DMDD DATA TO REMAIN ACCESSIBLE TO ALL

 

 

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


STAY UP TO DATE

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

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.

DATA RELEASE HIGHLIGHTS – JUNE 2018

Following today’s data release, the DMDD website now contains detailed phenotype data for nearly 700 embryos from 82 different knockout mouse lines. Highlights include the identification of limb defects and cysts in Col4a2 knockouts and replication of the major features of Meckel syndrome in B9d2 knockouts.

We have begun to add immunohistochemistry image data for the brain and spinal cord of some embryos at E18.5. These images give further information about lines in which the embryos appeared morphologically normal at E14.5, but were still not viable. We have also added viability data for every line at both E9.5 and E14.5.

Together with the placental phenotype data that we hold for more than 100 knockout lines, the DMDD website is a rich resource for those investigating the effect of gene mutations on embryo development, and may provide clues about the genetic basis of rare diseases.


LIMB DEFECTS SEEN IN Col4a2 KNOCKOUTS

In humans, COL4A2 mutations have been linked to porencephaly, a rare disorder with phenotypes that include the development of intracranial cysts. In the latest DMDD data, Col4a2 knockouts have a variety of nervous system disorders in line with porencephaly. However, all four embryos also show abnormal autopod morphology and cysts between the nasal septum and the oral cavity, as well as other morphological defects.

 

A Col4a2 knockout embryo has a cyst between the nasal septum and oral cavity (left) and abnormal autopod morphology (right). The individual fingers don’t diverge distally and can’t be discerned from an external view.

 


B9d2 KNOCKOUTS MODEL MECKEL SYNDROME

In humans, mutations of the gene B9D2 have been linked to Meckel syndrome, a severe disorder caused by dysfunction of the primary cilia during the early stages of embryogenesis. Meckel syndrome is characterised by multiple kidney cysts, occipital encephalocele (where a portion of the brain protrudes through an opening in the skull) and polydactyly, but it also commonly affects the brain and spinal cord, eyes, heart, lungs and bones.

B9d2 knockout mouse embryos included in our latest data release show the major features of Meckel syndrome, including polydactyly and defects in the brain, peripheral nervous system, heart and vascular system. They also display situs defects, where the left-right asymmetry of the body did not develop as expected. The image below shows a B9d2 knockout embryo with left pulmonary isomerism and symmetric branching of the principle bronchi from the trachea.

 

A B9d2 embryo showing situs defects (left). A magnified view (right) shows that both lungs have developed with a single-lobe structure. In mice the left lung usually has one lobe, while the right lung has four. In addition, the principle bronchi (red arrows) have branched symmetrically from the trachea. This branching would normally have a distinct asymmetry.

 


NEURAL IMAGE DATA NOW AVAILABLE

In around 20% of embryonic lethal lines, embryos appear morphologically normal at E14.5 but still go on to die before or shortly after birth. To understand more about why these embryos were not viable, DMDD colleagues Professor Corinne Houart and Dr Ihssane Bouybayoune at Kings College London analysed the lines at E18.5 – when embryo development is almost complete. They used immunohistochemistry to identify abnormalities in the brain and spinal cord that could not be picked up in our standard, whole-embryo morphological analyses. This data is now available for the line Trappc9, and additional lines will be added in future data releases.

 

Click to view larger image.
Immunohistochemistry analysis of the brains of two Trappc9 knockout mice. The calretinin (green) + neurofilament (red) combined stain highlights interneurons and axons, while Hoechst (blue) is a nuclear stain.

 

Neural images are available as 20-micron sections through the brain and spinal cord, and the images from different embryos can be compared side by side using the stack viewer. A separate Nissl stain was used to highlight neural death and these images can also be explored online.


 

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.

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.

 

MEET US AT ISDB 2017

 

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

 

 

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

 

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

 

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

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.

FIND DMDD PHENOTYPE DATA IN THE MGI DATABASE

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

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

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

 

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

 

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

 

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