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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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Engrailed gain of function involves the paracrine signalling properties of En. (A) Description of the two En2 mutants defective for paracrine signalling. (B) Diencephalon shortening requires En2 transfer. Activation of the En2(5E) transfer-deficient mutant following mRNA injection did not affect the size of the diencephalon. epi, epiboly. (C) Transcriptional activity of wild-type En2 and En2(5E). MAP1B:luciferase reporter plasmid was transfected into HeLa cells with an empty vector (ctrl) or together with the indicated constructs and analysed for luciferase activity after 24 h. a.u., arbitrary units. (D) In vivo ectopic expression of En proteins. Zebrafish embryos were injected at the one-cell stage with the indicated constructs. Following CYC addition at 50% epiboly, cell extracts from 90% epiboly embryos were prepared and analysed by western blotting with polyclonal (anti-En) or monoclonal (anti-tub) antibodies. (E) Inhibition of En2 transfer rescued the phenotype of En2 activation. Zebrafish embryos were injected at the one-cell stage with mRNA encoding En2ERT2 and injected again in the extracellular space at the blastula stage with two different anti-En antibodies. CYC was added at 50% epiboly in the water bath to activate the protein. Eye phenotypes were scored at 2 dpf. The error bars represent statistical errors. ***P<0.001.
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DEV114181F2: Engrailed gain of function involves the paracrine signalling properties of En. (A) Description of the two En2 mutants defective for paracrine signalling. (B) Diencephalon shortening requires En2 transfer. Activation of the En2(5E) transfer-deficient mutant following mRNA injection did not affect the size of the diencephalon. epi, epiboly. (C) Transcriptional activity of wild-type En2 and En2(5E). MAP1B:luciferase reporter plasmid was transfected into HeLa cells with an empty vector (ctrl) or together with the indicated constructs and analysed for luciferase activity after 24 h. a.u., arbitrary units. (D) In vivo ectopic expression of En proteins. Zebrafish embryos were injected at the one-cell stage with the indicated constructs. Following CYC addition at 50% epiboly, cell extracts from 90% epiboly embryos were prepared and analysed by western blotting with polyclonal (anti-En) or monoclonal (anti-tub) antibodies. (E) Inhibition of En2 transfer rescued the phenotype of En2 activation. Zebrafish embryos were injected at the one-cell stage with mRNA encoding En2ERT2 and injected again in the extracellular space at the blastula stage with two different anti-En antibodies. CYC was added at 50% epiboly in the water bath to activate the protein. Eye phenotypes were scored at 2 dpf. The error bars represent statistical errors. ***P<0.001.

Mentions: Specific motifs within Engrailed regulate the transfer of the protein, either through its secretion or its internalisation (Joliot et al., 1998; Maizel et al., 1999, 2002) (Fig. 2A), and En2 paracrine signalling activity is lost upon mutation of the internalisation motif (Brunet et al., 2005). A mutation preventing secretion, En2(5E)-ERT2 (Maizel et al., 2002), was introduced in the sequence of En2-ERT2, and its impact on Engrailed activity was analysed in the gain-of-function assay described above (Fig. 1). Activation of either mutated protein at the dome stage induced abnormal heart and axis development in ∼40% of the embryos (Fig. 2B), indicating that this widespread effect did not involve Engrailed 2 intercellular transfer. By contrast, En2(5E)-ERT2 was not able to induce an eye phenotype when photo-activated at 70% epiboly (Fig. 2B), suggesting that intercellular transfer of Engrailed 2 is involved in brain patterning. Besides the reported effect of this mutation on the transfer process, we could not exclude that other functions could be affected as well, including at the transcriptional level. To test this hypothesis, the transcriptional activities of Engrailed and of its mutated form were compared on the Map1b promoter fused to a luciferase reporter in HeLa cells (Fig. 2C). A 2 kilobase long fragment of the Map1b rat promoter is regulated by Engrailed in cell culture and in vivo in vertebrates (Montesinos et al., 2001). En2 and En2(5E) were equally competent for MAP1B activation, increasing luciferase expression by sevenfold. We also verified the expression of the two proteins in zebrafish. Following RNA injection at the one-cell stage and cyclofen activation, cell extracts from injected embryos expressing En2-ERT2 or the mutant were analysed by western blotting with a polyclonal anti-Engrailed antibody (Fig. 2D). En2-ERT2 and En2(5E)-ERT2 were expressed at similar levels.Fig. 2.


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)

Engrailed gain of function involves the paracrine signalling properties of En. (A) Description of the two En2 mutants defective for paracrine signalling. (B) Diencephalon shortening requires En2 transfer. Activation of the En2(5E) transfer-deficient mutant following mRNA injection did not affect the size of the diencephalon. epi, epiboly. (C) Transcriptional activity of wild-type En2 and En2(5E). MAP1B:luciferase reporter plasmid was transfected into HeLa cells with an empty vector (ctrl) or together with the indicated constructs and analysed for luciferase activity after 24 h. a.u., arbitrary units. (D) In vivo ectopic expression of En proteins. Zebrafish embryos were injected at the one-cell stage with the indicated constructs. Following CYC addition at 50% epiboly, cell extracts from 90% epiboly embryos were prepared and analysed by western blotting with polyclonal (anti-En) or monoclonal (anti-tub) antibodies. (E) Inhibition of En2 transfer rescued the phenotype of En2 activation. Zebrafish embryos were injected at the one-cell stage with mRNA encoding En2ERT2 and injected again in the extracellular space at the blastula stage with two different anti-En antibodies. CYC was added at 50% epiboly in the water bath to activate the protein. Eye phenotypes were scored at 2 dpf. The error bars represent statistical errors. ***P<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4440920&req=5

DEV114181F2: Engrailed gain of function involves the paracrine signalling properties of En. (A) Description of the two En2 mutants defective for paracrine signalling. (B) Diencephalon shortening requires En2 transfer. Activation of the En2(5E) transfer-deficient mutant following mRNA injection did not affect the size of the diencephalon. epi, epiboly. (C) Transcriptional activity of wild-type En2 and En2(5E). MAP1B:luciferase reporter plasmid was transfected into HeLa cells with an empty vector (ctrl) or together with the indicated constructs and analysed for luciferase activity after 24 h. a.u., arbitrary units. (D) In vivo ectopic expression of En proteins. Zebrafish embryos were injected at the one-cell stage with the indicated constructs. Following CYC addition at 50% epiboly, cell extracts from 90% epiboly embryos were prepared and analysed by western blotting with polyclonal (anti-En) or monoclonal (anti-tub) antibodies. (E) Inhibition of En2 transfer rescued the phenotype of En2 activation. Zebrafish embryos were injected at the one-cell stage with mRNA encoding En2ERT2 and injected again in the extracellular space at the blastula stage with two different anti-En antibodies. CYC was added at 50% epiboly in the water bath to activate the protein. Eye phenotypes were scored at 2 dpf. The error bars represent statistical errors. ***P<0.001.
Mentions: Specific motifs within Engrailed regulate the transfer of the protein, either through its secretion or its internalisation (Joliot et al., 1998; Maizel et al., 1999, 2002) (Fig. 2A), and En2 paracrine signalling activity is lost upon mutation of the internalisation motif (Brunet et al., 2005). A mutation preventing secretion, En2(5E)-ERT2 (Maizel et al., 2002), was introduced in the sequence of En2-ERT2, and its impact on Engrailed activity was analysed in the gain-of-function assay described above (Fig. 1). Activation of either mutated protein at the dome stage induced abnormal heart and axis development in ∼40% of the embryos (Fig. 2B), indicating that this widespread effect did not involve Engrailed 2 intercellular transfer. By contrast, En2(5E)-ERT2 was not able to induce an eye phenotype when photo-activated at 70% epiboly (Fig. 2B), suggesting that intercellular transfer of Engrailed 2 is involved in brain patterning. Besides the reported effect of this mutation on the transfer process, we could not exclude that other functions could be affected as well, including at the transcriptional level. To test this hypothesis, the transcriptional activities of Engrailed and of its mutated form were compared on the Map1b promoter fused to a luciferase reporter in HeLa cells (Fig. 2C). A 2 kilobase long fragment of the Map1b rat promoter is regulated by Engrailed in cell culture and in vivo in vertebrates (Montesinos et al., 2001). En2 and En2(5E) were equally competent for MAP1B activation, increasing luciferase expression by sevenfold. We also verified the expression of the two proteins in zebrafish. Following RNA injection at the one-cell stage and cyclofen activation, cell extracts from injected embryos expressing En2-ERT2 or the mutant were analysed by western blotting with a polyclonal anti-Engrailed antibody (Fig. 2D). En2-ERT2 and En2(5E)-ERT2 were expressed at similar levels.Fig. 2.

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.

Show MeSH
Related in: MedlinePlus