Neuromesodermal progenitors (NMPs) are bipotent progenitors, located at the caudal
end of the embryo and are essential for axis formation. These stem cell-like progenitors
possess the ability to self-renew and differentiate to both mesodermal and neural
lineages, such as skeletal muscle and spinal cord derivatives. These progenitors arise
at E8.5 and are localised in the caudal lateral epiblast (CLE), a posterior region of the
embryo near the primitive streak. Later in development, they reside in the tail bud until
cessation of axial elongation at E13.5. Throughout these stages NMPs are
characteristically marked by co-expression of T(Bra) (Brachyury) and Sox2. This
characteristic is also present in in vitro NMPs, which can be derived from Epiblast
Stem Cells (EpiSCs) through treatment with Wnt/β-catenin signalling agonists and
Fgf2, which simulates their in vivo environment.
Protein and mRNA profiling of NMPs and mutant phenotypes in vivo supports the
hypothesis that a non-canonical Wnt pathway, the Wnt/Planar Cell Polarity pathway
(PCP) could be involved in NMP fate decision and/or maintenance. This thesis focuses
on understanding more about the role of PCP by aiming to identify the spatio-temporal
profile of Wnt/PCP pathway components in NMP regions during axial elongation, as
well as determining its role in NMP behaviour through manipulation of this pathway
via in vivo and in vitro assays
Employing in situ hybridisation and immunohistochemistry techniques, key Wnt/PCP
components, including Pk1, Vangl2 and Ptk7, were confirmed to be present in in vivo
and in vitro NMPs, thus, providing strong evidence that Wnt/PCP may be involved
regulating NMP behaviour.
Disruption of Wnt/PCP signalling through overexpression of Wnt/PCP components
was tested in refined in vivo and in vitro assays. Overexpression of Vangl2 and Ptk7,
but not Pk1 in NMPs regions in vivo resulted in loss of contribution to neural lineages,
as well as lower contribution to NMP regions themselves. Similarly, Wnt/PCP
components were disrupted in vitro through generation of dox-inducible
overexpression cells lines for Wnt/PCP components. These lines were used to generate
NMPs from an optimised novel alternative source Epiblast-Like Cells (EpiLCs),
however no clear affect to lineage was observed.
Overall this work has successfully advanced our knowledge of Wnt/PCP mediated
control of NMP differentiation and maintenance, and provided a finer grained
description of the relationships between them