Additional Supporting Information may be found in the online version of this article.

sc-12-0505_sm_SupplFigure1.pdf160KFigure S1. Hes4 expression on whole mount embryos. Whole mount in situ hybridization analysis of Hes4 expression (lateral views of tailbud heads, anterior to the right). In addition to the eye, Hes4 expression is detected in several parts of the brain and spinal cord, in the branchial arches (white arrow in B-E) and in the pronephros (black arrowhead in B-D). For more details on Hes4 expression pattern in whole mount embryos, see [65]. Scale Bar: 300 μm.
sc-12-0505_sm_SupplFigure2.pdf241KFigure S2. Expression pattern of the retinal stem cell markers Id2 and Wnt8b during retinogenesis. In situ hybridization analysis of Id2 and Wnt8b expression during retinal development. Following expression in the presumptive RPE at the optic vesicle stage (stage 24), both genes get restricted to the peripheral RPE and forming CMZ in the optic cup (stage 32). Their expression in the CMZ of the mature retina (stage 41) is confined to the most peripheral region where neural stem cells reside (see magnifications of the dorsal CMZ on the right panels). Note that both Id2 and Wnt8b exhibit a more intense staining dorsally than ventrally. Scale Bar: 50 μm.
sc-12-0505_sm_SupplFigure3.pdf305KFigure S3. Hes4 is expressed in domains exhibiting high Wnt and low Hedgehog activity. Comparative in situ hybridization analysis of Hes4 expression profile with that of eGFP (in Wnt-responsive transgenic embryos) and Gli1 (as a readout of hedgehog signalling activity). Black and red dotted lines respectively delineate the RPE/NR border and the stem cell zone of the CMZ. Red and black arrows respectively point to the differentiating central RPE and the retinal margin. Drawings on the right schematize Hes4 expression and domains with Wnt or Hedgehog activity at stage 25 and 35. Scale Bar: 50 μm. Hes4 and retinal stemness
sc-12-0505_sm_SupplFigure4.pdf200KFigure S4. Hes4 represses its own transcription. (A) Whole mount in situ hybridization analysis of N-Tubulin and Hes4 expression on stage 23 embryos injected with Hes4VP16-GR mRNA. Arrows indicate ectopic expression in the epidermis (anterior on the right, dorsal side up). Of note, a similar result was obtained with a probe designed in the 3′UTR of the Hes4 gene, confirming that it was not due to crosshybridization with the Hes4VP16-GR mRNA. (B) Whole mount in situ hybridization analysis of Rx and Hes4 expression on stage 20 embryos, following injection of control or Hes4 morpholinos (Mo). Note the reduction of Rx staining in the eye field (black arrow) and the increase of Hes4 expression (white arrows) on the injected side (Inj). N indicates the proportion of embryos exhibiting the corresponding phenotype. Scale bar: 500 μm (A) or 300 μm (B).
sc-12-0505_sm_SupplFigure5.pdf300KFigure S5. Hes4 expression is insensitive to a 24-hour Notch signalling blockade. (A-E) In situ hybridization analysis following a 24- (from stage 25 to stage 35; A, B) or a 48- hour (from stage 25 to stage 39; C, D) treatment with the Notch inhibitor DAPT. (A, C) Expression of the Notch target gene HRT1 (lateral views of the head) serves as a control of DAPT efficiency [42]. Arrows point to the brain labelling, which is clearly decreased upon DAPT treatment. (B, D) Typical Hes4 staining on retinal sections from control or treated embryos as indicated. Shown on the right are higher magnifications of the dorsal CMZ, delineated with dotted lines. (E) Quantification of Hes4 staining area in the dorsal CMZ in each condition. (F) qPCR analysis of retinal Hes4 expression at stage 35 following a 24-hour DAPT treatment. N-Tubulin serves as a control of drug efficiency. Scale bar: 50 μm.
sc-12-0505_sm_SupplFigure6.pdf275KFigure S6. Hes4 overexpression prolongs proliferation of retinal progenitors. (A) Typical sections of stage 32 retinas lipofected with GFP and immunostained for both GFP and BrdU. White and yellow arrows point to a GFP+/BrdU- and to a GFP+/BrdU+ cell, Hes4 and retinal stemness respectively. (B) Quantification of BrdU incorporation (3-hour pulse) in retinal clones at stage 32 or 35, following Hes4 or Hes2 lipofection. Scale Bar: 50 μm.
sc-12-0505_sm_SupplFigure7.pdf301KFigure S7. Hes4-dependent reduction of neurogenesis can be rescued by forcing cell cycle exit. (A, B) Analysis of cell type distribution in stage 41 retinas, following Hes4, Xgadd-45γ or Hes4 plus Xgadd-45γ lipofection. (A) Typical sections of retinas transfected with the indicated construct. (B) Quantification of neurons and glia among transfected cells in each condition. (C) Birthdating experiments in retinas lipofected with Hes4, Xgadd-45γ or Hes4 plus Xgadd-45γ. GCL: ganglion cell layer; INL/ONL: inner/outer nuclear layer. Scale Bar: 25 μm.
sc-12-0505_sm_SupplTable1.pdf81KSupplementary Table 1. List of antibodies used.
sc-12-0505_sm_SupplTable2.pdf9KSupplementary Table 2. List of primers used in qPCR experiments.
sc-12-0505_sm_SupplTable3.pdf15KSupplementary Table 3. Labelling index and cell cycle parameters in the NR and NR/RPE border.

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