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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 results in reduction in both pax6 expression domain and eye size. (A-F) mRNA encoding En2ERT2 was injected at the one-cell stage and the protein was activated at different times of development. Eye size reduction (up to total disappearance) and axis abnormality phenotypes (A) were scored at different times of activation controlled by cyclofen release (B). WT, wild type; epi, epiboly. Diencephalons were measured on flat-mount embryos as the anterior pax6 expression domain (C) and compared to the eye phenotype (mean of the two eye sizes) for each embryo (D), demonstrating the correlation between these two parameters. White asterisks, eye position; red squares, control; blue squares, activated Engrailed. The dashed red line indicates the limit below which eyes were scored as reduced (or absent). All the embryos of this class (yellow box) have a small anterior pax6 expression domain. Additional effects of En2 activation were apparent at the 2-3 somite stage with the loss of the anterior pax6 expression domain (E) and at 4 dpf with the selective loss of the pretectal vmat2:GFP neuronal cluster (red asterisk) (F). Pr, pretectal cluster; Ra, raphe.
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DEV114181F1: Engrailed gain of function results in reduction in both pax6 expression domain and eye size. (A-F) mRNA encoding En2ERT2 was injected at the one-cell stage and the protein was activated at different times of development. Eye size reduction (up to total disappearance) and axis abnormality phenotypes (A) were scored at different times of activation controlled by cyclofen release (B). WT, wild type; epi, epiboly. Diencephalons were measured on flat-mount embryos as the anterior pax6 expression domain (C) and compared to the eye phenotype (mean of the two eye sizes) for each embryo (D), demonstrating the correlation between these two parameters. White asterisks, eye position; red squares, control; blue squares, activated Engrailed. The dashed red line indicates the limit below which eyes were scored as reduced (or absent). All the embryos of this class (yellow box) have a small anterior pax6 expression domain. Additional effects of En2 activation were apparent at the 2-3 somite stage with the loss of the anterior pax6 expression domain (E) and at 4 dpf with the selective loss of the pretectal vmat2:GFP neuronal cluster (red asterisk) (F). Pr, pretectal cluster; Ra, raphe.

Mentions: Injection of eng2b mRNA at the one-cell stage results in an anterior shift of the DMB (Scholpp et al., 2003). Given the multiple functions of Engrailed, we chose to perform inducible gain-of-function experiments by using the precisely controlled timing of Engrailed activation. Following injection of its mRNA at the one-cell stage, the protein of interest fused to ERT2 (Feil et al., 1997) was activated upon UV illumination of caged cyclofen ligand, added in the water bath. We first used an orthologous chicken Engrailed 2 (En2), similar to the zebrafish Engrailed 2 proteins (Eng2a and Eng2b), because its photoactivation has been well characterised in a previous study (Fournier et al., 2013). The amount of injected RNA was calibrated to produce less than 10% of diencephalon malformation in the absence of En2-ERT2 activation (whatever the readout). En2-ERT2 expressed in zebrafish embryos was activated at various stages of development by photorelease of cyclofen. Reduction of the expression domain of the Pax6 diencephalic marker at 1 day post-fertilisation (dpf) (Scholpp et al., 2003) and reduction of eye size (up to total disappearance) at 2 dpf (Ando et al., 2001) are two reported hallmarks of the En gain-of-function phenotype at the DMB. To determine the optimal time window, we first concentrated on eye size for simplicity. En2-ERT2 photo-activation prior to gastrulation induced a widespread insult resulting in abnormal axis and heart development defects in 40% of the embryos, whereas En2-ERT2 photoactivation at 50% and 70% epiboly impaired eye formation without any sign of axis abnormality (Fig. 1A,B). Activation of En2-ERT2 at the beginning of somitogenesis (1-2 somites) induced almost no phenotype (Fig. 1B). Activation at the 50-70% epiboly time window was thus used for all of the following experiments unless specified.Fig. 1.


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 results in reduction in both pax6 expression domain and eye size. (A-F) mRNA encoding En2ERT2 was injected at the one-cell stage and the protein was activated at different times of development. Eye size reduction (up to total disappearance) and axis abnormality phenotypes (A) were scored at different times of activation controlled by cyclofen release (B). WT, wild type; epi, epiboly. Diencephalons were measured on flat-mount embryos as the anterior pax6 expression domain (C) and compared to the eye phenotype (mean of the two eye sizes) for each embryo (D), demonstrating the correlation between these two parameters. White asterisks, eye position; red squares, control; blue squares, activated Engrailed. The dashed red line indicates the limit below which eyes were scored as reduced (or absent). All the embryos of this class (yellow box) have a small anterior pax6 expression domain. Additional effects of En2 activation were apparent at the 2-3 somite stage with the loss of the anterior pax6 expression domain (E) and at 4 dpf with the selective loss of the pretectal vmat2:GFP neuronal cluster (red asterisk) (F). Pr, pretectal cluster; Ra, raphe.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4440920&req=5

DEV114181F1: Engrailed gain of function results in reduction in both pax6 expression domain and eye size. (A-F) mRNA encoding En2ERT2 was injected at the one-cell stage and the protein was activated at different times of development. Eye size reduction (up to total disappearance) and axis abnormality phenotypes (A) were scored at different times of activation controlled by cyclofen release (B). WT, wild type; epi, epiboly. Diencephalons were measured on flat-mount embryos as the anterior pax6 expression domain (C) and compared to the eye phenotype (mean of the two eye sizes) for each embryo (D), demonstrating the correlation between these two parameters. White asterisks, eye position; red squares, control; blue squares, activated Engrailed. The dashed red line indicates the limit below which eyes were scored as reduced (or absent). All the embryos of this class (yellow box) have a small anterior pax6 expression domain. Additional effects of En2 activation were apparent at the 2-3 somite stage with the loss of the anterior pax6 expression domain (E) and at 4 dpf with the selective loss of the pretectal vmat2:GFP neuronal cluster (red asterisk) (F). Pr, pretectal cluster; Ra, raphe.
Mentions: Injection of eng2b mRNA at the one-cell stage results in an anterior shift of the DMB (Scholpp et al., 2003). Given the multiple functions of Engrailed, we chose to perform inducible gain-of-function experiments by using the precisely controlled timing of Engrailed activation. Following injection of its mRNA at the one-cell stage, the protein of interest fused to ERT2 (Feil et al., 1997) was activated upon UV illumination of caged cyclofen ligand, added in the water bath. We first used an orthologous chicken Engrailed 2 (En2), similar to the zebrafish Engrailed 2 proteins (Eng2a and Eng2b), because its photoactivation has been well characterised in a previous study (Fournier et al., 2013). The amount of injected RNA was calibrated to produce less than 10% of diencephalon malformation in the absence of En2-ERT2 activation (whatever the readout). En2-ERT2 expressed in zebrafish embryos was activated at various stages of development by photorelease of cyclofen. Reduction of the expression domain of the Pax6 diencephalic marker at 1 day post-fertilisation (dpf) (Scholpp et al., 2003) and reduction of eye size (up to total disappearance) at 2 dpf (Ando et al., 2001) are two reported hallmarks of the En gain-of-function phenotype at the DMB. To determine the optimal time window, we first concentrated on eye size for simplicity. En2-ERT2 photo-activation prior to gastrulation induced a widespread insult resulting in abnormal axis and heart development defects in 40% of the embryos, whereas En2-ERT2 photoactivation at 50% and 70% epiboly impaired eye formation without any sign of axis abnormality (Fig. 1A,B). Activation of En2-ERT2 at the beginning of somitogenesis (1-2 somites) induced almost no phenotype (Fig. 1B). Activation at the 50-70% epiboly time window was thus used for all of the following experiments unless specified.Fig. 1.

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