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De novo neurogenesis by targeted expression of atoh7 to Müller glia cells.

Lust K, Sinn R, Pérez Saturnino A, Centanin L, Wittbrodt J - Development (2016)

Bottom Line: The resulting neurogenic clusters differentiate in vivo into various retinal neurons.We use signaling reporters to demonstrate that the Atoh7-induced regeneration-like response of MG cells is mimicked by Notch, resembling the behavior of early progenitors during retinogenesis.Our results uncover a new role for Atoh7 as a universal neurogenic factor, and illustrate how signaling modules are re-employed in diverse contexts to trigger different biological responses.

View Article: PubMed Central - PubMed

Affiliation: Centre for Organismal Studies (COS) Heidelberg, Im Neuenheimer Feld 230, Heidelberg 69120, Germany The Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology (HBIGS), Heidelberg University, Heidelberg, Germany.

No MeSH data available.


Related in: MedlinePlus

Targeted atoh7 drivesneurogenic cluster formation ofclonal MG cells. (A-A″) Induction scheme (A) and constructs used for targeted induction of atoh7 (A′) and clonal labeling (A″). The rx2::ERT2Cre transgenic line mediates excision or inversion events in the Gaudírx2BBW2.1 cassette that result in three possible FP readouts (A″), which will be expressed by daughter cells that maintain the MG cell fate. (B-C″) In the central retina, recombination is targeted to MG cells and photoreceptors (B,C). In control retinae, MG cells display a compact nucleus and processes spanning from the apical to the basal domains of the neural retina (n=39 clones from three fish, data obtained from two independent experiments) (B′,B″). In atoh7-expressing fish, MG cells form clusters containing several nuclei (arrowheads) (n=41 clones, from four fish, data obtained from two independent experiments) (C-C″). (D) Quantification of numbers of nuclei per cluster shows that targeted induction of atoh7 results in clusters containing more nuclei than those of controls. (E) Constructs used for targeted induction of atoh7 and nuclear clonal labeling. The rx2::ERT2Cre transgenic line mediates excision of DSRed resulting in nuclear EGFP expression. (F-F″) Without induction of atoh7, single nuclear EGFP (white/green)-labeled GS-positive cells (white/magenta) (arrowhead) are present in the INL. (G-G″) Induction of atoh7 induces the formation of nuclear EGFP (white/green)-labeled clusters (open arrowheads). One nucleus (arrowhead) is positive for GS (white/magenta) (n=6 fish, data obtained from two independent experiments). Scale bars: 20 µm.
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DEV135905F6: Targeted atoh7 drivesneurogenic cluster formation ofclonal MG cells. (A-A″) Induction scheme (A) and constructs used for targeted induction of atoh7 (A′) and clonal labeling (A″). The rx2::ERT2Cre transgenic line mediates excision or inversion events in the Gaudírx2BBW2.1 cassette that result in three possible FP readouts (A″), which will be expressed by daughter cells that maintain the MG cell fate. (B-C″) In the central retina, recombination is targeted to MG cells and photoreceptors (B,C). In control retinae, MG cells display a compact nucleus and processes spanning from the apical to the basal domains of the neural retina (n=39 clones from three fish, data obtained from two independent experiments) (B′,B″). In atoh7-expressing fish, MG cells form clusters containing several nuclei (arrowheads) (n=41 clones, from four fish, data obtained from two independent experiments) (C-C″). (D) Quantification of numbers of nuclei per cluster shows that targeted induction of atoh7 results in clusters containing more nuclei than those of controls. (E) Constructs used for targeted induction of atoh7 and nuclear clonal labeling. The rx2::ERT2Cre transgenic line mediates excision of DSRed resulting in nuclear EGFP expression. (F-F″) Without induction of atoh7, single nuclear EGFP (white/green)-labeled GS-positive cells (white/magenta) (arrowhead) are present in the INL. (G-G″) Induction of atoh7 induces the formation of nuclear EGFP (white/green)-labeled clusters (open arrowheads). One nucleus (arrowhead) is positive for GS (white/magenta) (n=6 fish, data obtained from two independent experiments). Scale bars: 20 µm.

Mentions: To investigate the expansion and the lineage of the re-activated MG cells, we used the Gaudí toolkit, which allows multicolor labeling of progenitors, stem cells and their descendants via Cre/LoxP mediated recombination (Fig. 6A-A″) (Centanin et al., 2014; Livet et al., 2007). To follow the expansion of MG cells, we induced stochastic and sparse recombination by a mild tamoxifen induction (see scheme of treatment in Fig. 6A) of the rx2::ERT2Cre line in the background of an rx2-driven Gaudírx2BBW2.1 recombination reporter (Fig. 6A″). This approach labels individual MG cells and those descendants that maintained Rx2 expression. After a chase of 4 weeks, we observed predominantly single cells and clusters of two cells among the labeled MG cells of control retinae (Fig. 6B,D). By contrast, when clonal labeling was combined with the triggering of atoh7 expression, the majority of MG cells formed clonal clusters of three or more nuclei (Fig. 6C,D). To achieve exclusively nuclear labeling, we used rx2::ERT2GaudíRSG fish in combination with atoh7 inductions to analyze cluster formation (Fig. 6E). Supporting the findings of the previous experiment, control fish displayed single nuclear EGFP-labeled MG cells (Fig. 6F-F″), whereas upon targeted induction of atoh7 the formation of multicellular neurogenic clusters is triggered, as highlighted by nuclear-tagged EGFP (Fig. 6G-G″). These data demonstrate that the targeted induction of atoh7 in MG cells triggers the formation of neurogenic clusters highly reminiscent of the neurogenic clusters formed by zebrafish MG cells in response to intense light treatment or mechanical injuries (Fausett and Goldman, 2006; Kassen et al., 2007; Thummel et al., 2008).Fig. 6.


De novo neurogenesis by targeted expression of atoh7 to Müller glia cells.

Lust K, Sinn R, Pérez Saturnino A, Centanin L, Wittbrodt J - Development (2016)

Targeted atoh7 drivesneurogenic cluster formation ofclonal MG cells. (A-A″) Induction scheme (A) and constructs used for targeted induction of atoh7 (A′) and clonal labeling (A″). The rx2::ERT2Cre transgenic line mediates excision or inversion events in the Gaudírx2BBW2.1 cassette that result in three possible FP readouts (A″), which will be expressed by daughter cells that maintain the MG cell fate. (B-C″) In the central retina, recombination is targeted to MG cells and photoreceptors (B,C). In control retinae, MG cells display a compact nucleus and processes spanning from the apical to the basal domains of the neural retina (n=39 clones from three fish, data obtained from two independent experiments) (B′,B″). In atoh7-expressing fish, MG cells form clusters containing several nuclei (arrowheads) (n=41 clones, from four fish, data obtained from two independent experiments) (C-C″). (D) Quantification of numbers of nuclei per cluster shows that targeted induction of atoh7 results in clusters containing more nuclei than those of controls. (E) Constructs used for targeted induction of atoh7 and nuclear clonal labeling. The rx2::ERT2Cre transgenic line mediates excision of DSRed resulting in nuclear EGFP expression. (F-F″) Without induction of atoh7, single nuclear EGFP (white/green)-labeled GS-positive cells (white/magenta) (arrowhead) are present in the INL. (G-G″) Induction of atoh7 induces the formation of nuclear EGFP (white/green)-labeled clusters (open arrowheads). One nucleus (arrowhead) is positive for GS (white/magenta) (n=6 fish, data obtained from two independent experiments). Scale bars: 20 µm.
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Related In: Results  -  Collection

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DEV135905F6: Targeted atoh7 drivesneurogenic cluster formation ofclonal MG cells. (A-A″) Induction scheme (A) and constructs used for targeted induction of atoh7 (A′) and clonal labeling (A″). The rx2::ERT2Cre transgenic line mediates excision or inversion events in the Gaudírx2BBW2.1 cassette that result in three possible FP readouts (A″), which will be expressed by daughter cells that maintain the MG cell fate. (B-C″) In the central retina, recombination is targeted to MG cells and photoreceptors (B,C). In control retinae, MG cells display a compact nucleus and processes spanning from the apical to the basal domains of the neural retina (n=39 clones from three fish, data obtained from two independent experiments) (B′,B″). In atoh7-expressing fish, MG cells form clusters containing several nuclei (arrowheads) (n=41 clones, from four fish, data obtained from two independent experiments) (C-C″). (D) Quantification of numbers of nuclei per cluster shows that targeted induction of atoh7 results in clusters containing more nuclei than those of controls. (E) Constructs used for targeted induction of atoh7 and nuclear clonal labeling. The rx2::ERT2Cre transgenic line mediates excision of DSRed resulting in nuclear EGFP expression. (F-F″) Without induction of atoh7, single nuclear EGFP (white/green)-labeled GS-positive cells (white/magenta) (arrowhead) are present in the INL. (G-G″) Induction of atoh7 induces the formation of nuclear EGFP (white/green)-labeled clusters (open arrowheads). One nucleus (arrowhead) is positive for GS (white/magenta) (n=6 fish, data obtained from two independent experiments). Scale bars: 20 µm.
Mentions: To investigate the expansion and the lineage of the re-activated MG cells, we used the Gaudí toolkit, which allows multicolor labeling of progenitors, stem cells and their descendants via Cre/LoxP mediated recombination (Fig. 6A-A″) (Centanin et al., 2014; Livet et al., 2007). To follow the expansion of MG cells, we induced stochastic and sparse recombination by a mild tamoxifen induction (see scheme of treatment in Fig. 6A) of the rx2::ERT2Cre line in the background of an rx2-driven Gaudírx2BBW2.1 recombination reporter (Fig. 6A″). This approach labels individual MG cells and those descendants that maintained Rx2 expression. After a chase of 4 weeks, we observed predominantly single cells and clusters of two cells among the labeled MG cells of control retinae (Fig. 6B,D). By contrast, when clonal labeling was combined with the triggering of atoh7 expression, the majority of MG cells formed clonal clusters of three or more nuclei (Fig. 6C,D). To achieve exclusively nuclear labeling, we used rx2::ERT2GaudíRSG fish in combination with atoh7 inductions to analyze cluster formation (Fig. 6E). Supporting the findings of the previous experiment, control fish displayed single nuclear EGFP-labeled MG cells (Fig. 6F-F″), whereas upon targeted induction of atoh7 the formation of multicellular neurogenic clusters is triggered, as highlighted by nuclear-tagged EGFP (Fig. 6G-G″). These data demonstrate that the targeted induction of atoh7 in MG cells triggers the formation of neurogenic clusters highly reminiscent of the neurogenic clusters formed by zebrafish MG cells in response to intense light treatment or mechanical injuries (Fausett and Goldman, 2006; Kassen et al., 2007; Thummel et al., 2008).Fig. 6.

Bottom Line: The resulting neurogenic clusters differentiate in vivo into various retinal neurons.We use signaling reporters to demonstrate that the Atoh7-induced regeneration-like response of MG cells is mimicked by Notch, resembling the behavior of early progenitors during retinogenesis.Our results uncover a new role for Atoh7 as a universal neurogenic factor, and illustrate how signaling modules are re-employed in diverse contexts to trigger different biological responses.

View Article: PubMed Central - PubMed

Affiliation: Centre for Organismal Studies (COS) Heidelberg, Im Neuenheimer Feld 230, Heidelberg 69120, Germany The Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology (HBIGS), Heidelberg University, Heidelberg, Germany.

No MeSH data available.


Related in: MedlinePlus