Transcription factors play multiple and important roles during embryonic
development. The Pax gene family have been shown to be essential in these
processes, and mutations which abrogate their function disrupt the development of a
range of embryonic tissues. One member of this family, Pax3, is characterised by a
semidominant mutant phenotype and is involved in the proper formation of the brain,
central and peripheral nervous system, hypaxial musculature, and cardiac outflow
vessels. Pax3 is a transcription factor and its biological role involves the binding of
specific sequences in genomic DNA to regulate the expression of a set of target genes.
Defects observed in animals harbouring a mutation in Pax3 are assumed to arise from
the misregulation of the transcriptional targets of this gene.
In dissecting the precise biological function of Pax3, it is important to differentiate
between primary and secondary phenotypes in the mutant animal; specifically,
between defects caused by the direct misregulation of Pax3 targets and those
generated by the compounded knock -on effects of a dysfunctional master -regulator
such as Pax3. A simple comparison of expression profiles between wild type and
mutant animals, using microarray or differential display based methodologies, would
not achieve this aim. To date, no attempt to perform a comprehensive screen for direct
Pax3 targets in development has been performed.
This thesis presents a development of the tools necessary to perform the task of
identifying direct targets of Pax3 in vivo. Firstly, two candidate genes for direct Pax3
regulation are considered, Wntl and Pax7. These candidates are implicated from
previous work on Pax3 although no direct transcriptional link has ever been
established. Firstly, differences in the expression of these genes between the Pax3
mutant and wild type embryos is quantitatively analysed. In the case of Pax7, little
work had been performed to identify the regulatory elements controlling its
expression in the mouse. Described here is a series of experiments mapping the 5' end
of the transcript, the delineation of a putative promoter region and the identification
and cloning of a highly conserved enhancer element within the first intron. The Wntl
regulatory elements have been well described elsewhere. A series of experiments are
then performed to confirm the interaction between these regulatory regions and Pax3
and the identification and testing of specific Pax3 binding sites is reported.
To confirm these interactions in vivo, chromatin immunoprecipitation, using a novel
mono -specific anti -Pax3 antibody designed for the purposes of this thesis, is used.
This technique, having been verified on these two direct Pax3 targets in this way,
could then be used to screen for novel direct targets of Pax3 regulation in vivo and in
the wild type; expanding our understanding of the pathways and developmental
function of this gene in future.