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  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.
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.
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.
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.
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.
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.