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.


Dr David Adams.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 hear from David Adams, who oversees the production of embryonic lethal knockout mouse strains for the project.

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

I lead the Mouse Programme (MGP) at the Wellcome Trust Sanger Institute, which generates around 200 genetically modified mouse strains each year. The MGP explores the role of genes in a range of biological processes including in development, immunology and infection, and in metabolism and cancer. I am primarily a cancer geneticist interested in how the immune system controls tumour growth and the genetic wiring of cancer cells. Remarkably this had led me to explore aspects of developmental biology as we try and understand what these genes do.


What inspired you to devote your career to developing animal models of human disease

In all areas of medicine and biology, animal models have contributed significantly to our understanding of disease processes. For example, our understanding of the fundamentals of how the immune system recognises pathogens and cancer result from experiments in mice. The development of induced pluripotent stem cells, which appear to have huge potential in regenerative medicine, was also pioneered in mice. If that’s not enough, the role of literally thousands of mammalian genes in development has been elucidated in mouse model systems. The contribution has been huge. Further, virtually every drug approved for treating patients was tested in rodent models.


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

This has to be CRISPR. The ability to rapidly alter the genome with unprecedented precision makes generating new animal models significantly easier. In particular, we are able to introduce point mutations found in patients to essentially humanise the mouse genome.


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

There are still many many genes where we don’t have even the most basic understanding of their role in development or disease. Cell culture systems will undoubtedly contribute to further understanding, as will the analysis of human tissues, but to really understand what a gene does you need to manipulate it in the context of the whole organism and see what happens. We call this the post-genomics era but in fact we are still very much living in a time where we don’t know how the genome works and how individual genes function. I think there are many surprises and delights still to be found.


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

My role is to represent the Sanger Institute as a member of the DMDD programme. The DMDD is a group of world-leading investigators using cutting edge technology to explore processes involved in development and embryonic lethality. I find the idea of contributing to such a large-scale co-operative endeavour very compelling. It is also wonderful that the data is released to the research community to facilitate further discovery.


What do you hope the DMDD programme will achieve?

So far there have been some big surprises. In particular, the significance of the placenta in development and the high frequency of cardiac malformations in developmental disorders have been a surprise to me. Large-scale programmes such as the DMDD that make no assumptions about how genes work or what they do have the potential to challenge dogma, and that’s what I think the DMDD is achieving.


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

I have a particular interest in how the immune system regulates the growth of cancers. In the last few years there have been substantial advances in understanding how T-cells control tumour growth. I would love to have been involved in, or seen first-hand, the development of T-cell checkpoint therapies because these are truly changing people’s lives and a proportion of patients with advanced disease are being cured.


What are you most proud of achieving outside of science?

I have two delightful children, which is a surprise given my genetic contribution. I am also a keen runner like other members of the DMDD (Tim Mohun and Jim Smith) and routinely run 20 km a week. I am currently training for the Cambridge Half Marathon.


Tell us a surprising fact about yourself

I used to breed, raise, and show chickens as a child. I guess this was probably the basis for my interest in genetics. Like everything I do I was extremely competitive.


David Adams is a Senior Group Leader at the Wellcome Trust Sanger Institute, and a joint grant holder for the DMDD programme.


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.



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.