Identification and characterisation of genes which are critical for murine gastrulation
Date
2017
Authors
Alzahrani, Alaa
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Abstract
Gastrulation, a critical stage of embryogenesis, converts a group
of relatively undifferentiated cells into the body plan of the
growing foetus. Embryonic cells are allocated to one of three
definitive germs layers and are organised with respect to three
embryonic axes (anterior-posterior, dorsal-ventral and
left-right). By the end of gastrulation the primordia of most
organs and physiological systems have been established. In
mammals, gastrulation occurs just after the embryo implants into
the uterine wall. The small size of the mammalian gastrula and
its inaccessibility means that, despite its central importance to
embryonic development, gastrulation remains one of the most
challenging and underexplored areas of mammalian biology. The
study of other complex biological processes has been greatly
facilitated by the use of genetic screens, which employ a
powerful mutagen to introduce mutations into the germline of an
organism and then examine descendants for altered biology (i.e.
an abnormal phenotype) followed by post-hoc identification of the
underlying genetic alteration. The utility of these screens is
dependent upon quick, robust assays to identify pheno-deviants
and it has long been thought that practical issues prevent this
approach being applied to mammalian gastrulation. In this thesis,
I examine this notion. In the first part of the thesis, I use
phenotype analysis to infer the function of the gene altered in
the katun mouse strain which was identified in a screen for
mutations that alter embryonic development and was found to have
a gastrulation phenotype. I show that the left-right axis defects
in this strain are likely caused by dysregulated Wnt signalling
at gastrulation which interferes with endoderm development. This
confirms that mutants recovered from genetic screens can be used
to infer gene function at gastrulation. In the second part of the
thesis, I report results form a prototype, genome-wide, screen
for recessive mutations that alter gastrulation. The screen was
able to overcome the perceived difficulties of small litter
sizes, of isolating a large number of gastrulation stage embryos,
of distinguishing phenotypes from natural embryonic loss and of
working with limiting amounts of genomic DNA. I demonstrate that
mutant pedigrees can be identified, they can be maintained in the
absence of genetic markers via test breeding protocols and that
low-resolution meiotic mapping can be combined with next
generation sequencing technologies to identify the causative
mutation. This
confirms that gastrulation is amenable to genetic dissection by
mutagenesis and I suggest a protocol that could be used to
conduct a larger scale, efficient screen to identify the genes
that direct mammalian gastrulation.
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Keywords
gastrulation, heart, heterotaxy, laterality disorder, mouse, node, ENU, WES, Wnt signalling pathway
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Thesis (PhD)
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