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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 Regulates Nodal Target Gene Expression and Phenocopies Smad2 in DevelopmentIn situ hybridization with Nodal probe on 6.5 dpc (A and B) and 8.5 dpc (C–H) embryos; (C–H) embryos are shown as ventral views with anterior to the top and left toward the right; (A, C, and G) wild-type (+/+); (B, E, and F) Arkadia−/− (−/−); and (D and H) tetraploid chimeras (TC). Compare (A) and (B) to see reduced Nodal expression in the mutant embryo. The TC shows normal expression of Nodal around the node (D) and reduced expression in the left-LPM (H), while the −/− embryo has no node or Nodal expression (E and F). Lefty2 probe on 8.5 dpc embryos (I and J) showing loss of expression in the left-LPM in the TC; Pitx2 (K and L) on 9.5 dpc embryos showing reduction of expression in the TC; and Hex (M and N) on 8.5 dpc showing loss of expression in the foregut pocket of the TC. Heart-level sections from 9.5 dpc (O) and 8.5 dpc (P) mosaic embryos (chimeras) stained for β-galactosidase activity (blue). Note selective contribution of wild-type cells in the foregut. Black arrowheads point at the visceral endoderm; black arrow, points at the node; red dots indicate the left-LPM; red arrows point at the head folds; red arrowheads indicate the definitive endoderm at the level of the foregut. Bars, 0.1 mm.
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pbio-0050067-g007: Arkadia Regulates Nodal Target Gene Expression and Phenocopies Smad2 in DevelopmentIn situ hybridization with Nodal probe on 6.5 dpc (A and B) and 8.5 dpc (C–H) embryos; (C–H) embryos are shown as ventral views with anterior to the top and left toward the right; (A, C, and G) wild-type (+/+); (B, E, and F) Arkadia−/− (−/−); and (D and H) tetraploid chimeras (TC). Compare (A) and (B) to see reduced Nodal expression in the mutant embryo. The TC shows normal expression of Nodal around the node (D) and reduced expression in the left-LPM (H), while the −/− embryo has no node or Nodal expression (E and F). Lefty2 probe on 8.5 dpc embryos (I and J) showing loss of expression in the left-LPM in the TC; Pitx2 (K and L) on 9.5 dpc embryos showing reduction of expression in the TC; and Hex (M and N) on 8.5 dpc showing loss of expression in the foregut pocket of the TC. Heart-level sections from 9.5 dpc (O) and 8.5 dpc (P) mosaic embryos (chimeras) stained for β-galactosidase activity (blue). Note selective contribution of wild-type cells in the foregut. Black arrowheads point at the visceral endoderm; black arrow, points at the node; red dots indicate the left-LPM; red arrows point at the head folds; red arrowheads indicate the definitive endoderm at the level of the foregut. Bars, 0.1 mm.

Mentions: All the above analysis shows that in ES cells Arkadia functions as a coactivator of P-Smad2/3 transcription. To address whether this also occurs in other embryonic cells and if this is the underlying cause of the Arkadia−/− phenotype in the embryo, we examined the expression of known Smad2 target genes in Arkadia−/− embryos. The FoxH1/P-Smad2 complex directly upregulates the Nodal gene and is responsible for its tissue-specific expression in the visceral endoderm (VE) at pre-gastrulation stages [10,12,54]. Whole mount in situ hybridization, as expected, revealed that in Arkadia−/− embryos (n = 10) Nodal expression is dramatically reduced in the epiblast and almost lost in the VE (Figure 7A and 7B).


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 Regulates Nodal Target Gene Expression and Phenocopies Smad2 in DevelopmentIn situ hybridization with Nodal probe on 6.5 dpc (A and B) and 8.5 dpc (C–H) embryos; (C–H) embryos are shown as ventral views with anterior to the top and left toward the right; (A, C, and G) wild-type (+/+); (B, E, and F) Arkadia−/− (−/−); and (D and H) tetraploid chimeras (TC). Compare (A) and (B) to see reduced Nodal expression in the mutant embryo. The TC shows normal expression of Nodal around the node (D) and reduced expression in the left-LPM (H), while the −/− embryo has no node or Nodal expression (E and F). Lefty2 probe on 8.5 dpc embryos (I and J) showing loss of expression in the left-LPM in the TC; Pitx2 (K and L) on 9.5 dpc embryos showing reduction of expression in the TC; and Hex (M and N) on 8.5 dpc showing loss of expression in the foregut pocket of the TC. Heart-level sections from 9.5 dpc (O) and 8.5 dpc (P) mosaic embryos (chimeras) stained for β-galactosidase activity (blue). Note selective contribution of wild-type cells in the foregut. Black arrowheads point at the visceral endoderm; black arrow, points at the node; red dots indicate the left-LPM; red arrows point at the head folds; red arrowheads indicate the definitive endoderm at the level of the foregut. Bars, 0.1 mm.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-0050067-g007: Arkadia Regulates Nodal Target Gene Expression and Phenocopies Smad2 in DevelopmentIn situ hybridization with Nodal probe on 6.5 dpc (A and B) and 8.5 dpc (C–H) embryos; (C–H) embryos are shown as ventral views with anterior to the top and left toward the right; (A, C, and G) wild-type (+/+); (B, E, and F) Arkadia−/− (−/−); and (D and H) tetraploid chimeras (TC). Compare (A) and (B) to see reduced Nodal expression in the mutant embryo. The TC shows normal expression of Nodal around the node (D) and reduced expression in the left-LPM (H), while the −/− embryo has no node or Nodal expression (E and F). Lefty2 probe on 8.5 dpc embryos (I and J) showing loss of expression in the left-LPM in the TC; Pitx2 (K and L) on 9.5 dpc embryos showing reduction of expression in the TC; and Hex (M and N) on 8.5 dpc showing loss of expression in the foregut pocket of the TC. Heart-level sections from 9.5 dpc (O) and 8.5 dpc (P) mosaic embryos (chimeras) stained for β-galactosidase activity (blue). Note selective contribution of wild-type cells in the foregut. Black arrowheads point at the visceral endoderm; black arrow, points at the node; red dots indicate the left-LPM; red arrows point at the head folds; red arrowheads indicate the definitive endoderm at the level of the foregut. Bars, 0.1 mm.
Mentions: All the above analysis shows that in ES cells Arkadia functions as a coactivator of P-Smad2/3 transcription. To address whether this also occurs in other embryonic cells and if this is the underlying cause of the Arkadia−/− phenotype in the embryo, we examined the expression of known Smad2 target genes in Arkadia−/− embryos. The FoxH1/P-Smad2 complex directly upregulates the Nodal gene and is responsible for its tissue-specific expression in the visceral endoderm (VE) at pre-gastrulation stages [10,12,54]. Whole mount in situ hybridization, as expected, revealed that in Arkadia−/− embryos (n = 10) Nodal expression is dramatically reduced in the epiblast and almost lost in the VE (Figure 7A and 7B).

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