Investigating the differential instructive roles of WT1's isoforms
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Date
02/07/2016Author
Petrovich, Giulia
Metadata
Abstract
The Wilms' tumour suppressor gene Wt1 is a key regulator of embryonic development
and tissue homeostasis. In humans, mutation in the gene may lead to childhood kidney
cancer, severe glomerular kidney diseases, gonadal dysgenesis and, in rare cases,
heart diseases. The importance of WT1 in embryonic development is related to its
crucial function in the regulation of two cellular plasticity processes: the epithelial to
mesenchymal transition (EMT) and the reverse process, the mesenchymal to epithelial
transition (MET). WT1 expression persists during the waves of EMT and MET that
generate certain mesodermal tissues. In fact, WT1 is a major regulator of both transitions
and it is essential for the survival of mesenchyme progenitors. Furthermore, it
has been proposed that WT1 is required for the derivation of progenitors from different
mesothelia, possibly through an EMT. Progenitors expressing WT1 are believed to differentiate
into different cell types, giving rise to coronary vasculature, adipocytes and
hepatic stellate cells.
In my PhD I aimed to investigate the instructive role of different WT1 isoforms. To
address this, the first goal was to generate a single plasmid that would accommodate
all necessary components of an inducible system, in order to derive cellular models for
the inducible expression of WT1 single isoforms. Second, I aimed to understand the
processes that the single variants were sufficient to drive.
Therefore, I started with the establishment of two cellular models for the inducible expression
of the main four isoforms of WT1 (with or without the exon 5 and/or the KTS,
here referred as +/+, +/-, -/+ and -/-). I cloned different plasmids carrying doxycycline
inducible WT1 isoforms and derived single stable clones in two epithelial kidney
cell lines that do not express WT1: the MDCK and the IMCD3 cells. I then analysed
the expression profiles of the clones, using either microarray or RNA sequencing, and
performed cellular assays to characterize the cells after WT1 induction.
Overall, WT1 induction did not affect cell growth, cell cycle, cell migration or anchorage
independent growth, suggesting that the expression of WT1 in these two cell lines does
not change their oncogenic potential.
The expression analysis of the MDCK cells suggested that the induction of WT1 isoforms
activates an inflammatory response, leading to the overexpression of several cytokines.
Moreover, the -/+ isoform speciffically caused the upregulation of fibrotic
markers and the rearrangement of the actin cytoskeleton. Interestingly, the expression
of the mesothelial marker UPK3B increased following the induction of the -/+ isoform.
Because the expression of the -/+ variant led to the most signifficant isoform-specific
changes in both cell lines, I focused on this isoform for the validation of the transcriptomic
data of the IMCD3 cells. I confirmed that the induction of WT1 -/+ in the
IMCD3 cells leads to the upregulation of fibrotic markers, increases cell adhesion and
activates the AKT and MAPK pathways. Moreover, there was a significant overexpression
of different mesothelial markers and, importantly, of key regulators and markers of
developmental processes, such as adipogenesis, skeletal and cartilage development, as
well as angiogenesis. I then dissected the timing of expression of some specific markers
and regulators, analysing the levels of the genes at different time points after WT1 -/+
induction. The preliminary results intimate that WT1 -/+ might induce epithelial cells
in the direction of cartilage-skeletal tissue and fat, possibly through a mesothelial intermediate.
The data also suggest that the induction of this isoform initiates an EMT,
possibly followed by an MET, as the levels of expression of the differentiation markers
and regulators increase.
To validate the proposed instructive role of WT1, it will be of crucial importance to
determine both RNA and protein levels of markers and regulators at even later time
points, both in IMCD3 cells and in a model of inducible embryonic stem cells, which
is currently under development. In the future, it will be important to address the
relevance of these findings in vivo and to dissect the molecular mechanisms.