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.

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.

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.

 

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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 DMDD TEAM – MYRIAM HEMBERGER

In a series of interviews we’re hearing from members of the DMDD programme. Who are they? What inspires them? And what do they hope that DMDD will achieve? This month we speak to Myriam Hemberger, who leads the team’s analysis of placentas from embryonic lethal knockout mouse lines.


What has been your main area of research in your career so far?

Ever since my PhD I have been interested in studying the mechanisms that underpin placental development. Over the years we have worked on identifying chromosome “hotspots” harbouring genes that are important for placentation, studying the physiological processes that ensure the normal function of the placenta, and exploring mechanisms for gene regulation known as “epigenetic mechanisms” that make placental cells different to any other cell type found in an embryo .

 

What inspired you to devote your career to understanding placental development?

The placenta is absolutely essential for reproduction. The earliest cells of the placental “trophoblast” lineage allow the embryo to implant, while later on in pregnancy the placenta is the organ solely responsible for providing nutrient and gas supply to the embryo as it grows. The importance of the placenta is reflected by the fact that defects in its function can cause some of the most common and serious pregnancy complications, such as preeclampsia, fetal growth restriction, or even miscarriage. Even increased risk of childhood and adult diseases, such as cardiovascular disease or diabetes, may originate in a malfunctional placenta. The placenta has a long–lasting impact on our health and wellbeing, but it has often been under-appreciated in research.

I have always been fascinated by the types of cell that make up the placenta. Some of them have truly extraordinary capacities: they can become gigantic in size by amplifying their genome dramatically and invade into foreign tissue, completely remodelling the structure of blood vessels (an ability only shared by metastatic tumour cells). In the placenta, invasive behaviour is needed to attach the embryo to the wall of the uterus and to access maternal blood supply. These (and many other) intriguing features captured my imagination, sparking my research in this area.

 

What do you think is the most exciting recent development in your field?

A major milestone was Professor Janet Rossant’s work to successfully isolate and maintain stem cells from the early mouse embryo that are specific for the placenta (so-called trophoblast stem cells). The field of placental research has really been propelled forwards by the possibility to grow and manipulate these cells in culture. Recent advances mean that we now understand trophoblast stem cells and their differentiation capacity in much more detail, but there is still much to learn about what makes these cells so special and distinct from any other cell type in the embryo itself.

 

What is the biggest outstanding problem in your field that you wish could be solved?

Perhaps the biggest question in the field is whether such a stem cell population exists in the early human placenta and, if so, how to propagate it. This would open up the possibility to derive patient-specific stem cells and identify, in unprecedented detail, precisely which aspects of placental development are failing in specific cases of complicated pregnancy.

 

Why did you decide to become involved with the DMDD programme?

DMDD was proposed as a systematic screen of embryonic lethal mouse knockouts – one of the most detailed screens of its kind. But instead of screening only the embryos, a key part of the proposal was to consider the placenta as an essential (and often overlooked) organ system that must form during early development in order for an embryo to reach full gestation. It meant that we would also screen for placental phenotypes in addition to embryo phenotypes. It was a hugely exciting opportunity to make an impact on the field, as it allowed us to determine just how many genes contribute to the formation of the placenta and are therefore important to ensure normal development of the embryo.

 

What do you hope the DMDD programme will achieve?

Our placental analysis has already highlighted that a far greater number of genes than previously known are necessary for placental development . We find that an extraordinarily high proportion of embryonic lethal knockouts show a placental phenotype and, at least in some cases, this will mean that the placenta was either the cause or a contributing factor of embryonic lethality. One of the most important and personally rewarding achievements of the programme would be to raise awareness of this result, and to encourage others to include the placenta in studies aimed at finding the causes of developmental disorders.

 

What are you most proud of achieving outside of your research?

I am proud to think that some of my outreach work might have inspired young people to be fascinated by biology in the broadest sense. Ultimately, the precise field that sparks their interest is secondary – what’s important is that young people discover and develop an admiration of biological processes, for example the development of a fertilised egg to become a complete embryo and placenta. Engaging a new generation of scientists is personally rewarding, but it’s also really important to ensure the advancement of science in the future.

 

Myriam Hemberger is a group leader in epigenetics research at the Babraham Institute.

MEET THE DMDD TEAM – LIZ ROBERTSON

 

In a new series of interviews we’ll hear from members of the DMDD team. Who are they? What inspires them? And what do they hope that the DMDD programme will achieve? We kick things off with joint grant-holder Liz Robertson.

Photograph of Professor Elizabeth Robertson

 

What has been your main area of research during your career?

My lab studies how our body plan is established in the early embryo using mice as a model. We focus on one particular signal known as TGFb, which cells use to communicate with each other. This turns out to be fundamental to why different tissues form in different parts of the early embryo in a predictable and reliable pattern. A consistent theme emerging from our work is that the way TGFb works is to activate master regulator genes in different parts of the embryo. These master regulators then trigger a programme of gene activity that results in differentiation of an individual tissue.

 

What inspired you devote your career to understanding embryonic development?

I actually started out as a PhD student back in the late 70s, early 80s studying tumour-derived embryonal carcinoma cells in a dish. It was only with the advent of embryo-derived stem cells, and our discovery that they would reproducibly colonise the germ-line, that I became really interested in early embryos.

Back in the days when only a few genes had been identified molecularly, I was fortunate enough to stumble on a mutation that gave a very early post-implantation defect and completely derailed the embryo. The first dissections were a real “wow” moment for me. I knew I was on to something really interesting when I showed the mutant embryos to my good friend the late Rosa Beddington, a card-carrying embryologist, and even she was suitably puzzled about why they were so disturbed!

 

What do you think is the most exciting recent development in your field?

Most people would probably respond to this by saying CRISPR-mediated engineering but, as a mouse geneticist, I’ve had the ability to manipulate the mouse genome in vivo for decades. So whilst CRISPR has become an incredibly useful tool to manipulate other model organisms, for me it’s just a nice addition to my tool kit. What’s caught my attention the most is the emergence of wonderful imaging technologies and new methods for looking at the organisation of the genome (although my lab are very much amateurs in both areas).

 

What is the biggest outstanding problem in your field that you wish could be solved?

There are so many I don’t know where to start!

 

Why did you decide to become involved with the DMDD programme?

Sitting on the sidelines of the international KOMP and EUCOMM consortia over the last decade, I’ve seen the enormous effort and investment that has been put into knockout mouse projects. Given my long-held interest in studying embryo phenotypes, I was delighted to be included in the initial discussions with Jim Smith, Tim Mohun and David Adams to plan DMDD – an in-depth phenotype screen for a sub-set of 240 embryonic lethal lines. In particular, I was interested to see how much new information we could find out about later developmental patterning defects using the High Resolution Episcopic Microscopy (HREM) imaging technique that Tim has developed.

 

In the long-term what do you hope that DMDD will achieve?

Given that around one third of protein coding genes are now known to be essential to sustain development of the embryo, it’s a huge challenge to understand their many and varied roles. The pipeline that generates knockout mice for DMDD is largely unbiased, and many of the phenotypes we’ve detected are related to previously uncharacterised genes. Hopefully some of the mutations will match up with genes that have emerging associations with human developmental defects, such as those being uncovered by the Deciphering Developmental Disorders (DDD) study.

I also hope that after exploring our phenotype data on the DMDD database, individual investigators will feel compelled to call up specific null or conditional alleles from KOMP or EMMA for further study. We’re carrying out a primary screen, but I hope it will spark further research to determine the roles these genes play at the molecular level, and exactly why they are essential for embryo development.

 

If you could have been a fly on the wall during any scientific discovery in history, which would you choose?

My lab is based at the Dunn School of Pathology, where the first mass quantities of penicillin were made. It was here that the initial trials on animals and humans were carried out 75 years ago. The dawn of the antibiotic age must have been a very exciting time.

 

What are you most proud of achieving outside of science?

In my next life I’m going to try and have a life outside work!

 

Liz Robertson is Professor of Developmental Biology at the University of Oxford and a grant-holder for the DMDD Programme.

3 NEW RESULTS FROM KNOCKOUT MOUSE SCREENS

Around a third of mammalian genes are essential for life, and the recent Nature paper from the IMPC  ‘High-throughput discovery of novel developmental phenotypes‘ [1] describes some achievements from sytematic study of these genes in knockout mice.

Screens like those of the IMPC and DMDD are vital to understand gene function on a genome-wide scale and, based on the results recently published in Nature, here are some reasons why.


FURTHER EVIDENCE THAT MOUSE DATA IS RELEVANT TO CLINICAL STUDIES

Lethal genes in the mouse are known to be enriched for human disease genes [2,3]. When additional data from the IMPC was included on the genes essential for survival of the embryo, this enrichment was increased even further. More than half of the human disease genes considered were essential for mouse embryo survival. The study also found a remarkable correlation between the core essential genes in humans and mice.

Systematic knockout mouse screens provide data that could not be derived from human patients. These new results further underline the importance of mouse models in the study of human disease, and their relevance in a clinical setting.


INCOMPLETE PHENOTYPE PENETRANCE

A suprising observation from knockout mouse screens is the incomplete penetrance of phenotypes for many lines.

One example of this is the sub-viability of lines. The IMPC has found that in around 11% of knockout lines some homozygous pups were observed, but fewer than the 1 in 4 pups predicted by Mendelian genetics. Some pups were able to survive with the homozygous gene knockout, but some weren’t.

Incomplete penetrance is a result also echoed in DMDD data. For example, in the seven Adamts3 knockout embryos studied, all display subcutaneous edema and absent lymph sac, while only two display a bifid ureter.

Click to view larger image.
Subcutaneous edema and bifid ureter (left side) observed in an Adamts3 mutant embryo. The red arrows highlight a single ureter on the right side, but two branches on the left side.

Data from systematic screens of knockout mice is showing, on an unprecedented scale, that even for a complete gene knockout, the observed phenotypes can vary from embryo to embryo. Given the standardised background and allele construction, this is a suprising result and could suggest an underlying stochastic process.


CANDIDATES GENES FOR UNDIAGNOSED HUMAN DISEASE

As part of its systematic screen, the IMPC has identified 22 essential mouse genes with human orthologs that are not known to be associated with any human disease. These are potential candidates for undiagnosed diseases and could shine new light on the causes of genetic disorders.

Efforts are continuing to study knockouts of every gene in the mouse genome. As these datasets grow in size, so too does the potential for them to help us understand gene expression and the genetic basis of human disease.

The DMDD database of embryonic-lethal mouse knockouts can be found at dmdd.org.uk.

The IMPC database of knockout mice can be found at www.mousephenotype.org.


REFERENCES

[1] The IMPC Collaboration (2016)
High-throughput discovery of novel developmental phenotypes
Nature  doi:10.1038/nature19356

[2] B. Georgi1, B. F. Voight1, M. Bućan1 (2013)
From mouse to human: evolutionary genomics analysis of human orthologs of essential genes
PLoS Genet 9(5): e1003484. doi: 10.1371/journal.pgen.1003484

1 Department of Genetics, Perelman School of Medicine, University of Pennsylvania, USA

[3] J. E. Dickerson 1, A. Zhu1, D. L. Robertson1 K. E. Hentges1 (2011)
Defining the role of essential genes in human disease
PLoS ONE, 6(11), e27368. http://doi.org/10.1371/journal.pone.0027368

1 Faculty of Life Sciences, University of Manchester, UK