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.

EDEMA – A SIGN OF UNDERLYING DEVELOPMENTAL DEFECTS

In around 30% of the knockout lines studied by DMDD, the embryos have edema – swelling due to fluid trapped in the embryo’s tissues. Most of us have experienced edema at some point, after a bee sting, an infection or perhaps from hitting your thumb with a hammer. One reason that it happens is if small blood vessels start to leak, releasing fluid into the surrounding tissue. And in some cases it can be helpful – for example the additional white blood cells in the fluid can help fight infection more quickly.

In embryos from our embryonic lethal knockout lines, however, edema is more generalised, often surrounding the brain, abdomen or the entire body. We spot it in the initial checks of the embryo, where we look for major abnormalities that are visible without detailed imaging. In the image below, a Traf6 knockout embryo clearly shows edema around the skull and back.

A Traf6 homozygous knockout embryo shows edema around the back of the skull and body.

 

In adult humans, we know that more generalised edema can be caused by heart failure. If the heart is not able to pump blood around the body quickly enough then fluid can build up in the legs, lungs and abdomen. It can also be caused by liver and kidney diseases, as well as many other critical conditions.

The equivalent phenotype in human foetuses is known as hydrops fetalis. It’s an excessive accumulation of fluid in the body cavities, and can have a root cause in the foetus itself, in the placenta or in the mother. It’s the end stage of many different disorders. [1]

 

It’s almost never the edema that kills, but the underlying etiology. (Dr Fowzan Alkuraya, King Faisal Hospital, Riyadh).

 

The edema we see in embryonic lethal knockout mice is therefore unlikely to be a direct cause of lethality. As we might expect, many DMDD embryos with edema also show a wide range of other abnormalities, like this Ssr2 knockout that has a ventricular septal defect as well as subcutaneous edema.

 

Click to view larger image.
An Ssr2 knockout embryo has a ventricular septal defect as well as subcutaneous edema.

 

So when we see this general type of edema in our initial checks, it’s a huge clue that there may be serious underlying abnormalities that have led to the embryo’s demise. And to identify these we need detailed imaging.

[1] C. Bellini et al., Etiology of non-immune fetal hydrops: a systematic review, Am Gen Med Genet A, 149A (2009) p844-851.

 

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.

 

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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.

TEA, DINNER OR SUPPER?

Like most big biomedical research projects, DMDD is gathering huge amounts of data. The latest count is more than 5 million images of developing embryos, and thousands of abnormalities (or phenotypes) that have been identified within them.

So we need to be organised. All that time spent collecting and analysing images is wasted if the phenotypes we find aren’t described and recorded in a consistent, unambiguous way – it would be hard for us to share, search or analyse the results. To organise our data properly, we need an ontology.

 

WHAT IS AN ONTOLOGY?

Imagine your friend invites you over for tea. You might be expecting a hot drink, and perhaps a biscuit or two. But if you grew up in the north of England you could be forgiven for expecting an evening meal. It’s possible that you could even be imagining an afternoon tea of crust-less sandwiches and cream cakes. Without more information in advance it’s hard to tell, as the word ‘tea’ has several different meanings. Then add to the mix that tea as an evening meal can also be described as ‘dinner’ or ‘supper’, and there’s a lot of room for confusion.

If you’re now completely baffled by the intricacies of British mealtimes, Wikipedia has a good explanation! But this does highlight a problem with language. Sometimes the same word can have different meanings, and the same meaning can be expressed with different words.

We can deal with this problem by defining a ‘controlled vocabulary’ where each word has a single, specific meaning. We can also define the relationships between the words, creating a hierarchy of terms that become increasingly specific as we move down the chain. In our simple example, we might say that every time we eat during the day, we’ll call this sustenance. We can categorise sustenance as either a meal or a snack. But then we can then classify the meals even further, by saying that the possible options are called breakfast, lunch or dinner.

 

 

An ontology is then the language made up of words from the controlled vocabulary. If we consistently speak the language of the ontology, there’s no longer any room for confusion.

 

AN ONTOLOGY TO DESCRIBE MOUSE PHENOTYPES

There are already lots of different ontologies out there, so fortunately we don’t need to define our own. For example, the Gene Ontology (GO) has been designed to describe the functions of genes, while the Disease Ontology (DO) exists to describe human diseases.

DMDD data is recorded using the Mammalian Phenotype (MP) ontology, a language developed by the Jackson Lab (JAX) to describe developmental abnormalities (phenotypes) found in mammals. The abnormalities can be described by high-level categories, such as ‘nervous system phenotype’ or ‘respiratory system phenotype’, which are then sub-categorised to give increasing detail. Moving down several levels in the chain we can describe abnormalities as specific as individual nerves that are either missing, misplaced or unusually thin.

Occasionally, we find phenotypes that aren’t described by MP in its current form. When this happens we work with JAX to incorporate the new terms into the ontology. That way the terms are there for everyone to use, and our data is still fully described by the MP ontology.

Without this approach, phenotypes identified by different DMDD members could be inconsistent, ambiguous or conflicting. By sticking to the language of MP we avoid these pitfalls, whilst also making it easy for others to search and analyse the data.

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.

 

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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.

 

THE ZIKA VIRUS AND DMDD

The Zika virus has raised global awareness of birth defects more than at any time in the last 50 years [1]. A recent Nature Editorial explores the opportunities this presents to increase support for vaccination programmes, compulsory fortification of food staples and investment in population-scale databases to mine information on the causes of birth defects. But what does it mean for research into the genetic basis of developmental disorders and rare diseases?


HOW RARE IS RARE?

Some birth defects, such as Zika-linked microcephaly, are infectious in origin. But many are related to genetic mutations, which can also prevent an embryo from developing as it should. The rare diseases resulting from genetic mutations can be chronic and life threatening, and 30% of rare disease patients die before their fifth birthday [2]. Many mutations can also lead to miscarriage. Genetic cardiac conditions affect around 1% of newborn babies [3], but it’s estimated that these defects prevent around ten times as many foetuses from surviving until birth.

A rare disease is difficult to study, because of the relatively small group of patients – by definition less than 1 in 2000 of the general population are affected. But with more than 6000 known rare diseases, 80% of these with a genetic component, it’s likely you or someone you know has a rare disease. 7% of the population will be affected at some point in their lives, which equates to around 3.5 million people in the UK alone [3].


CLUES FROM SYSTEMATIC SCREENS

We have a huge challenge to find and prevent the causes of rare diseases. For those diseases linked to genetic mutations it’s vital that we understand their genetic basis, and basic research in developmental biology is fundamental to this.

Mouse research in particular offers a wealth of information, thanks to systematic studies that would not be possible in human patients. The mouse genome can be manipulated to delete (knock out) a specific gene. The resulting embryos and adult mice can then be studied to look for abnormalities in the way they develop. It’s a unique way to understand the role that a specific gene plays in development from embryo through to adult, and the developmental abnormalities that may arise from a fault with the gene. Around 30% of gene deletions cause abnormalities so severe that the embryo does not survive until birth, so study of these genes also provides clues about the genetic basis of miscarriage.

Systematic screens by the DMDD and IMPC are working to study the effects of individually knocking out every gene in the mouse genome. With all data freely available online, they are a rich resource for those researching human birth defects, miscarriage and developmental disorders.

The Zika virus has put birth defects back on the political agenda, and scientists have a challenge to find and prevent their causes. In this context, systematic screens of mouse gene knockouts and other gene mutations are more important than ever.


REFERENCES

[1] Use Zika to renew focus on birth-defect research, Nature Editorial, Nature 535, 8 (7 July 2016)

[2] About Rare Diseases, Rare Disease UK

[3] Congenital Heart Disease, Centres for Disease Control and Prevention

WHAT IS NORMAL?

At the DMDD one of our projects is to measure the amount of variation in wild-type embryos. But this led us to ask the question – what is normal? We asked some leading scientists for their opinions and found that it can be quite hard to define.

What does normal mean in your research field? Comment on this post or join the debate on Twitter #dmddnormal

'Define normal' cartoon

 


 Tim Mohun, DMDD Programme

‘Normal’ is what we expect – so the real answer turns on what drives our expectation – Experience? Prior knowledge? Inference? Prejudice?

For embryo structure and development, we rely on experience and prior knowledge (our own, or more commonly that of acknowledged experts like Kaufman). But even reference atlases are incomplete in lots of ways. They show only individual embryos, using a limited number of stages and based on a particular 2D imaging technique.

Embryos, like people, will lie in a bell-shaped distribution – we really need to study and compare many embryos and many stages to appreciate the extent of the distribution we call ‘normal’. The atlases also cannot encompass all the many variations we know can be observed between different mouse strains and genetic backgrounds – so there are many subtly different ‘normals’.

What you see depends on how you look, which affects what you understand as ‘normal’. 3D analysis shows things that are difficult or impossible to see or interpret by 2D. So ‘normal’ is often a misleading shorthand that can hide enormous range and variation. Perhaps its more useful to ask what is ‘abnormal’ – how do we set the limits of what we understand as ‘normal’?


Robin Lovell-Badge, the Francis Crick Institute

Of course, I think I am (fairly) normal – but others might disagree. I also suspect a C57BL/6J mouse might think a 129S8 mouse is abnormal and vice versa. They certainly behave differently. And from studies on sex determination, we know that, in the early gonad, the balance of gene expression relevant to ovary versus testis differentiation is skewed in favour of the former in C57BL6/J compared to that seen in 129S8.

This explains why C57BL/6J mice are far more sensitive than many other strains to mutations or allelic variants of genes involved in testis differentiation and are far more likely to show XY female sex reversal. This means that ‘wild-type’ is very context dependent and in the context of an experiment the term relates to the particular strain used. And of course, no inbred mouse strain is directly comparable to any mouse found in the wild.


John Skehel, the Francis Crick Insitute

We work with RNA viruses, which have high rates of mutation, about 10-4 to 10-5, without repair mechanisms. So the product of a replication cycle is viewed as a quasi-species and normal, genetically and phenotypically, as a consensus.


Jacqui White, Wellcome Trust Sanger Institute

I confess we have migrated away from the using the term ‘normal’, as what is normal for one strain of mouse is entirely different for another.

The inbred strain 129 does not have a corpus callosum (the white matter tracks linking the left and right hemispheres of the brain), but agenesis of that structure in a C57BL/6 sub-strain is an interesting find. Even within sub-strains ‘normal’ is different. For example, C57BL/6J mice are virtually resistant to seizures and retain a normal retina into adulthood, whilst C57BL/6N mice are more susceptible to seizures and will develop retinal degeneration as adults.

We have moved away for using the term normal and now use ‘as expected’ in its place. ‘As expected’ describes the observation on the basis of a well-established baseline of control data, and deviation from that baseline can then be described as abnormal.


Dorota Szumska, University of Oxford

In analysing mouse embryos, ‘normal’ means ‘developing as expected’ or ‘developing in a way so to form a healthy animal’ and is based on thousands of observations and the anatomical knowledge of the studied object. It means that all embryonic structures are formed as expected to provide a shape and a function characteristic for the given organism (in this case, a mouse). This would depend on the developmental stage, for example unfused palatal shelves are absolutely ‘normal’ at the early stages while it would be considered a malformation later on in development.

‘Normal’ can also mean ‘typical’ for the genetic background or the mouse line, as these may differ in some features like coat colour or eye pigmentation. But variability of a given feature can also be ‘normal’ within a mouse strain, for example the number of splits in some nerves or the number of canals in some bones.


Janet Rossant, Hospital for Sick Kids

Normal embryogenesis means that phenotypic variation falls within a fairly narrow range and is compatible with full development to term.


Antonella Galli, Wellcome Trust Sanger Institute

Normal is independent of any phenotype observed, as it is relative to your point of comparison.  Sometimes we may say that it’s normal for a particular mouse mutation to show a certain phenotype – they might have no kidneys or no limbs. Then the embryos not displaying that phenotype would be abnormal.


 

Whether you agree or disagree, tell us what you think! Comment on this post or join the debate on Twitter #dmddnormal