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.
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.
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.