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Arkadia enhances Nodal/TGF-beta signaling by coupling phospho-Smad2/3 activity and turnover.

Mavrakis KJ, Andrew RL, Lee KL, Petropoulou C, Dixon JE, Navaratnam N, Norris DP, Episkopou V - PLoS Biol. (2007)

Bottom Line: Consistent with this dual function, introduction of Arkadia in homozygous (-/-) embryonic stem cells activates the accumulated and hypoactive P-Smad2/3 at the expense of their abundance.Arkadia-/- cells, like Smad2-/- cells, cannot form foregut and prechordal plate in chimeras, confirming this functional interaction in vivo.As Arkadia overexpression never represses, and in some cells enhances signaling, the degradation of P-Smad2/3 by Arkadia cannot occur prior to their activation in the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Mammalian Neurogenesis, Medical Research Council, Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom.

ABSTRACT
Regulation of transforming growth factor-beta (TGF-beta) signaling is critical in vertebrate development, as several members of the TGF-beta family have been shown to act as morphogens, controlling a variety of cell fate decisions depending on concentration. Little is known about the role of intracellular regulation of the TGF-beta pathway in development. E3 ubiquitin ligases target specific protein substrates for proteasome-mediated degradation, and several are implicated in signaling. We have shown that Arkadia, a nuclear RING-domain E3 ubiquitin ligase, is essential for a subset of Nodal functions in the embryo, but the molecular mechanism of its action in embryonic cells had not been addressed. Here, we find that Arkadia facilitates Nodal signaling broadly in the embryo, and that it is indispensable for cell fates that depend on maximum signaling. Loss of Arkadia in embryonic cells causes nuclear accumulation of phospho-Smad2/3 (P-Smad2/3), the effectors of Nodal signaling; however, these must be repressed or hypoactive as the expression of their direct target genes is reduced or lost. Molecular and functional analysis shows that Arkadia interacts with and ubiquitinates P-Smad2/3 causing their degradation, and that this is via the same domains required for enhancing their activity. Consistent with this dual function, introduction of Arkadia in homozygous (-/-) embryonic stem cells activates the accumulated and hypoactive P-Smad2/3 at the expense of their abundance. Arkadia-/- cells, like Smad2-/- cells, cannot form foregut and prechordal plate in chimeras, confirming this functional interaction in vivo. As Arkadia overexpression never represses, and in some cells enhances signaling, the degradation of P-Smad2/3 by Arkadia cannot occur prior to their activation in the nucleus. Therefore, Arkadia provides a mechanism for signaling termination at the end of the cascade by coupling degradation of P-Smad2/3 with the activation of target gene transcription. This mechanism can account for achieving efficient and maximum Nodal signaling during embryogenesis and for rapid resetting of target gene promoters allowing cells to respond to dynamic changes in extracellular signals.

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Arkadia Facilitates All Major Nodal-Dependent Developmental Events(A–F) In situ hybridization on 6.5 dpc embryos (mid- to late-streak stage) shown as lateral views with anterior to the left and posterior to the right, with probes (A–C), Hex and Brachyury; (D–F), Cer-l. In (C), Akd−/− Nodal+/− embryo showing lack of elongation and proximal positioning of the Brachyury–expressing primitive streak, as well as loss of Hex expression indicating absence of AVE. (E) An Akd−/− embryo with normal Cer-l expression in the AVE but no Cer-l expressing ADE. (F) An Akd−/−, Nodal+/− embryo showing a distal Cer-l-expressing domain indicating lack of AVE migration (three other embryos do not express Cer-l, not shown). Red arrowheads point to a constriction between extraembryonic and embryonic regions indicating incomplete anterior-posterior axis specification.(G–I) Nkx2.5 probe on four-somite stage, 8.5 dpc embryo showing expression in the cardiac precursor tissue in the wild-type (G) and Akd−/− (H), but not in the Akd−/−, Nodal+/− embryo (I).(J–L) Shh probe on 12-somite stage embryos 9.5 dpc viewed from the ventral side with anterior toward the top. In the Akd−/− Nodal+/−embryo (L), note the reduction of Shh expression, the increased severity of the anterior truncation, and the absence of a morphologically distinguishable heart tube. PS, primitive streak; T, Brachyury; CP, cardiac precursors; HF, head folds; NC, notochord; GE, gut endoderm. Bars, 0.1 mm.
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pbio-0050067-g001: Arkadia Facilitates All Major Nodal-Dependent Developmental Events(A–F) In situ hybridization on 6.5 dpc embryos (mid- to late-streak stage) shown as lateral views with anterior to the left and posterior to the right, with probes (A–C), Hex and Brachyury; (D–F), Cer-l. In (C), Akd−/− Nodal+/− embryo showing lack of elongation and proximal positioning of the Brachyury–expressing primitive streak, as well as loss of Hex expression indicating absence of AVE. (E) An Akd−/− embryo with normal Cer-l expression in the AVE but no Cer-l expressing ADE. (F) An Akd−/−, Nodal+/− embryo showing a distal Cer-l-expressing domain indicating lack of AVE migration (three other embryos do not express Cer-l, not shown). Red arrowheads point to a constriction between extraembryonic and embryonic regions indicating incomplete anterior-posterior axis specification.(G–I) Nkx2.5 probe on four-somite stage, 8.5 dpc embryo showing expression in the cardiac precursor tissue in the wild-type (G) and Akd−/− (H), but not in the Akd−/−, Nodal+/− embryo (I).(J–L) Shh probe on 12-somite stage embryos 9.5 dpc viewed from the ventral side with anterior toward the top. In the Akd−/− Nodal+/−embryo (L), note the reduction of Shh expression, the increased severity of the anterior truncation, and the absence of a morphologically distinguishable heart tube. PS, primitive streak; T, Brachyury; CP, cardiac precursors; HF, head folds; NC, notochord; GE, gut endoderm. Bars, 0.1 mm.

Mentions: The phenotype of Arkadia−/− embryos consists of loss of anterior primitive streak derivatives (node, notochord, prechordal plate, and anterior definitive endoderm [ADE]/foregut) leading to anterior patterning defects including head truncations [39,40]. We have shown previously that while Arkadia or Nodal heterozygous (+/−) mice are normal, a small number of double heterozygotes for Arkadia and Nodal recapitulate the Arkadia−/− phenotype [39,40]. This suggested a functional interaction between Arkadia and Nodal. To investigate the extent of Arkadia's role in Nodal signaling, and in additional Nodal-dependent developmental events, we generated Arkadia−/− embryos with only one wild-type copy of the Nodal gene (Akd−/−, Nodal+/−). The majority of Akd−/−, Nodal+/− embryos that were analyzed (n = 19/33) exhibited phenotypes never observed in Arkadia−/− embryos. Using whole mount in situ hybridization, we performed marker analysis to define whether these phenotypes are Nodal-dependent (Figure 1).


Arkadia enhances Nodal/TGF-beta signaling by coupling phospho-Smad2/3 activity and turnover.

Mavrakis KJ, Andrew RL, Lee KL, Petropoulou C, Dixon JE, Navaratnam N, Norris DP, Episkopou V - PLoS Biol. (2007)

Arkadia Facilitates All Major Nodal-Dependent Developmental Events(A–F) In situ hybridization on 6.5 dpc embryos (mid- to late-streak stage) shown as lateral views with anterior to the left and posterior to the right, with probes (A–C), Hex and Brachyury; (D–F), Cer-l. In (C), Akd−/− Nodal+/− embryo showing lack of elongation and proximal positioning of the Brachyury–expressing primitive streak, as well as loss of Hex expression indicating absence of AVE. (E) An Akd−/− embryo with normal Cer-l expression in the AVE but no Cer-l expressing ADE. (F) An Akd−/−, Nodal+/− embryo showing a distal Cer-l-expressing domain indicating lack of AVE migration (three other embryos do not express Cer-l, not shown). Red arrowheads point to a constriction between extraembryonic and embryonic regions indicating incomplete anterior-posterior axis specification.(G–I) Nkx2.5 probe on four-somite stage, 8.5 dpc embryo showing expression in the cardiac precursor tissue in the wild-type (G) and Akd−/− (H), but not in the Akd−/−, Nodal+/− embryo (I).(J–L) Shh probe on 12-somite stage embryos 9.5 dpc viewed from the ventral side with anterior toward the top. In the Akd−/− Nodal+/−embryo (L), note the reduction of Shh expression, the increased severity of the anterior truncation, and the absence of a morphologically distinguishable heart tube. PS, primitive streak; T, Brachyury; CP, cardiac precursors; HF, head folds; NC, notochord; GE, gut endoderm. Bars, 0.1 mm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1808117&req=5

pbio-0050067-g001: Arkadia Facilitates All Major Nodal-Dependent Developmental Events(A–F) In situ hybridization on 6.5 dpc embryos (mid- to late-streak stage) shown as lateral views with anterior to the left and posterior to the right, with probes (A–C), Hex and Brachyury; (D–F), Cer-l. In (C), Akd−/− Nodal+/− embryo showing lack of elongation and proximal positioning of the Brachyury–expressing primitive streak, as well as loss of Hex expression indicating absence of AVE. (E) An Akd−/− embryo with normal Cer-l expression in the AVE but no Cer-l expressing ADE. (F) An Akd−/−, Nodal+/− embryo showing a distal Cer-l-expressing domain indicating lack of AVE migration (three other embryos do not express Cer-l, not shown). Red arrowheads point to a constriction between extraembryonic and embryonic regions indicating incomplete anterior-posterior axis specification.(G–I) Nkx2.5 probe on four-somite stage, 8.5 dpc embryo showing expression in the cardiac precursor tissue in the wild-type (G) and Akd−/− (H), but not in the Akd−/−, Nodal+/− embryo (I).(J–L) Shh probe on 12-somite stage embryos 9.5 dpc viewed from the ventral side with anterior toward the top. In the Akd−/− Nodal+/−embryo (L), note the reduction of Shh expression, the increased severity of the anterior truncation, and the absence of a morphologically distinguishable heart tube. PS, primitive streak; T, Brachyury; CP, cardiac precursors; HF, head folds; NC, notochord; GE, gut endoderm. Bars, 0.1 mm.
Mentions: The phenotype of Arkadia−/− embryos consists of loss of anterior primitive streak derivatives (node, notochord, prechordal plate, and anterior definitive endoderm [ADE]/foregut) leading to anterior patterning defects including head truncations [39,40]. We have shown previously that while Arkadia or Nodal heterozygous (+/−) mice are normal, a small number of double heterozygotes for Arkadia and Nodal recapitulate the Arkadia−/− phenotype [39,40]. This suggested a functional interaction between Arkadia and Nodal. To investigate the extent of Arkadia's role in Nodal signaling, and in additional Nodal-dependent developmental events, we generated Arkadia−/− embryos with only one wild-type copy of the Nodal gene (Akd−/−, Nodal+/−). The majority of Akd−/−, Nodal+/− embryos that were analyzed (n = 19/33) exhibited phenotypes never observed in Arkadia−/− embryos. Using whole mount in situ hybridization, we performed marker analysis to define whether these phenotypes are Nodal-dependent (Figure 1).

Bottom Line: Consistent with this dual function, introduction of Arkadia in homozygous (-/-) embryonic stem cells activates the accumulated and hypoactive P-Smad2/3 at the expense of their abundance.Arkadia-/- cells, like Smad2-/- cells, cannot form foregut and prechordal plate in chimeras, confirming this functional interaction in vivo.As Arkadia overexpression never represses, and in some cells enhances signaling, the degradation of P-Smad2/3 by Arkadia cannot occur prior to their activation in the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Mammalian Neurogenesis, Medical Research Council, Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom.

ABSTRACT
Regulation of transforming growth factor-beta (TGF-beta) signaling is critical in vertebrate development, as several members of the TGF-beta family have been shown to act as morphogens, controlling a variety of cell fate decisions depending on concentration. Little is known about the role of intracellular regulation of the TGF-beta pathway in development. E3 ubiquitin ligases target specific protein substrates for proteasome-mediated degradation, and several are implicated in signaling. We have shown that Arkadia, a nuclear RING-domain E3 ubiquitin ligase, is essential for a subset of Nodal functions in the embryo, but the molecular mechanism of its action in embryonic cells had not been addressed. Here, we find that Arkadia facilitates Nodal signaling broadly in the embryo, and that it is indispensable for cell fates that depend on maximum signaling. Loss of Arkadia in embryonic cells causes nuclear accumulation of phospho-Smad2/3 (P-Smad2/3), the effectors of Nodal signaling; however, these must be repressed or hypoactive as the expression of their direct target genes is reduced or lost. Molecular and functional analysis shows that Arkadia interacts with and ubiquitinates P-Smad2/3 causing their degradation, and that this is via the same domains required for enhancing their activity. Consistent with this dual function, introduction of Arkadia in homozygous (-/-) embryonic stem cells activates the accumulated and hypoactive P-Smad2/3 at the expense of their abundance. Arkadia-/- cells, like Smad2-/- cells, cannot form foregut and prechordal plate in chimeras, confirming this functional interaction in vivo. As Arkadia overexpression never represses, and in some cells enhances signaling, the degradation of P-Smad2/3 by Arkadia cannot occur prior to their activation in the nucleus. Therefore, Arkadia provides a mechanism for signaling termination at the end of the cascade by coupling degradation of P-Smad2/3 with the activation of target gene transcription. This mechanism can account for achieving efficient and maximum Nodal signaling during embryogenesis and for rapid resetting of target gene promoters allowing cells to respond to dynamic changes in extracellular signals.

Show MeSH
Related in: MedlinePlus