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Experiment details for ascl1

A nutrient-sensitive restriction point is active during retinal progenitor cell differentiation.

A nutrient-sensitive restriction point is active during retinal progenitor cell differentiation.

Gene Clone Species Stages Anatomy
ascl1.L laevis NF stage 41 retina , ciliary marginal zone

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  Fig. 2. Nutrients are necessary for acquisition of the committed neural progenitor fate but not for the maintenance of the most immature stem/progenitor cells. (A) Proneural gene expression levels decreased following ND, as did levels of the progenitor markers sox2 and n-Myc (B). Dashed line in B indicates the central domain of the CMZ. n=8, 32 embryos/condition. (C) Expression of the eye field transcription factor gene rx1 remained unchanged. n=4, 16 embryos/condition. (C) c-Myc gene expression levels remained unchanged; however, the domain of c-Myc gene expression was reduced in the central CMZ of ND embryos. n=8, 32 embryos/condition. (D) Expression levels of the stem cell marker genes hes1 and hes2 remained unchanged in ND conditions. n=4, 16 embryos/condition. Scale bars: 50 μm (A,B,D); 50 μm (C; rx1) 25 μm (C; c-Myc).

Gene Clone Species Stages Anatomy
ascl1.L laevis NF stage 41 retina , ciliary marginal zone

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  Fig. 4. Re-feeding promotes reconstitution of the CMZ. (A,B) Explanted retinas from ND embryos grown in L15 medium containing sugars and amino acids for 24 hours showed an increase in CMZ proliferation after a 2-hour pulse with EdU. n=3, 18 retinas/condition. (C) This increase in proliferation varied with nutrient levels, suggesting that nutrients can regulate proliferation within a dynamic range. n=3, 18 retinas/condition. (D,E) Proliferation was initiated in the CMZ as early as 20 minutes after re-feeding (D), with robust proliferation being observed 2 hours after the addition of nutrients (E). (F,G) After culture for 24 hours in L15, a strong upregulation of Cyclin D1 and the proneural genes ascl1 and atoh7 was observed in re-fed retinas. n=3, 18 retinas/condition. (B,C,G) Error bars show 95% confidence intervals; ***P≤0.001 (unpaired t-test). Scale bars: 50 μm (A,D,E,F).

Gene Clone Species Stages Anatomy
ascl1.L laevis NF stage 45 retina , ciliary marginal zone

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  Fig. 7. Activation of mTOR signalling is sufficient to maintain the progenitor pool. (A,B) Activation of mTOR with bpV rescues pS6 to a level 70% of that of controls in ND retinas (top row), specifically in CMZ progenitor cells (bottom row). Dashed lines encircle the CMZ. n=4, 16 embryos/condition. (C-F) bpV treatment rescued the downregulation of genes observed following ND, without affecting the expression of stem cell markers. n=4, 16 embryos/ condition. (B-E) Error bars show 95% confidence intervals; ***P≤0.001, *P≤0.05 (unpaired t-test). Scale bars: 100 μm (A, top panel); 50 μm (A, bottom panel); 50 μm (F).