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The retina of fish and amphibian contains genuine neural stem cells located at the most peripheral edge of the ciliary marginal zone (CMZ). However, their cell-of-origin as well as the mechanisms that sustain their maintenance during development are presently unknown. We identified Hes4 (previously named XHairy2), a gene encoding a bHLH-O transcriptional repressor, as a stem cell-specific marker of the Xenopus CMZ that is positively regulated by the canonical Wnt pathway and negatively by Hedgehog signaling. We found that during retinogenesis, Hes4 labels a small territory, located first at the pigmented epithelium (RPE)/neural retina (NR) border and later in the retinal margin, that likely gives rise to adult retinal stem cells. We next addressed whether Hes4 might impart this cell subpopulation with retinal stem cell features: inhibited RPE or NR differentiation programs, continuous proliferation, and slow cell cycle speed. We could indeed show that Hes4 overexpression cell autonomously prevents retinal precursor cells from commitment toward retinal fates and maintains them in a proliferative state. Besides, our data highlight for the first time that Hes4 may also constitute a crucial regulator of cell cycle kinetics. Hes4 gain of function indeed significantly slows down cell division, mainly through the lengthening of G1 phase. As a whole, we propose that Hes4 maintains particular stemness features in a cellular cohort dedicated to constitute the adult retinal stem cell pool, by keeping it in an undifferentiated and slowly proliferative state along embryonic retinogenesis.
Figure 1. Hes4 expression during retinogenesis. Comparative in situ hybridization analysis of Hes4 expression profile (B, K–R) with that of Pax6(A), Rx(C–F)Mitf1A(G–J), and Pax2 (inset in L). White arrow in (L) point to the OS. Black arrows in (K–M) indicate the RPE/NR border (K, L) and the retinal margin (M). (Q) corresponds to a magnification of the CMZ delineated in (R). White and red arrows, respectively, indicate stem cell and progenitor zones of the CMZ. Scale bars = 300 μm (A, B) or 50 μm (C–R). Abbreviations: CMZ, ciliary marginal zone; CR, central retina; L: lens; NR, neural retina; OS, optic stalk; RPE, retinal pigmented epithelium.
Figure 2. Wnt and Hedgehog signalings affect Hes4 expression in an opposite manner. (AâE):In situ hybridization analysis of Hes4 expression on retinal sections, following a 24-hour (from stage 25 to stage 35; A, CâE) or a 15-hour (from stage 25 to stage 32; B, C) treatment with the indicated drug. Shown beneath each retinal section is a higher magnification of the dorsal CMZ delineated with dotted lines. (C, E): Quantification of Hes4 staining area in the dorsal CMZ in each condition. (F): Quantitative polymerase chain reaction analysis of retinal Hes4 expression at stage 35 following 24 hours of Hedgehog or Wnt signaling inhibition. Ptc1 and CyclinD1 serve as controls of drug efficiency. Scale bar = 50 μm. Abbreviations: BIO, 6-bromoindirubin-3â²-oxime; CMZ, ciliary marginal zone; CR, central retina; Cyclo, cyclopamine; IWR1, inhibitor of wnt response 1; L, lens; N, number of analyzed sections; Purm, purmorphamine.
Figure 3. Hes4 is required for proper eye formation. (A, B): Analysis of Hes4 loss of function. Hes4 or Ctrl Mo were injected at the indicated dose with or without Hes4-GR mRNA. (A): Typical pictures of stage 37 injected embryos (lateral view of the head). Note that embryos injected with Hes4-GR mRNA alone exhibit a defective RPE pigmentation and a slightly smaller eye. (B): Quantification of morphant and rescued embryos exhibiting the indicated phenotypes. (C): Transverse sections of a stage 25 morphant embryo (injected with 8 ng Hes4 Mo) immunostained with anti-active caspase3 and anti-GFP (to visualize the injected side). Scale bars = 300 μm (A) or 100 μm (C). Abbreviations: GR, glucocorticoid receptor; GFP, green fluorescent protein; Inj, injected side; NT, neural tube; R, retina; Uninj, uninjected side.
Figure 4. Hes4 misexpression inhibits neuronal and RPE differentiation. (A): Immunofluorescence analysis of cell-type-specific marker expression in stage 37 retinas, following Hes4-GR mRNA injection. Arrows point to the staining in the RPE (Xar-1), photoreceptors (calbindin) or interneuron and ganglion cell fibers (syntaxin). The optic nerve is shown in insets. (B):In situ hybridization analysis with the indicated probe in stage 25 retinas, following Hes4-GR mRNA injection. (C, D): Analysis of cell type distribution in stage 39 or 41 retinas, following Hes4 lipofection. (C) Typical sections of retinas transfected with GFP alone (control) or GFP plus Hes4. Arrows indicate NE at stage 39 or Müller glial cells at stage 41. Respective morphologies of these cells are illustrated on the schematics below. (D) Quantification of NE, neurons, and glia among transfected cells at stage 39 or 41. Scale bars = 50 μm (A, B) or 25 μm (C). Abbreviations: CMZ, ciliary marginal zone; CR, central retina; GR, glucocorticoid receptor; GCL, ganglion cell layer; INL/ONL, inner/outer nuclear layer; L, lens; NE, neuroepithelial cell; NR, neural retina; RPE, retinal pigmented epithelium.
Figure 5. Hes4 overexpression delays cell cycle exit. (A, B): BrdU incorporation assay (3-hour pulse) at stage 37, following Hes4-GR mRNA injection. (A): Typical retinal sections immunostained with anti-BrdU. (B): Corresponding quantification. (C, D): Birthdating experiments in Hes4 lipofected retinas. (C): Typical retinal section immunostained for both GFP and BrdU, following continuous BrdU exposure from stage 34 to 41. (D): Percentage of BrdU+ cells among transfected cells. Scale bar = 50 μm. Abbreviations: BrdU, 5-bromo-2â²-deoxyuridine; CMZ, ciliary marginal zone; CR, central retina; GR, glucocorticoid receptor; GFP, green fluorescent protein; L, lens.
Figure 6. Hes4 overexpression slows down cell cycle speed of retinal precursors. (A): Quantification of the mitotic index in the retinal neuroepithelium at stage 25, following Hes4-GR mRNA injection. (B): Evaluation of the EdU labeling index in the NR of control and Hes4-GR-injected embryos, after cumulative EdU labeling starting from stage 25. (C, D): G2 length evaluation in vivo, following Hes4-GR mRNA injection. (C) Typical retinal section (stage 25) stained for both phospho-Histone H3 (P-H3) and EdU, following a 1.5-hour EdU pulse. White and yellow arrows point to P-H3+/EdU− and to P-H3+/EdU+ cells, respectively. (D) Proportion of EdU-labeled mitosis along with increasing EdU exposure times. (E, F): Proportion of EdU-labeled nuclei after cumulative EdU labeling, starting from stage 22. EdU detection was performed following in situ hybridization with a Hes4 probe. Shown in (E) are typical retinal sections stained for both Hes4 and EdU, following a 1-hour EdU pulse. Labeling index was measured in the Hes4-expressing NR/RPE border (delineated by white dotted lines in E) or in the Hes4-negative NR (yellow dotted lines) of control embryos. Scale bar = 50 μm. Abbreviations: EdU, 5-ethynyl-2′-deoxyuridine; GR, glucocorticoid receptor; NR, neural retina; RPE, retinal pigmented epithelium.
Figure 7. A proposed model for retinal stem cell (RSC) cell-off-origin and Hes4 function during embryonic retinogenesis. (A): Schema highlighting the dynamics of Hes4 expression during retinogenesis. Adult RSCs likely originate from the most dorsal Hes4-expressing part of the optic vesicle (red dotted lines). (B): Schematic illustration of Hes4 regulation and function in the presumptive adult RSCs. Abbreviations: CMZ, ciliary marginal zone; GCL, ganglion cell layer; INL/ONL: inner/outer nuclear layer; NR, neural retina; RPE, retinal pigmented epithelium.
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