CHRAT Vacation Studentship- Summer 2015
Student: Sara Bartlome
Supervisors: Dr Evanthia Nikolopoulou, Prof Nicholas Greene
Title of project: Defining the role of grainyhead-like proteins in regulation of proliferation during early
development of the central nervous system.
Aim of Project: To determine the effect of loss or gain of Grhl2 function on proliferation during the formation
of the neural tube in overexpression/ knockout mouse embryo models.
Brief description of project and results. Please also comment on the value of your experience:
Grainyhead like (Grhl) proteins are transcription factors that regulate some of the many crucial genes
regulating the formation of the neural tube. In the developing mammalian embryo the neural tube is the
precursor that later forms the organism’s brain and spinal cord. Failure in neural tube closure leads to severe
birth defects (termed neural tube defects; NTDs), these include spina bifida and anencephaly and arise in
approximately 1 in 1000 pregnancies worldwide. Homozygous Grhl2 or Grhl3 mouse knockouts demonstrate
the importance of these transcription factors’ functions during neural development as these all developed
NTDs. The mouse model used in the experiments we performed was the Axial defects (Axd) mouse model,
which overexpresses Grhl2 and also exhibits spina bifida. Grhl2 is over-expressed in some cancer cells and has
been linked to poor patient diagnosis, suggesting a pro-proliferative role.
Not much is known about the surface ectoderm, the layer of cells that covers the dorsal neural tube. Grhl2 is
expressed in this cell layer and we asked specifically whether Grhl2 function affects its proliferation during the
formation of the neural tube. The aim of this project was, by using the proliferation markers: phospho-histone
H3 (PH3), a mitosis marker, and BrdU, a thymidine analogue used to measure DNA synthesis, to determine the
proliferation of the surface ectoderm cells and in relation to the expression level of Grhl2.
For both PH3 and BrdU experiments, wild type and Axd/Axd mutant embryos were collected at embryonic
day 9.5, fixed in 4% PFA, embedded in wax and transversely sectioned at 4µm thickness before staining with
antibodies to the chosen markers. For the BrdU experiment, dams were injected with 50mg/ml BrdU in PBS
for 15 minutes (time decided after initial optimisation experiments) before the embryo collection. Sections in
both the PH3 and BrdU immunofluorescence experiments were double stained with E-cadherin as a surface
ectoderm marker and DAPI for the calculation of the total number of nuclei. This combination meant that the
proportion of surface ectoderm cells in mitosis could be identified. The stained cells from 4 samples (2
wildtype and 2 mutants) were thereafter counted after images obtained using a Leica fluorescence
microscope. Finally, an immunofluorescence experiment was also performed on whole embryos for PH3 and
E-cadherin.
Figure 1: Immunofluorescence and whole mount immunofluorescence of wild type and Axd/Axd mutant mice,
at the 15 and 14 somite stage respectively. Sections are at the level of the closure point of the neural folds in
the posterior neuropore (PH3 in red). Whole mount images show a dorsal view of the embryo encompassing
the point of closure of the neural folds.
Immunofluorescence x40 (DAPI blue, E- Cadherin white)
Wildtype
15 S
Axd/Axd
15S
Whole mount immunofluorescence
Wildtype
14S
Axd/Axd
15S
From the whole mount immunofluorescence experiments completed by my supervisors, it was apparent that
a difference in the number of PH3 stained cells in the surface ectoderm of the wildtype and mutant may exist.
In order to investigate this we counted the total number of stained cells on sections, which was around 10
cells per section. We thereafter chose different regions of the spinal neural tube in relation to the closure
point. The samples collected however contained lots of variability, therefore this experiment needs to be
repeated with more samples.
Although initial optimisation experiments were successful the BrdU staining did not work as all the cells
seemed to be stained in the samples, suggesting that embryo collection was not optimal or a problem with the
staining protocol. This error was therefore unexpected.
This experience has been a fantastic introduction to work in an academic research lab. Having had no previous
experience before, this was a great opportunity for me to learn some techniques. For instance, I learnt how to
dissect mouse embryos, from removing them from the womb to removing the amniotic sac, how to use a
microtome to section embryos embedded in paraffin wax, how to perform an immunohistochemistry stain
and how to use a fluorescence microscope. Moreover, simply being immersed in the lab taught me many
things such as how to keep a lab book, how to be organised, clean and to never hesitate to ask questions. This
has been a brilliant experience and I highly recommend it to anyone who has the opportunity.