Epigenetic profiling of the developing zebrafish embryo, and technical developments towards cloning zebrafish and isolating pluripotent stem cells
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Date
2009Author
Thakrar, Sanjay
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Abstract
In normal embryonic development, cells generated from a fertilised oocyte lose their
pluripotent status and become restricted to a particular differentiation pathway. This
production of functionally distinct cell lineages is thought to be mediated by
epigenetic processes that help control gene expression both temporally and spatially
without any changes to the DNA sequence. These epigenetic changes consist of posttranslational
modifications of the N-terminal tails of histones and differential DNA
methylation. Together these act by altering local chromatin structure, which in turn
directs gene transcription by regulating the accessibility of the underlying DNA. To
examine the potential developmental roles of these modifications, we determined the
global cellular patterns of DNA methylation, as well as histone H3 lysine 9 (H3K9)
and histone H4 lysine 20 (H4K20) methylation in the developing zebrafish embryo.
These modifications are seen as hallmarks of heterochromatin, which consists of
DNA that is tightly packaged, gene-poor and transcriptionally silent. Thus using
immunostaining techniques, we confirmed the occurrence of genome-wide DNA
methylation changes during zebrafish embryogenesis, as well as observing the
unique localisation of this mark around the nuclear periphery in conjunction with
pericentric heterochromatin. For mono-, di- and tri-methylated H3K9, it was
observed by both immunostaining and immunoblotting that these marks became
apparent after the onset of zygotic transcription. Ultimately their levels increased as
development progressed, in a fashion similar to that of DNA methylation, consistent
with a link between these epigenetic marks. Using the same methodology, the three
methylation states of H4K20 were seen to vary differentially during zebrafish
development, where in particular the levels of H4K20me1 decreased in concert with
a potentially sumoylated form. In contrast, the levels of H4K20me2 increased
progressively during embryogenesis, while those of H4K20me3 decreased rapidly
after the mid-blastula transition. Together, these findings demonstrate that both DNA
and histone lysine methylation take place in a highly dynamic manner, further
supporting their roles in augmenting chromatin structure and directing cellular
differentiation, while also providing a valuable comparison to the developmental epigenetics of other model organisms characterised to date. Preparatory work for
somatic cell nuclear transfer in zebrafish was also undertaken. In future studies, the
dynamics of these marks could be compared with those of cloned embryos, so that
the specific epigenetic profiles necessary for development can be elucidated.
Epigenetically, a homologous process occurs within pluripotent embryonic stem cells
(ESCs), which can differentiate into any cell type or undergo indefinite self-renewal.
Advantageously, we were able to derive zebrafish ESC-like clusters which were
morphologically similar to those derived from mice. These clusters were alkaline
phosphatase-positive and expressed key ESC markers as detected by RT-PCR and
immunofluorescence. In pilot studies, GFP-expressing ESC-like clusters have so far
also contributed to ectodermal tissues when transplanted into wild type zebrafish
embryos. Subsequently, these ESC-like clusters were epigenetically profiled using
immunofluorescence, which showed that they had a similar complement of
modifications to ESCs derived from mice. The derivation and initial characterisation
of these ESC-like clusters from zebrafish, in addition to the development of somatic
cell nuclear transfer in this species, will help pave the way for future studies
involving tissue repair and regeneration, as well as opening up the potential of
targeted genetic manipulation in this valuable model organism.