Body axis formation in Xenopus laevis : positive and negative regulation of canonical Wnt-mediated transcription
Thesis (Ph.D.)--Memorial University of Newfoundland, 2010. Medicine Includes bibliographical references (leaves 6-2-6-28) Establishment of dorsoventral polarity in Xenopus embryos requires activation of the canonical Wnt signal transduction pathway. Accumulated evidence has indicated that the key ef...
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2009
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Online Access: | http://collections.mun.ca/cdm/ref/collection/theses4/id/41211 |
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Memorial University of Newfoundland: Digital Archives Initiative (DAI) |
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English |
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Cadherins Cellular signal transduction Pattern formation (Biology) Wnt proteins Xenopus laevis--Embryos Embryonic Development Signal Transduction beta Catenin Xenopus laevis |
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Cadherins Cellular signal transduction Pattern formation (Biology) Wnt proteins Xenopus laevis--Embryos Embryonic Development Signal Transduction beta Catenin Xenopus laevis Kennedy, Mark, 1980- Body axis formation in Xenopus laevis : positive and negative regulation of canonical Wnt-mediated transcription |
topic_facet |
Cadherins Cellular signal transduction Pattern formation (Biology) Wnt proteins Xenopus laevis--Embryos Embryonic Development Signal Transduction beta Catenin Xenopus laevis |
description |
Thesis (Ph.D.)--Memorial University of Newfoundland, 2010. Medicine Includes bibliographical references (leaves 6-2-6-28) Establishment of dorsoventral polarity in Xenopus embryos requires activation of the canonical Wnt signal transduction pathway. Accumulated evidence has indicated that the key effector of canonical Wnt signaling, the β-catenin transcriptional activator, is localized in nuclei of dorsally fated cells of the early embryo and is required for dorsal development. The importance of β-catenin as a key element in body axis formation implies that factors which influence β-catenin expression and activity play important roles during dorsal development. Our understanding, however, of the mechanism(s) that govern β-catenin activity, for example, during embryonic development, is incomplete. Therefore, there is a need to identify factors that both inhibit and promote its activity. To this end, I have identified several novel proteins that interact with β-catenin to modulate its transcriptional activity in Xenopus embryos. -- I first determined that the Xenopus Rel/NF-KB proteins, XRelA and XRel3, function as inhibitors of β-catenin activity in embryos. Using gain-of-function assays, I found that both XRelA and XReB perturbed dorsal development by repressing the expression of multiple Xenopus nodal-related (Xnr) genes. Since dorsal development is a canonical Wnt-dependent process and the timing and level of Xnr expression is regulated by canonical Wnt signaling, I hypothesized that XRelA/XRel3 inhibits Canonical Wnt activity in embryos to regulate axis formation. Co-expression of either XRelA or XReB efficiently antagonized ectopic β-catenin activity, as measured by their ability to prevent supernumerary axis formation in embryos injected at the 2-cell stage with β-catenin and a constitutively active β-catenin mutant. Furthermore, XReB directly interacted with β-catenin, using in vitro co-immunoprecipitation assays. These results suggest a mechanism whereby Xenopus Rel proteins negatively regulate Xnr expression by inhibiting β-catenin-dependent transcription thus controlling dorsal axis development. -- In a second set of experiments, I explored the role of a component of the β-catenin transcriptional activation complex called B-cell lymphoma 9 (Bcl9), which is the orthologue to Legless (Lgs) of Drosophila and mammals. In Drosophila embryos, Lgs/Bcl9 was identified as a bridging protein between the downstream component, Pygopus, and β-catenin. Furthermore, both Lgs/Bcl9 and Pygopus were demonstrated to be indispensable for β-catenin-dependent embryonic patterning in Drosophila. Unlike Pygopus, however, the role of Lgs/Bcl9 in vertebrate development is unknown. I determined that like its fly counterpart, Xenopus Bcl9 (XBcl9) directly interacted in vitro, via conserved peptide sequences with the co-activator proteins, Pygopus and β-catenin. Interestingly, XBcl9 preferentially accumulated in dorsal nuclei at a stage in development later than that reported for β-catenin and just prior to Wnt target gene activation. Gain-of-function assays demonstrated that XBcl9 was dependent on Pygopus to ectopically promote β-catenin target gene transcription, and that β-catenin was dependent on its interaction with XBcl9 for dorsal axis formation. Additionally, loss-of-function assays determined that XBcl9 was required for body axis formation during Xenopus development. These results implied that the timing of XBcl9 nuclear localization may indicate an important step in dorsal cell fate determination. -- The role of XBcl9 in axis formation suggested that its regulation is important for normal development. In my final set of experiments, I determined that XBcl9 is post-transcriptionally regulated in Xenopus embryos. The inhibition of XBcl9 translation is dependent on a minimal 29nt element in the 5’UTR, proximal to the putative start of translation, and is well conserved in human Bcl9. The minimal repression element is predicted to form a stable secondary structure, posing as a possible block to constitutive translation. Due to the dependence of β-catenin on XBcl9 for axis development in Xenopus embryos, these results suggest a novel mechanism regulating β-catenin-dependent transcription. |
author2 |
Memorial University of Newfoundland. Faculty of Medicine |
format |
Thesis |
author |
Kennedy, Mark, 1980- |
author_facet |
Kennedy, Mark, 1980- |
author_sort |
Kennedy, Mark, 1980- |
title |
Body axis formation in Xenopus laevis : positive and negative regulation of canonical Wnt-mediated transcription |
title_short |
Body axis formation in Xenopus laevis : positive and negative regulation of canonical Wnt-mediated transcription |
title_full |
Body axis formation in Xenopus laevis : positive and negative regulation of canonical Wnt-mediated transcription |
title_fullStr |
Body axis formation in Xenopus laevis : positive and negative regulation of canonical Wnt-mediated transcription |
title_full_unstemmed |
Body axis formation in Xenopus laevis : positive and negative regulation of canonical Wnt-mediated transcription |
title_sort |
body axis formation in xenopus laevis : positive and negative regulation of canonical wnt-mediated transcription |
publishDate |
2009 |
url |
http://collections.mun.ca/cdm/ref/collection/theses4/id/41211 |
genre |
Newfoundland studies University of Newfoundland |
genre_facet |
Newfoundland studies University of Newfoundland |
op_source |
Paper copy kept in the Centre for Newfoundland Studies, Memorial University Libraries |
op_relation |
Electronic Theses and Dissertations (23.16 MB) -- http://collections.mun.ca/PDFs/theses/Kennedy_Mark.pdf a3315239 http://collections.mun.ca/cdm/ref/collection/theses4/id/41211 |
op_rights |
The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. |
_version_ |
1766113233478877184 |
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ftmemorialunivdc:oai:collections.mun.ca:theses4/41211 2023-05-15T17:23:33+02:00 Body axis formation in Xenopus laevis : positive and negative regulation of canonical Wnt-mediated transcription Kennedy, Mark, 1980- Memorial University of Newfoundland. Faculty of Medicine 2009. 1 v. (various foliations) : col. ill. Image/jpeg; Application/pdf http://collections.mun.ca/cdm/ref/collection/theses4/id/41211 Eng eng Electronic Theses and Dissertations (23.16 MB) -- http://collections.mun.ca/PDFs/theses/Kennedy_Mark.pdf a3315239 http://collections.mun.ca/cdm/ref/collection/theses4/id/41211 The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Paper copy kept in the Centre for Newfoundland Studies, Memorial University Libraries Cadherins Cellular signal transduction Pattern formation (Biology) Wnt proteins Xenopus laevis--Embryos Embryonic Development Signal Transduction beta Catenin Xenopus laevis Text Electronic thesis or dissertation 2009 ftmemorialunivdc 2015-08-06T19:21:57Z Thesis (Ph.D.)--Memorial University of Newfoundland, 2010. Medicine Includes bibliographical references (leaves 6-2-6-28) Establishment of dorsoventral polarity in Xenopus embryos requires activation of the canonical Wnt signal transduction pathway. Accumulated evidence has indicated that the key effector of canonical Wnt signaling, the β-catenin transcriptional activator, is localized in nuclei of dorsally fated cells of the early embryo and is required for dorsal development. The importance of β-catenin as a key element in body axis formation implies that factors which influence β-catenin expression and activity play important roles during dorsal development. Our understanding, however, of the mechanism(s) that govern β-catenin activity, for example, during embryonic development, is incomplete. Therefore, there is a need to identify factors that both inhibit and promote its activity. To this end, I have identified several novel proteins that interact with β-catenin to modulate its transcriptional activity in Xenopus embryos. -- I first determined that the Xenopus Rel/NF-KB proteins, XRelA and XRel3, function as inhibitors of β-catenin activity in embryos. Using gain-of-function assays, I found that both XRelA and XReB perturbed dorsal development by repressing the expression of multiple Xenopus nodal-related (Xnr) genes. Since dorsal development is a canonical Wnt-dependent process and the timing and level of Xnr expression is regulated by canonical Wnt signaling, I hypothesized that XRelA/XRel3 inhibits Canonical Wnt activity in embryos to regulate axis formation. Co-expression of either XRelA or XReB efficiently antagonized ectopic β-catenin activity, as measured by their ability to prevent supernumerary axis formation in embryos injected at the 2-cell stage with β-catenin and a constitutively active β-catenin mutant. Furthermore, XReB directly interacted with β-catenin, using in vitro co-immunoprecipitation assays. These results suggest a mechanism whereby Xenopus Rel proteins negatively regulate Xnr expression by inhibiting β-catenin-dependent transcription thus controlling dorsal axis development. -- In a second set of experiments, I explored the role of a component of the β-catenin transcriptional activation complex called B-cell lymphoma 9 (Bcl9), which is the orthologue to Legless (Lgs) of Drosophila and mammals. In Drosophila embryos, Lgs/Bcl9 was identified as a bridging protein between the downstream component, Pygopus, and β-catenin. Furthermore, both Lgs/Bcl9 and Pygopus were demonstrated to be indispensable for β-catenin-dependent embryonic patterning in Drosophila. Unlike Pygopus, however, the role of Lgs/Bcl9 in vertebrate development is unknown. I determined that like its fly counterpart, Xenopus Bcl9 (XBcl9) directly interacted in vitro, via conserved peptide sequences with the co-activator proteins, Pygopus and β-catenin. Interestingly, XBcl9 preferentially accumulated in dorsal nuclei at a stage in development later than that reported for β-catenin and just prior to Wnt target gene activation. Gain-of-function assays demonstrated that XBcl9 was dependent on Pygopus to ectopically promote β-catenin target gene transcription, and that β-catenin was dependent on its interaction with XBcl9 for dorsal axis formation. Additionally, loss-of-function assays determined that XBcl9 was required for body axis formation during Xenopus development. These results implied that the timing of XBcl9 nuclear localization may indicate an important step in dorsal cell fate determination. -- The role of XBcl9 in axis formation suggested that its regulation is important for normal development. In my final set of experiments, I determined that XBcl9 is post-transcriptionally regulated in Xenopus embryos. The inhibition of XBcl9 translation is dependent on a minimal 29nt element in the 5’UTR, proximal to the putative start of translation, and is well conserved in human Bcl9. The minimal repression element is predicted to form a stable secondary structure, posing as a possible block to constitutive translation. Due to the dependence of β-catenin on XBcl9 for axis development in Xenopus embryos, these results suggest a novel mechanism regulating β-catenin-dependent transcription. Thesis Newfoundland studies University of Newfoundland Memorial University of Newfoundland: Digital Archives Initiative (DAI) |