Notch activity permits retinal cells to progress through multiple progenitor states and acquire a stem cell property

Abstract

Signaling through the Notch pathway regulates multiple aspects of development. The vertebrate retina allows an investigation of the basis for these various effects, because the major cell types of the retina arise from a common progenitor that expresses Notch1. The Notch pathway was constitutively activated in distinct populations of retinal cells during development. Prolonged Notch activity in progenitor cells maintained cells in the progenitor state without perturbing temporal identity, promoting early progenitor characteristics early in development and late progenitor characteristics later in development. Eventually, constitutive Notch activation led these cells to acquire characteristics of glial and stem cells. In contrast, reactivating the Notch pathway in newly postmitotic retinal cells promoted mature glial cell formation in a subset of cells. These data suggest that prolonged Notch activity does not disrupt the normal progression of progenitor temporal states, and that down-regulating or overcoming Notch activity is required for proper formation of both neuronal and glial cell fates.

Fig. 1.

Directed misexpression of NIC in Chxlo expression cells. Section ISH on WT (A and C) and Chx10^N1-IC (B and D) retinae at P10. (A and B) Notch1. (C and D) clusterin. Immunostaining on WT (E and I) and Chx10^N1-IC (F–H and J–L) retinae at P10. (E and F) Merge of DAPI, nGFP, and β tubulin III (β tub III). (I and J) merge of DAPI, nGFP, and glutamine synthetase (glut syn). (G and K) nGFP. (H) β tubulin III. (L) glut syn.

Fig. 2.

Gene expression changes in activated Notch retinal progenitor cells at P10. Section ISH on WT (A and C) and Chx10^N1-IC (B and D) retinae at P10. (A and B) FGF15. (C and D) cyclin D1. (E–H) Immunostaining on WT (E) and Chx10^N1-IC retinae with anti-Pax6 antibody at P10 (F–H). (E–H) Merge of DAPI, GFP, and Pax6 (E and F), nGFP (G), and Pax6 (H).

Fig. 3.

Gene expression changes in activated Notch retinal progenitor cells at E13.5. Fold changes are averaged from a color-swap experiment. (A–J) Section ISH on WT (A–E) and Chx10^N1-IC (F–J) retinae at E13.5. (A and F) Notch1. (B and G) FGF15. (C and H) Dio3as. (D and I) Sfrp2. (E and J) clusterin. A black line is drawn to indicate the vitreal edge of the retina.

Fig. 4.

Neurosphere assay of Chx10^N1-IC retinal cells. Immunostaining and merge of DAPI and GFP on WT (A) and Chx10^N1-IC (D) retinae at P21 is shown. Adult retinae from WT (B and C) and Chx10^N1-IC (E and F) were dissociated and cultured in serum-free media with EGF and basic FGF in vitro and visualized by light microscopy at low (B and E) and high (C and F) magnification. (G–I) Spheres derived from Chx10^N1-IC retinae contained GFP+ cells.

Fig. 5.

Directed misexpression of NIC in Shh-expressing cells. (A) Diagram of transgenic constructs. The Shh-CRE mice have a CreGFP fusion under control of the Shh promoter (27). Shh^N1-IC mice were generated by crossing the Shh-CRE allele into Rosa^N1-IC/Rosa^N1-IC mice. Fate mapping of recombined cells was possible by additionally crossing these mice to ROSA26-R (R26R) mice. R26R is a Cre-recombinase reporter comprising a loxP flanked transcriptional STOP preceding a β-gal coding region (lacZ) (29). (B and C) X-Gal staining of P11 retinae from control (B) and Shh^N1-IC (C) mice. (D–I) Immunostaining of Shh^N1-IC retinae at P11. (F) Merge of DAPI, anti-β-galactosidase (β Gal), and anti-glutamine synthetase (glut syn). (I) merge of DAPI, anti-β-gal (β Gal), and anti-Pax6. (D and G) β Gal. (E) glut syn. (H) Pax 6. For control staining of WT retinae, see Figs. 1 (glutamine synthetase) and 2 (Pax6).