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Control of brain patterning by Engrailed paracrine transfer: a new function of the Pbx interaction domain.

Rampon C, Gauron C, Lin T, Meda F, Dupont E, Cosson A, Ipendey E, Frerot A, Aujard I, Le Saux T, Bensimon D, Jullien L, Volovitch M, Vriz S, Joliot A - Development (2015)

Bottom Line: In light of recent reports on the paracrine activity of homeoproteins, including Engrailed, we asked whether Engrailed intercellular transfer was also involved in brain patterning and boundary formation.Both zebrafish Eng2a and Eng2b are competent for intercellular transfer in vivo, but only extracellular endogenous Eng2b, and not Eng2a, participates in DMB positioning.In addition, disruption of the Pbx-interacting motif in Engrailed, known to strongly reduce the gain-of-function phenotype, also downregulates Engrailed transfer, thus revealing an unsuspected participation of the Pbx interaction domain in this pathway.

View Article: PubMed Central - PubMed

Affiliation: Université Paris Diderot, Sorbonne Paris Cité, Paris 75205, Cedex 13, France Center for Interdisciplinary Research in Biology (CIRB) - CNRS UMR 7241, INSERM U1050, Labex MemoLife, PSL Research University, Collège de France, Paris F-75005, France.

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Zebrafish Engrailed 2a and 2b are able to transfer between cells in vivo and ex vivo. (A,B) In vivo intercellular transfer of En2 protein. (A) The En2ERT2-P2A-mCherry plasmid was injected at the one-cell stage (10 ng/µl). Embryos were fixed at 90% epiboly and immunostained for En2ERT2 (green) and mCherry (red). En2 transfer was visualised by the presence of En2ERT2 staining (green) in non-injected cells (negative for mCherry staining). (B) Cells expressing mCherry (red) were transplanted into 30% epiboly embryos ubiquitously expressing En2ERT2. En2ERT2 transfer was revealed by the detection of EnERT2 signal (green) in mCherry-positive cells (yellow cells, arrow). Scale bars: 20 µm. (C,D) Internalisation of Eng2a and 2b in HEK293 cells. Following extracellular addition, internalisation of fluorescein-labelled En2, Eng2a or Eng2b protein (green) was visualised (C) and quantified (D) after 1 h incubation at 37°C in the presence of 0.4% Trypan Blue (red) to quench extracellular fluorescence. MFI, mean fluorescence intensity. (E) Secretion of Eng2a and 2b. HEK293 cells expressing the indicated proteins under the control of doxycycline were cultured for 24 h and cell surface accumulation of the secreted protein was monitored by flow cytometry. ***P<0.001. (F) Paracrine activity of Eng2a and Eng2b proteins. mRNA encoding En2ERT2, Eng2aERT2 or Eng2bERT2 were injected at the one-cell stage, the protein was activated with CYC at 50% epiboly and eye defects were scored at 30 hpf. The error bars represent statistical errors (F) or s.e.m. (D,E).
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DEV114181F3: Zebrafish Engrailed 2a and 2b are able to transfer between cells in vivo and ex vivo. (A,B) In vivo intercellular transfer of En2 protein. (A) The En2ERT2-P2A-mCherry plasmid was injected at the one-cell stage (10 ng/µl). Embryos were fixed at 90% epiboly and immunostained for En2ERT2 (green) and mCherry (red). En2 transfer was visualised by the presence of En2ERT2 staining (green) in non-injected cells (negative for mCherry staining). (B) Cells expressing mCherry (red) were transplanted into 30% epiboly embryos ubiquitously expressing En2ERT2. En2ERT2 transfer was revealed by the detection of EnERT2 signal (green) in mCherry-positive cells (yellow cells, arrow). Scale bars: 20 µm. (C,D) Internalisation of Eng2a and 2b in HEK293 cells. Following extracellular addition, internalisation of fluorescein-labelled En2, Eng2a or Eng2b protein (green) was visualised (C) and quantified (D) after 1 h incubation at 37°C in the presence of 0.4% Trypan Blue (red) to quench extracellular fluorescence. MFI, mean fluorescence intensity. (E) Secretion of Eng2a and 2b. HEK293 cells expressing the indicated proteins under the control of doxycycline were cultured for 24 h and cell surface accumulation of the secreted protein was monitored by flow cytometry. ***P<0.001. (F) Paracrine activity of Eng2a and Eng2b proteins. mRNA encoding En2ERT2, Eng2aERT2 or Eng2bERT2 were injected at the one-cell stage, the protein was activated with CYC at 50% epiboly and eye defects were scored at 30 hpf. The error bars represent statistical errors (F) or s.e.m. (D,E).

Mentions: Homeoprotein paracrine activity cannot be dissociated from its ability to transfer between cells, thus it was crucial to confirm the actual intercellular transfer of the inducible form of En2-ERT2 in the fish. We used two complementary approaches to directly visualise the transfer process in vivo. In the first one, En2-ERT2-expressing cells were specifically labelled by tandem translation of mCherry from the same mRNA molecule (En2-ERT2-P2A-mCherry). Injection of plasmid DNA at the one-cell stage led to mosaic expression of the injected construct (encoding both mCherry and En2-ERT2), as classically reported. Embryos treated with cyclofen at 50% epiboly to activate En2-ERT2 were fixed at 90% epiboly and processed for immunodetection of En2-ERT2 and mCherry. En2-ERT2 was detected in non-expressing cells characterised by the absence of mCherry staining, suggesting En2-ERT2 intercellular transfer (Fig. 3A; supplementary material Fig. S2 for separate channels). In the same experimental set-up, following injection of a plasmid DNA expressing the mutated protein En2(5E)-ERT2-P2A-mCherry, the in vivo intercellular transfer of the mutated form of En2 was drastically reduced (supplementary material Fig. S2). To unambiguously confirm in vivo intercellular transfer of En2 with the reverse strategy, mCherry-expressing cells were grafted into En2-ERT2-expressing embryos (Fig. 3B). Cells co-labelled with mCherry and En2-ERT2 were detected (Fig. 3B). This unambiguously demonstrates the intercellular transfer of En2-ERT2 between 50% and 90% epiboly, at the time of its paracrine action on brain patterning.Fig. 3.


Control of brain patterning by Engrailed paracrine transfer: a new function of the Pbx interaction domain.

Rampon C, Gauron C, Lin T, Meda F, Dupont E, Cosson A, Ipendey E, Frerot A, Aujard I, Le Saux T, Bensimon D, Jullien L, Volovitch M, Vriz S, Joliot A - Development (2015)

Zebrafish Engrailed 2a and 2b are able to transfer between cells in vivo and ex vivo. (A,B) In vivo intercellular transfer of En2 protein. (A) The En2ERT2-P2A-mCherry plasmid was injected at the one-cell stage (10 ng/µl). Embryos were fixed at 90% epiboly and immunostained for En2ERT2 (green) and mCherry (red). En2 transfer was visualised by the presence of En2ERT2 staining (green) in non-injected cells (negative for mCherry staining). (B) Cells expressing mCherry (red) were transplanted into 30% epiboly embryos ubiquitously expressing En2ERT2. En2ERT2 transfer was revealed by the detection of EnERT2 signal (green) in mCherry-positive cells (yellow cells, arrow). Scale bars: 20 µm. (C,D) Internalisation of Eng2a and 2b in HEK293 cells. Following extracellular addition, internalisation of fluorescein-labelled En2, Eng2a or Eng2b protein (green) was visualised (C) and quantified (D) after 1 h incubation at 37°C in the presence of 0.4% Trypan Blue (red) to quench extracellular fluorescence. MFI, mean fluorescence intensity. (E) Secretion of Eng2a and 2b. HEK293 cells expressing the indicated proteins under the control of doxycycline were cultured for 24 h and cell surface accumulation of the secreted protein was monitored by flow cytometry. ***P<0.001. (F) Paracrine activity of Eng2a and Eng2b proteins. mRNA encoding En2ERT2, Eng2aERT2 or Eng2bERT2 were injected at the one-cell stage, the protein was activated with CYC at 50% epiboly and eye defects were scored at 30 hpf. The error bars represent statistical errors (F) or s.e.m. (D,E).
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DEV114181F3: Zebrafish Engrailed 2a and 2b are able to transfer between cells in vivo and ex vivo. (A,B) In vivo intercellular transfer of En2 protein. (A) The En2ERT2-P2A-mCherry plasmid was injected at the one-cell stage (10 ng/µl). Embryos were fixed at 90% epiboly and immunostained for En2ERT2 (green) and mCherry (red). En2 transfer was visualised by the presence of En2ERT2 staining (green) in non-injected cells (negative for mCherry staining). (B) Cells expressing mCherry (red) were transplanted into 30% epiboly embryos ubiquitously expressing En2ERT2. En2ERT2 transfer was revealed by the detection of EnERT2 signal (green) in mCherry-positive cells (yellow cells, arrow). Scale bars: 20 µm. (C,D) Internalisation of Eng2a and 2b in HEK293 cells. Following extracellular addition, internalisation of fluorescein-labelled En2, Eng2a or Eng2b protein (green) was visualised (C) and quantified (D) after 1 h incubation at 37°C in the presence of 0.4% Trypan Blue (red) to quench extracellular fluorescence. MFI, mean fluorescence intensity. (E) Secretion of Eng2a and 2b. HEK293 cells expressing the indicated proteins under the control of doxycycline were cultured for 24 h and cell surface accumulation of the secreted protein was monitored by flow cytometry. ***P<0.001. (F) Paracrine activity of Eng2a and Eng2b proteins. mRNA encoding En2ERT2, Eng2aERT2 or Eng2bERT2 were injected at the one-cell stage, the protein was activated with CYC at 50% epiboly and eye defects were scored at 30 hpf. The error bars represent statistical errors (F) or s.e.m. (D,E).
Mentions: Homeoprotein paracrine activity cannot be dissociated from its ability to transfer between cells, thus it was crucial to confirm the actual intercellular transfer of the inducible form of En2-ERT2 in the fish. We used two complementary approaches to directly visualise the transfer process in vivo. In the first one, En2-ERT2-expressing cells were specifically labelled by tandem translation of mCherry from the same mRNA molecule (En2-ERT2-P2A-mCherry). Injection of plasmid DNA at the one-cell stage led to mosaic expression of the injected construct (encoding both mCherry and En2-ERT2), as classically reported. Embryos treated with cyclofen at 50% epiboly to activate En2-ERT2 were fixed at 90% epiboly and processed for immunodetection of En2-ERT2 and mCherry. En2-ERT2 was detected in non-expressing cells characterised by the absence of mCherry staining, suggesting En2-ERT2 intercellular transfer (Fig. 3A; supplementary material Fig. S2 for separate channels). In the same experimental set-up, following injection of a plasmid DNA expressing the mutated protein En2(5E)-ERT2-P2A-mCherry, the in vivo intercellular transfer of the mutated form of En2 was drastically reduced (supplementary material Fig. S2). To unambiguously confirm in vivo intercellular transfer of En2 with the reverse strategy, mCherry-expressing cells were grafted into En2-ERT2-expressing embryos (Fig. 3B). Cells co-labelled with mCherry and En2-ERT2 were detected (Fig. 3B). This unambiguously demonstrates the intercellular transfer of En2-ERT2 between 50% and 90% epiboly, at the time of its paracrine action on brain patterning.Fig. 3.

Bottom Line: In light of recent reports on the paracrine activity of homeoproteins, including Engrailed, we asked whether Engrailed intercellular transfer was also involved in brain patterning and boundary formation.Both zebrafish Eng2a and Eng2b are competent for intercellular transfer in vivo, but only extracellular endogenous Eng2b, and not Eng2a, participates in DMB positioning.In addition, disruption of the Pbx-interacting motif in Engrailed, known to strongly reduce the gain-of-function phenotype, also downregulates Engrailed transfer, thus revealing an unsuspected participation of the Pbx interaction domain in this pathway.

View Article: PubMed Central - PubMed

Affiliation: Université Paris Diderot, Sorbonne Paris Cité, Paris 75205, Cedex 13, France Center for Interdisciplinary Research in Biology (CIRB) - CNRS UMR 7241, INSERM U1050, Labex MemoLife, PSL Research University, Collège de France, Paris F-75005, France.

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