Title:
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A chemical genetic and functional approach to studying cell migration in Xenopus development
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Xenopus embryos have long been used as a developmental model
organism, their potential as an in vivo model for single cell migration has yet
to be fully explored. The PhD study presented used two cell types,
macrophage and melanophores. Xenopus melanophores were assayed in a
chemical genetic screen to identify novel small molecule compounds effecting
their migration. With respect to macrophage migration, a set of three identified
XMMPs showed expression in the migrating macrophages their functional
role was unknown. The function of three Xenopus MMPs was characterised.
by loss-of-function. Analysis of each XMMP shows that they are all functional.
The MMPs were shown to act synergistically when knocked down with each
other. Localisation of the XMMPs in an embryonic wound healing model was
also characterised.
The highest model organism used in chemical genetic studies to date
is the developing zebrafish embryo. Xenopus embryos offer a higher
evolutionary model and an alternative to zebrafish embryos. Xenopus
embryos were characterised as a potential developmental chemical genetic
model. This proof of principle study assayed penetrance, efficacy, genetic
variation and throughput. From these studies, a large-scale screen was
undertaken to identify novel compounds modulating melanophore migration.
Compounds effecting a number of other areas of embryonic development
were identified including pigmentation, edema formation, eye development,
general morphology and melanophore morphology were also identified.
One compound (NCI 84093) has a striking pigment phenotype,
causing a vertical banding pattern and effecting only one pigment stripe. The
mode of action of NCI 84093 was further characterised. Application of the
compound disrupts the formation of the dorsal pigment stripe at an early stage in development, before migration is normally observed. By molecular,
biochemical and computational analysis NCI 84093 was discovered to have
the pharmacological properties of a matrix metalloproteinase (MMP) inhibitor.
A set of novel small molecule compounds effecting melanophore
migration was identified and the mode of action of one of the compounds
characterised. The set of three macrophage expressed XMMPs are shown to
be functional, evidence suggests they act in combination. The application of
NCI 84093 and the loss-of-function of an XMMP (XMMP-14) previously
identified as being expressed by the migrating melanophores were used to
characterise the migration routes of embryonic melanophores.
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