Notch signaling differentially regulates Atoh7 and Neurog2 in the distal mouse retina

Abstract

Notch signaling regulates basic helix-loop-helix (bHLH) factors as an evolutionarily conserved module, but the tissue-specific mechanisms are incompletely elucidated. In the mouse retina, bHLH genes Atoh7 and Neurog2 have distinct functions, with Atoh7 regulating retinal competence and Neurog2 required for progression of neurogenesis. These transcription factors are extensively co-expressed, suggesting similar regulation. We directly compared Atoh7 and Neurog2 regulation at the earliest stages of retinal neurogenesis in a broad spectrum of Notch pathway mutants. Notch1 and Rbpj normally block Atoh7 and Neurog2 expression. However, the combined activities of Notch1, Notch3 and Rbpj regulate Neurog2 patterning in the distal retina. Downstream of the Notch complex, we found the Hes1 repressor mediates Atoh7 suppression, but Hes1, Hes3 and Hes5 do not regulate Neurog2 expression. We also tested Notch-mediated regulation of Jag1 and Pax6 in the distal retina, to establish the appropriate context for Neurog2 patterning. We found that Notch1;Notch3 and Rbpj block co-expression of Jag1 and Neurog2, while specifically stimulating Pax6 within an adjacent domain. Our data suggest that Notch signaling controls the overall tempo of retinogenesis, by integrating cell fate specification, the wave of neurogenesis and the developmental status of cells ahead of this wave.

Keywords: Atoh7; Jagged1; Mouse; Neurog2; Notch signaling; Retinal neurogenesis.

Fig. 1.

Comparison of Atoh7^lacZ, Neurog2, Rbpj and Hes1 retinal expression. (A-A″) In Atoh7^lacZ/+ eyes, every β-gal+ cell co-expresses Rbpj (arrows). (B-B″) Similarly, all Neurog2+ cells co-express Rbpj protein (arrows). (C-C″) No β-gal+Hes1+ cells were observed (arrowheads indicate β-gal+ cells). (D-E″) At E11.5, Neurog2 and Hes1 proteins are extensively co-expressed (arrows), but by E13.5, their patterns are mutually exclusive (E-E″, arrowheads). Scale bar: 100 µm for all panels; insets on the right are higher magnifications of the panels on the left.

Fig. 2.

Differential Rbpj-mediated regulation of Atoh7^lacZ and Neurog2. (A-F) E13.5 Hes1+ RPCs, Pou4f+ RGCs and Crx+ photoreceptor precursors RPCs were autonomously reduced (D), whereas RGCs (E) and photoreceptor precursors (F) were autonomously increased. Inset arrows indicate α-Cre lineage cells (GFP+) co-labeled with anti-Hes1 (A,D), anti-Pou4f (B,E) or anti-Crx (C,F). (G-G‴″) Overlapping Atoh7^lacZ (anti-β-gal) and Neurog2 expression in the α-Cre (GFP+) lineage. Arrow and arrowhead in G⁗,G‴″. (H-H‴″) In the absence of Rbpj, Atoh^lacZ+ cells are increased (bracket in H‴″). Although Neurog2+ and the β-gal+Neurog2+ double-positive cohorts increased (J), Neurog2 expression was also mispatterned (H-H‴,H‴″). In H‴″, the arrow indicates an abnormal accumulation of Neurog2+ cells, yet the arrowhead denotes an area with no Neurog2+ cells. Insets show co-expression of GFP, β-gal and/or Neurog2 (arrows). (I,J) Quantification of co-labeling between the white lines in G and H. Insets in G-G‴ and H-H‴ are boxed areas shown at higher magnification. Scale bar: 100 µm; n≥3 embryos per genotype; ***P≤0.001; *P=0.05; error bars represent s.e.m.

Fig. 3.

Hes1 does not regulate Neurog2 in the prenatal retina. Pou4f+ RGCs (A,D) are expanded and Crx+ photoreceptor precursors are reduced (B,E) in E13.5 Hes1^−/− retinas, consistent with previous reports (Takatsuka et al., 2004; Riesenberg et al., 2009b). (C,F) The Neurog2 expression pattern is not altered by loss of Hes1. (G) There was no significant change in the percentage of Neurog2+ cells. Scale bar: 100 µm; n≥3 embryos per genotype; NS, not significant; error bars represent s.e.m.

Fig. 4.

Notch1 regulates Atoh7^lacZ and Neurog2 identically. (A-F) The loss of Notch1 autonomously reduced Hes1+ RPCs (D) and expanded Crx+ photoreceptor precursors (F), with no effect on Pouf4+ RGCs (E) (Jadhav et al., 2006b; Yaron et al., 2006). (G-G‴″) Highly overlapping domains for Atoh7^lacZ(β-gal) and Neurog2, in α-Cre; Notch1^CKO/+;Z/EG;Atoh7^lacZ/+ retinas. (H-J) In α-Cre;Notch1^CKO/CKO; Z/EG;Atoh7^lacZ/+ retinas, however (H-H‴″), the Atoh7^lacZ- and Neurog2-expressing cells are autonomously increased (H‴′, H‴″, brackets, quantified in I,J). (K-T′) Notch1 ICD overexpression induced an autonomous increase of Hes1+ RPCs, and nearly abolished Pou4f+ RGCs (P) and Crx+ photoreceptor precursors (R). Both β-gal+ (S) and Neurog2+ cell populations (T,T′) were dramatically reduced (arrowhead). Scale bar: 100 µm; insets show boxed areas at higher magnification. Arrows indicate n≥3 embryos per genotype; errors bars show s.e.m.; ***P≤0.001.

Fig. 5.

Notch3 is not required for Atoh7 and Neurog2 retinal expression. (A-F) At E16.5, the normal and germline mutant Notch3 lineages (β-gal+ vesicles) contain Hes1+ RPCs (A,D), Pou4f+ RGCs (B,E) and Crx+ photoreceptor precursors (C,F). To monitor Atoh7 expression in Notch3^lacZ/+ (G) and Notch3^lacZ/lacZ (I) retinas, an Atoh7^GFP Tg was crossed into the Notch3 background, while anti-Neurog2 labeling identified the Neurog2+ population. (G-K) Loss of Notch3 had no cell-autonomous effect on Atoh7-GFP+ or Neurog2+ cells, quantified in K. Scale bar: 100 µm; boxed areas are at higher magnification in insets; arrows indicate β-gal+marker+ cells. n≥3 embryos per age and genotype, error bars show s.e.m.

Fig. 6.

Notch1;Notch3 double mutants phenocopy the loss of Rbpj. (A-C,F-H,K-M,P-R) Notch1;Notch3 allelic series generated by conditionally deleting Notch1 in the genetic background of Notch3 germline mutants. Double heterozygous retinas (A-C α-Cre; Notch1^CKO/+;Z/EG; Notch3^lacZ/+) and those with one wild-type Notch1 allele (F-H,U-W; α-Cre;Notch1^CKO/+;Z/EG; Notch3^lacZ/lacZ) had normal distribution of Hes1+ RPCs, Pou4f+ RGCs and Crx+ photoreceptor precursors, quantified in U-W. (K-M,U-W) α-Cre;Notch1^CKO/CKO;Z/EG; Notch3^lacZ/+ retinas had reduced Hes1+ RPCs, increased Crx+ cells, but Pou4f+ RGCs were unaffected (green bars in U-W). (P-R,U-W) Notch1;Notch3 double mutants had a loss of RPCs and increase in Crx+ cells (red bars in U-W). There was a unique derepression of nascent RGCs, quantified in U-W. (D,E,I,J,N,O,S,T) Notch1;Notch3 double heterozygotes had no change in Neurog2 protein levels or in Atoh7 mRNA expression (D,E), neither did retinas with one wild-type allele of Notch1 (I,J). Both bHLH factors were derepressed when only the wild-type allele of Notch3 remained (N,O). Receptor double mutants phenocopied Rbpj mutants, including Neurog2 mispatterning (S). (X) Quantification of Neurog2+ cells between white lines in D,I,N,S. Scale bars: 100 µm; boxed areas are at higher magnification in insets. Arrows indicate GFP+marker+ cells, arrowheads indicate marker+GFP-negative cells. Brackets in E,J,O,T define retinal zones 1+2 and Atoh7 mRNA expression. *P≤0.05, ***P≤0.001; n≥3 embryos per age and genotype; error bars indicate s.e.m.

Fig. 7.

Notch signaling maintains distal retinal patterning. (A,A′,C,C′) Hes1 and Jag1 are normally not co-expressed in the E13.5 distal retina. Loss of Rbpj derepresses Jag1 in zone 1 (white bracket in C,C′). Ectopic Jag1 in the cell membrane does not colocalize with nuclear Hes1 (C′). (B,D) In E13.5 Hes1 mutants, Jag1 is broadly derepressed (white bracket in D). (E-F″) Ectopic Jag1 and Neurog2 are co-expressed within zone 1 of Rbpj mutants. (G-J′,N). In the absence of Rbpj, Pax6 expression is reduced only in zone 2, accompanied by ectopic Crx expression (J,J′, arrow). (K-L′) Co-labeling for Jag1 and Pax6 clearly shows zone-specific downregulation of Pax6 in Rbpj conditional mutants, with ectopic Jag1 (bracket in L,L′) distal to Pax6-negative cells (arrow in L′). (M-M‴) Significant loss of Pax6+ cells in α-Cre;Notch1^CKO/CKO;Z/EG; Notch3^lacZ/+(M″) and α-Cre; Notch1^CKO/CKO; Z/EG;Notch3^lacZ/lacZ (M‴) retinas (quantified in O). Scale bars: 100 µm; boxed areas are at higher magnification in the insets. Brackets in C, F and L indicate distal zone 1, whereas in D the bracket denotes zones 1+2. White lines in G,I,M-M‴ delineate area of quantification. *P≤0.05,***P≤0.001; n≥3 embryos per age and genotype; error bars indicate s.e.m.

Fig. 8.

Notch pathway regulation during early retinal neurogenesis. (A) Atoh7 and Neurog2 ‘salt-and-pepper’ expression in the E13.5 retina. Pouf+ RGCs and Crx+ bipotential photoreceptor precursor domains are indicated. (B) Rbpj^−/− zone 1 cells have increased Jag1, Neurog2 and Atoh7^lacZ expression. Mutant zone 2 has extra Atoh7^lacZ+ cells, but fewer Neurog2+ cells. Pax6 expression is also lost in cells that then ectopically express Crx. (C) Schematic of the regulatory relationship among the canonical Notch pathway genes Pax6, Atoh7 and Neurog2.