Ascl1 as a Novel Player in the Ptf1a Transcriptional Network for GABAergic Cell Specification in the Retina

Mazurier, Nicolas; Parain, Karine; Parlier, Damien; Pretto, Silvia; Hamdache, Johanna; Vernier, Philippe; Locker, Morgane; Bellefroid, Éric; Perron, Muriel

doi:10.1371/journal.pone.0092113

Cited by 12 publications

(9 citation statements)

References 81 publications

(121 reference statements)

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“…Ptf1a‐committed precursors are further specified by other transcription factors (Liu et al, ; Nakhai et al, ): Nr4a2 (orphan nuclear receptor subfamily 4, group A, member 2) is essential for GABAergic and dopaminergic amacrines; and Tfap2a (AP‐2 alpha) marks a subset of glycinergic and GABAergic amacrines. Studies in xenopus retinal progenitors have suggested that Ascl1, acting in concert with Ptf1a, is sufficient and necessary for amacrine genesis (Mazurier et al, ). We detected Ascl1, Ptf1a, and Nr4a2 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Age-dependent Müller glia neurogenic competence in the mouse retina

Löffler

Patrick

Völkner

et al. 2015

Glia

105

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The mechanisms limiting neuronal regeneration in mammals and their relationship with reactive gliosis are unknown. Müller glia (MG), common to all vertebrate retinas, readily regenerate neuron loss in some species, but normally not in mammals. However, experimental stimulation of limited mammalian retina regeneration has been reported. Here, we use a mouse retina organ culture approach to investigate the MG responses at different mouse ages. We found that MG undergo defined spatio-temporal changes upon stimulation. In EGF-stimulated juvenile postmitotic retinas, most MG upregulate cell-cycle regulators (Mcm6, Pcna, Ki67, Ccnd1) within 48 h ex vivo; some also express the neurogenic factors Ascl1, Pax6, and Vsx2; up to 60% re-enter the cell cycle, some of which delaminate to divide mostly apically; and the majority cease to proliferate after stimulation. A subpopulation of MG progeny starts to express transcription factors (Ptf1a, Nr4a2) and neuronal (Calb1, Calb2, Rbfox3), but not glial, markers, indicating neurogenesis. BrdU-tracking, genetic lineage-tracing, and transgenic-reporter experiments suggest that MG reprogram to a neurogenic stage and proliferate; and that some MG progeny differentiate into neuronal-like cells, most likely amacrines, no photoreceptors; most others remain in a de-differentiated state. The mouse MG regeneration potential becomes restricted, dependent on the age of the animal, as observed by limited activation of the cell cycle and neurogenic factors. The stage-dependent analysis of mouse MG revealed similarities and differences when compared with MG-derived regeneration in fish and chicks. Therefore, the mouse retina ex vivo approach is a potential assay for understanding and overcoming the limitations of mammalian MG-derived neuronal regeneration. Postmitotic MG in mouse retina ex vivo can be stimulated to proliferate, express neurogenic factors, and generate progeny expressing neuronal or glial markers. This potential regenerative competence becomes limited with increasing mouse age.

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Section: Resultsmentioning

confidence: 99%

Age-dependent Müller glia neurogenic competence in the mouse retina

Löffler

Patrick

Völkner

et al. 2015

Glia

105

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“…A sizeable number of proneural bHLH transcription factors have been identified that act at different stages in the specification, differentiation and maturation of a neuron [ 1 ], but how their different roles are controlled and co-ordinated is still poorly understood. We have previously shown that two bHLH proteins are controlled by multi-site phosphorylation on serine-proline sites; Ascl1, which regulates autonomic neuron formation in Xenopus [ 41 ] and generally a GABAergic phenotype (among other roles) [ 42 ], and Neurogenin2, an inducer of primary neuron formation in the developing Xenopus embryo [ 43 ] and generally of glutamatergic neurons [ 42 ]. Multi-site phosphorylation by cdks regulates the ability of Ascl1 and Ngn2 to control the proliferation versus differentiation decision; when cells are rapidly cycling, high cdk levels suppress the ability of these proneural proteins to drive differentiation [ 14 – 16 ].…”

Section: Discussionmentioning

confidence: 99%

Multi-site phosphorylation regulates NeuroD4 activity during primary neurogenesis: a conserved mechanism amongst proneural proteins

Hardwick

Philpott

2015

Neural Dev

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BackgroundBasic Helix Loop Helix (bHLH) proneural transcription factors are master regulators of neurogenesis that act at multiple stages in this process. We have previously demonstrated that multi-site phosphorylation of two members of the proneural protein family, Ngn2 and Ascl1, limits their ability to drive neuronal differentiation when cyclin-dependent kinase levels are high, as would be found in rapidly cycling cells. Here we investigate potential phospho-regulation of proneural protein NeuroD4 (also known as Xath3), the Xenopus homologue of Math3/NeuroM, that functions downstream of Ngn2 in the neurogenic cascade.ResultsUsing the developing Xenopus embryo system, we show that NeuroD4 is expressed and phosphorylated during primary neurogenesis, and this phosphorylation limits its ability to drive neuronal differentiation. Phosphorylation of up to six serine/threonine-proline sites contributes additively to regulation of NeuroD4 proneural activity without altering neuronal subtype specification, and number rather than location of available phospho-sites is the key for limiting NeuroD4 activity. Mechanistically, a phospho-mutant NeuroD4 displays increased protein stability and enhanced chromatin binding relative to wild-type NeuroD4, resulting in transcriptional up-regulation of a range of target genes that further promote neuronal differentiation.ConclusionsMulti-site phosphorylation on serine/threonine-proline pairs is a widely conserved mechanism of limiting proneural protein activity, where it is the number of phosphorylated sites, rather than their location that determines protein activity. Hence, multi-site phosphorylation is very well suited to allow co-ordination of proneural protein activity with the cellular proline-directed kinase environment.Electronic supplementary materialThe online version of this article (doi:10.1186/s13064-015-0044-8) contains supplementary material, which is available to authorized users.

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“…Ascl1 is another class II bHLH transcription factor particularly associated with GABAergic and noradrenergic neurogenesis in Xenopus embryos [ 18 , 19 ], while NeuroD1 is a proneural protein acting downstream of Ngn2 in the primary neurogenesis cascade. All three factors are able to induce ectopic primary neurons when over-expressed [ 7 , 12 ].…”

Section: Resultsmentioning

confidence: 99%

N-terminal phosphorylation of xHes1 controls inhibition of primary neurogenesis in Xenopus

Hardwick

Philpott

2019

Biochemical and Biophysical Research Communications

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The processes of cell proliferation and differentiation are intimately linked during embryogenesis, and the superfamily of (basic) Helix-Loop-Helix (bHLH) transcription factors play critical roles in these events. For example, neuronal differentiation is promoted by class II bHLH proneural proteins such as Ngn2 and Ascl1, while class VI Hes proteins act to restrain differentiation and promote progenitor maintenance. We have previously described multi-site phosphorylation as a key regulator of tissue specific class II bHLH proteins in all three embryonic germ layers, and this enables coordination of differentiation with the cell cycle. Hes1 homologues also show analogous conserved proline directed kinase sites. Here we have used formation of Xenopus primary neurons to investigate the effects of xHes1 multi-site phosphorylation on both endogenous and ectopic proneural protein-induced neurogenesis. We find that xHes1 is phosphorylated in vivo , and preventing phosphorylation on three conserved SP/TP sites in the N terminus of the protein enhances xHes1 protein stability and repressor activity. Mechanistically, compared to wild-type xHes1, phospho-mutant xHes1 exhibits greater repression of Ngn2 transcription as well as producing a greater reduction in Ngn2 protein stability and chromatin binding. We propose that cell cycle dependent phosphorylation of class VI Hes proteins may act alongside similar regulation of class II bHLH proneural proteins to co-ordinate their activity.

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Ascl1 as a Novel Player in the Ptf1a Transcriptional Network for GABAergic Cell Specification in the Retina

Cited by 12 publications

References 81 publications

Age-dependent Müller glia neurogenic competence in the mouse retina

Age-dependent Müller glia neurogenic competence in the mouse retina

Multi-site phosphorylation regulates NeuroD4 activity during primary neurogenesis: a conserved mechanism amongst proneural proteins

N-terminal phosphorylation of xHes1 controls inhibition of primary neurogenesis in Xenopus

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