change rather rapidly in embryos around E14.5, even within a single Theiler stage. So on some occasions the Theiler stages don’t give enough granularity. The most striking example that we found was the appearance of the palatal shelves, which completely reorient themselves during this period of development. We found that the changes happen in a predictable order, but extremely quickly. In some embryos harvested at E14.5 the shelves are positioned laterally to the tongue, in others they have already elevated and are positioned above the tongue. In a few embryos, elevation is in progress and the shelves are arranged asymmetrically, one laterally to the tongue, the other above it. If we want to diagnose cleft palate with confidence in E14.5 embryos, exact staging is essential.

We also think that our technique simplifies the staging process, as it’s based only on one parameter – the amount of webbing between the fingers. Theiler staging is based on the appearance of several features, so sometimes you can get conflicting results.

 

Could your new technique also impact other areas of developmental biology?

It would be useful for any study that compares mutant and control embryos around E14.5. This isn’t limited to gene knockouts, it’s relevant to any mutation that is being studied to look for resulting abnormalities in the embryo. Relying on simple comparisons between mutant and control embryos harvested at the same time point might falsely diagnose a malformation. For example an embryo at stage S22- still shows an interventricular foramen in the heart (a temporary gap between the developing ventricles), which is normal for his developmental stage. In a embryo at stage S23 the interventricular foramen has disappeared. Therefore comparing a mutant at stage S22- to a control embryo at stage S23 is problematic. Only precise staging allows us to identify true malformations.

 

What is next on the horizon for you?

We are now able to tell the stage of an embryo very precisely. But we don’t know exactly how every organ develops during each of our new substages. To really understand how an embryo’s organs develop I want to understand at what a ‘normal’ mouse looks like and exactly what structures are changing between stages 21 and 23.  Our research group will do statistical analyses of different structures, to find what a normal range of variation looks like. We also plan to look at variations in different strains of mouse. I believe that phenotype screens would really benefit from such a comprehensive study of a ‘normal’ control mouse.

 


The original article described in this post is SH Geyer et al., ‘A staging system for correct phenotype interpretation of mouse embryos harvested on embryonic day 14 (E14.5)‘, J. Anat., 10.1111/joa.12590.

Stefan Geyer is Assistant Professor in the Division of Anatomy, Center for Anatomy and Cell Biology at the Medical University of Vienna.

WHY ARE GENETICISTS MEASURING THE WEBBING BETWEEN MOUSE EMBRYO FINGERS?

A new paper published in Journal of Anatomy shows that measuring the amount of inter-digital webbing in mouse embryos between 14 and 15 days gestation is the best way to find out their exact stage of development.

So why is this important to a geneticist? If we want to discover a causal link between a gene mutation and a developmental abnormality in a mouse, we need to be really sure that any abnormalities we see aren’t just standard features of embryo development. The morphology of an embryo can change rapidly as it grows, and things that seem unusual at first glance might be completely typical for a given stage of development. Understanding and avoiding these potential pitfalls is hugely important when studying embryos for the effects of a gene mutation.

Using the new technique described by the authors, it’s now possible to find an embryo’s exact developmental stage simply by looking at its fingers.


NORMAL OR ABNORMAL?

To be sure that an embryo has developed abnormally, we need to compare it to one that we know has developed normally. But this can be a complicated comparison to make when an embryo’s ‘normal’ appearance is changing dramatically as it develops.

Cleft palate is a serious abnormality, as it can prevent a mouse pup from suckling and eventually lead to death. But the palate is also one of many tissues that changes its structure and orientation dramatically between E14 and E15 (days 14 and 15 of gestation). The image below shows three snapshots of a typical mouse embryo palate during this period.

 

During days 14 – 15 of embryo development, the palatine plates change their orientation rapidly. Initially, both plates are lateral to the tongue (left). They begin to elevate, but do so asymmetrically (centre), ending with both shelves above the tongue (right) at which point the plates fuse together.

 

Not only do the palatine plates completely change their orientation, they also move one at a time. Without knowing the precise developmental stage of an embryo, it would be impossible to tell whether the palate was developing normally, or whether a cleft palate had developed as a resust of a gene mutation.

Dramatic changes within a similarly short timeframe can also be seen in both the heart and the intestine. It’s vital to measure the developmental stage of the embryo as precisely as possible, so that it can be compared to what is normal at that exact stage.


MEASURING INTER-DIGITAL WEBBING

In the past, the development of a mouse embryo has been described using a set of 26 Theiler stages. Days 14 – 15 of gestation are typically described by the stages 21, 22 and 23.

However, the authors found that for embryos of this age many organs and tissues developed rapidly even within a single Theiler stage. The stages weren’t granular enough to be sure that tissues like the palate, heart and intestines were really showing abnormal development. To address this the article defines 6 alternative stages, together with a novel technique to identify the stage solely by measuring the amount of webbing between the embryo’s fingers.

 

An image of each of the six newly defined stages, and the appearance of the forelimb at each stage.
Six individual stages were defined, each of which can be identified by measuring the amount of inter-digital webbing.

 

During this time, the forelimb develops from a paddle to a hand with separate fingers. The full set of new stages S21, S22-, S22, S22+, S23- and S23 allow the developmental stage of the embryo to be determined much more precisely, meaning that developmental abnormalities can be identified with confidence.


This blog post is based on the original article SH Geyer et al., ‘A staging system for correct phenotype interpretation of mouse embryos harvested on embryonic day 14 (E14.5)‘, J. Anat., doi:10.1111/joa.12590.