JOIN OUR HREM AND PHENOTYPING WORKSHOP

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

 

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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)

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.

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.

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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.

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.

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.

 

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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!

NEW PHENOTYPE DATA FOR EMBRYOS AND PLACENTAS

Today, DMDD has released many new images and phenotypes for embryos and placentas from embryonic lethal knockout mouse lines. We now hold data on 70 mutant lines that have been phenotyped in detail using the Mammalian Phenotype ontology. The resulting data is freely available to the scientific community and is a potential goldmine of information about the genetic basis of developmental disorders.

The new data is accompanied by several exciting updates to our website. These include the ability to search for phenotypes by anatomy terms and the release of additional data about gene knockouts that are lethal very early in embryonic development. Highlights of the release, including examples of interesting phenotypes, can be found below.

 

SEARCH FOR PHENOTYPES USING ANATOMY TERMS

Following a major update to our search tool, users of the DMDD database can now search for phenotypes by anatomy term. This new functionality is designed to help researchers of specific organ or tissue types to quickly identify all phenotypes that are relevant to their studies. Choose from embryo and adult anatomy terms for both humans and mice.

 

Image of the DMDD search box
New search functunality gives users the option to search for phenotypes by anatomy term.

 

FIND GENE KNOCKOUTS THAT ARE LETHAL BEFORE E9.5

Around a third of the knockout lines studied by the DMDD programme have been found to cause lethality before 9.5 days of gestation. Although it is not possible for us to image and phenotype embryos from these lines, we have added them to our database and they can be found using the ‘search’ tool.

For a full list of lines that are lethal before E9.5, visit our Early lethals page.

 

EXPLORE THE NEW EMBRYO PHENOTYPE DATA

In our latest release we’ve made phenotypes available for 7 new knockout lines. These include Cfap53, which is known to be involved in left-right asymmetric patterning in humans. In mouse embryos we identified the phenotype ‘abdominal situs ambiguus’, in which the abdominal organs have neither the usual nor the mirror-image arrangement.

We have also released data on Fut8. This gene is linked to Leukocyte Adhesion Deficiency, a syndrome with symptoms including microcephaly and abnormality of the tongue and palate. In the mouse we identified various phenotypes related to the hypoglossal nerve, which controls movements of the tongue.

Some further highlights from the phenotypes released today include spinal haemorrhage in a Fut8 knockout embryo, a perimembraneous ventricular septal defect in an Arid1b knockout embryo and abnormal lens epithelium morphology in an Actn4 knockout embryo.

 

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A Fut8 knockout embryo found to have a spinal haemorrhage.

 

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Abnormal lens epithelium morphology in an Actn4 embryo. Lens epithelial cells are the parental cells responsible for growth and development of the lens.

 

 

In total, 162 distinct phenotypes were identified across 91 new mutant and wild-type control embryos. Phenotype data for a total of 81 new placentas has also been released.

 

A FULL LIST OF THE NEW DATA

HREM embryo image stacks added for Dennd4c, Dnajc8 and Pigf.

Embryo phenotypes added for Actn4, Arid1b, Cfap53, Cyp11a1, Dmxl2, Fut8 and Mfsd7c.

Placenta images and phenotypes added for B9d2, Cbx6, Commd10, Coq4, Dcx, Dhx35, Fam160a1, Gpatch1, Mfsd7c, Oaz1 and Smg1.

 

All image and phenotype data from the DMDD programme can be accessed at dmdd.org.uk. For assistance, 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.

NEW EMBRYO PHENOTYPE DATA AVAILABLE

New image and phenotype data for embryos and placentas from embryonic lethal knockout mouse lines has been made available on the DMDD website today. The knockout data includes the ciliary gene Rpgrip1l as well as Atg16l1, a gene encoding a protein that forms part of a larger complex needed for autophagy. In total we have added HREM image data for 10 new lines, embryo phenotypes for 11 lines and placenta image and phenotype data for 6 lines.

The new data was released at the same time as enhancements to our website, which have been described in a separate blog post. Keep reading to see some highlights from the phenotype data.


DETAILED EMBRYO PHENOTYPES REVEALED

The comprehensive and detailed nature of DMDD embryo phenotyping means that we are able to identify a wide range of abnormalities. In the data released today, a total of 423 phenotypes were scored across 78 embryos. These included gross morphological defects such as exencephaly and edema, but also abormalities on a much smaller scale such as an unusually small dorsal root ganglion, absent hypoglossal nerve and narrowing of the semicircular ear canal.

In the image below, a Trim45 embryo at E14.5, was found to have abnormal optic cup morphology and aphakia (a missing lens).

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HREM imaging of a Trim45 knockout embryo reveals abnormal optic cup morphology and aphakia on the left side.

3D modelling of the exterior of an Rpgrip1l knockout embryo at E14.5 revealed a cleft upper lip, as well as polydactyly.

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A 3D HREM model of an Rpgrip1l embryo shows a cleft upper lip.

All phenotypes are searchable on the DMDD website, highlighted on relevant images, and the full-resolution image data is available to explore online.


SYSTEMATIC PLACENTAL ANALYSIS

DMDD also carries out systematic phenotyping of the placentas from knockout lines. The image below shows a Cfap53 knockout placenta at E14.5, which was found to have an aberrant fibrotic lesion. The density of fetal blood vessels was also considerably reduced, the overall effect being to reduce the nutrient flow from mother to embryo.

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Placental histology for the line Cfap53 shows a fibrotic lesion (large arrow) and several regions of reduced blood vessel density (small arrows).

 


GENE EXPRESSION PROFILES

Work is underway to measure the gene expression profiles for embryos from embryonic lethal knockout lines, a study that complements the morphological phenotype data we are gathering. One of our ultimate goals is to allow data users to explore correlations between gene, morphological phenotype and gene expression profile. The first part of this dataset was released recently – a temporal baseline gene expression profile for wild type embryos.

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Example expression profiles of Nacad and Pdzk1 with increasing somite number. The data shows that, at this depth of sequencing, Nacad is switched on during somitogenesis and Pdzk1 is switched off.

 

The expression data is now accessible via a dedicated wild type gene expression profiling page on the DMDD website, which also gives background information about the analysis. Mutant expression data will follow in the new year.


LINKS BETWEEN DMDD GENES AND HUMAN DISEASE

Many of the genes studied by the DMDD programme are known to have links to human disease, including several new lines that have been made available in this release.

Rpgripl1: in humans, mutations in RPGRIPL1 are known to cause Joubert Syndrome (type 7) and Meckel Syndrome (type 5), a rare disorder affecting the cerebellum.

Cfap53: the human ortholog of this gene is known to be associated with visceral heterotaxy-6, in which organs have an abnormal placement and/or orientation.

Arhgef7: in humans the ortholog is associated with Borjeson-Forssman-Lehmann Syndrome.

Arid1b: in humans, mutations in ARID1B are associated with Coffin-Siris Syndrome.

Embryonic lethal lines with no known links to human disease may also be novel candidate genes for undiagnosed genetic disorders. Visit the DMDD website to explore the phenotype data.


A FULL LIST OF NEW DATA

HREM embryo image data has been added for Actn4, Arid1b, Cfap53, Crim1, Cyp11a1, Dmxl2, Fut8, Gas2l2, Mfsd7c, Rala.

Embryo phenotype data has been added for Atg16l1, Capza2, Coro1c, Crim1, Cyfip2Gas2l2, Gm5544Rala, Rpgrip1l, Syt1, Trim45.

Placenta image and phenotype data has been added for Arhgef7, Arid1b, Fam21, Fut8, Med23, Timmdc1.

If you have questions about the DMDD programme or our data, please email contact@dmdd.org.uk.