|dc.description.abstract||miRNAs are short, non-coding RNA molecules that regulate gene expression posttranscriptionally
through inhibition of translation and/or mRNA degradation.
Mammalian development is a complex series of developmental transitions, which
relies on accurate spatial and temporal regulation of gene expression and we are
interested in the role that miRNAs may play in these developmental transitions.
An initial objective was to establish which, if any, miRNAs were dynamically
regulated in a cell model of an early developmental transition, and to establish
whether differential expression of any particular miRNA played a functional role
in this developmental process. Having established a role for specific miRNAs,
further objectives were to assess the reliability of current miRNA-mRNA target
identification procedures and to assess the general role of miRNAs in cellular
In order to explore the roles of miRNAs during an early developmental transition,
an embryonic stem (ES) cell model of trophectoderm differentiation was used. In
this model system the expression of the key ES cell regulatory gene, Oct4, can be
conditionally repressed, which induces the ES cells to differentiate down the trophectoderm
lineage. The expression of microRNAs was profiled in this model system
by cloning and sequencing of small RNAs. This approach identified miRNAs
that were dynamically regulated during differentiation. The expression patterns
of differentially regulated miRNAs were confirmed by miRNA northern analysis.
The miRNA profiling data showed that mmu-miR-294 and mmu-mir-295 are
expressed at similar levels in ES cells and differentiated cells, which disagrees
with previous reports that these miRNAs are ES cell specific. Several of the
miRNAs with higher expression levels in differentiated cells are encoded within
a placental-enriched polycomb group gene, Sfmbt2, suggesting an important role
for these miRNAs in extraembryonic development. One of the miRNAs that was
expressed at higher levels in ES cells than in differentiated cells, mmu-miR-92a,
was shown to play a role in regulation of cell proliferation.
Three current methods of identifying miRNA targets were assessed. A sequencebased
method using the web-based utility miRecords, which amalgamates results
from numerous target prediction databases, was used to generate lists of potential
targets of the Sfmbt2 miRNA cluster and of mmu-miR-92a. Amalgamating results from multiple target prediction programs may improve the likelihood that
the predicted targets are real. Exemplifying this, the single mmu-miR-92a target
that was predicted by six different target prediction programs had been previously
experimentally verified. An experimental method of identifying direct
miRNA targets, PAR-CLIP, was investigated but proved technically limiting for
routine use in the laboratory. A proteome-based experimental method for identifying
potential miRNA targets, called SILAC, was successfully used to identify
proteins that were differentially expressed in the cell model of trophectoderm differentiation.
Differential expression of two of these proteins, CTBP2 and CKB,
was confirmed by western analysis. miRecords was then used to assess whether
the differentially expressed proteins were likely to be targets of the differentially
expressed miRNAs that had been identified in the miRNA profiling analysis.
The general role of miRNAs in cell differentiation was investigated using a cell
line that does not express miRNAs. This ES cell line is deficient for the miRNAprocessing
enzyme DGCR8, which results in loss of expression of mature miRNAs
in these cells. Compared to wild type ES cells, miRNA-deficient ES cells
expressed normal levels of the ES cell marker genes Oct4 and Sox2 but elevated
levels of Nanog. In contrast to wild type ES cells, miRNA-deficient ES cells
did not upregulate the mesoderm marker gene Brachyury during embryoid body
differentiation and showed reduced upregulation of the endoderm marker gene
Gata6. These findings suggest that miRNAs are not required for maintenance of
pluripotency, but are essential for proper ES cell differentiation.
The results presented in this thesis show that miRNAs are dynamically expressed
during a mammalian developmental transition and are involved in regulating early
developmental processes. We believe that miRNAs act as an additional level of
genetic regulation to ensure canalisation during embryonic development.||en