CHRAT Vacation Studentship - Summer 2014
Student: Amy Cutler
Supervisor(s): Dr. Catherine Roberts & Professor Pete Scambler
Title of Project: Cyp26b1 in Proepicardial and Coronary Artery Development
Aim of Project: To investigate the role of retinoic acid-metabolising enzyme CYP26B1 in cardiac development
Brief description of project and results. Please also comment on the value of your experience:
The Cyp26 gene products are retinoic acid (RA) metabolising enzymes. They are therefore important during
development, for maintenance and balance of RA levels. This is a crucial role as both an excess and a lack of RA can
cause serious developmental abnormalities, including cardiac, pharyngeal/craniofacial and limb defects. So far, three
cytochrome P450 26 (CYP26) enzymes have been identified - CYP26A1, CYP26B1 and CYP26C1. For the purposes
of this project, we focused on the cardiac role of Cyp26b1.
Perhaps my most important experiment was to examine epithelial to mesenchyme transition (EMT) in the epicardium
and in endocardial valve cushions of wild type and null-mutant Cyp26b1 mouse embryos. To do this I used antibody
staining on both whole-mount and cryo-section embryos. Antibodies were used against Podoplanin (PDPN) and
SLUG proteins. PDPN is expressed in epicardial cells whilst SLUG is specifically expressed in cells undergoing
epithelial/endothelial mesenchyme transition. Confocal microscopy of the whole-mounts demonstrated downregulation of both markers, but particularly SLUG, in Cyp26b1 mutants compared with wild-types - therefore
suggesting EMT is abnormal. This may explain the epicardial, coronary vessel and valve defects associated with the
mutation. This finding led to the cryo-section staining, of which the analysis is still in progress. However, so far the
data does appear to support the confocal observations. Such a discovery may one day lead to publication.
Furthermore, I continued some of Dr. Robert’s previous work by fluorescently staining cryo-sections with antibodies
against neuropilin 2 (NRP2) and alpha-smooth-muscle actin (α-SMA). This was due to preliminary data suggesting
abnormal vascular vesicles to be present on the ventricular surface in some Cyp26b1 mutants. These vesicles were
hypothesised to be of venous identity and so the aim of the experiment was to confirm this. Unfortunately, the
antibody staining was not optimal. However, I obtained data showing at least some of the epicardial vesicles were
NRP2 positive, so this experiment should be repeated with a more reliable antibody in the future.
In addition to my antibody staining experiments, I also performed In Situ hybridisation to study Cyp26a1 expression
in Cyp26b1 mutants. The theory behind this experiment is that in a number of genetic mutations, alternative gene
family members may compensate for the loss of a gene. We wanted to confirm that this is not the case in Cyp26b1
mutants, and that abnormal expression of Cyp26a1 is not responsible for any of the defects seen. We did not see any
ectopic or over-expression of Cyp26a1 in the mutants, thus suggesting that abnormal expression is not contributing to
the phenotype of Cyp26b1 mutants. Additionally, wild-type expression in the pharyngeal region of E9-10 embryos
and in older E12.5 heart was confirmed.
Finally, I carried out antibody staining of whole-mount, wild-type mouse embryos and adult-heart cryo-sections, using
CYP26B1 and TBX1 antibodies, in order to confirm their normal expression pattern. Unfortunately, results
demonstrated that the CYP26B1 antibody had degraded and so this experiment must be repeated in the future.
The opportunity to work at the Institute of Child Health this summer has been one of inspiration and I realise just how
lucky I have been to study the most recent, up and coming research in a centre of excellence. Until now I was naive as
to how much time and work goes in to discovering every tiny detail within science. The laboratory experience was
invaluable in itself as I was given space to work independently and make my own mistakes (which enables you to
learn much faster), but was supported patiently throughout by someone willing to answer all my questions, no matter
how ridiculous. I have learnt skills that will be beneficial to whatever I may do in the future - from perseverance to
time-management, and have been able to advance my cardiac anatomy more than I ever could from a textbook.
Finally, I have come to understand how a research-individual can become so passionate about their topic of work and
its applications. I would recommend the experience to any science undergraduate of the future.