Vax1/2 genes counteract Mitf-induced respecification of the retinal pigment epithelium

Abstract

During vertebrate eye development, the transcription factor MITF acts to promote the development of the retinal pigment epithelium (RPE). In embryos with Mitf mutations, the future RPE hyperproliferates and is respecified as retinal tissue but only in a small portion of the dorsal RPE. Using a series of genetic crosses, we show that this spatial restriction of RPE respecification is brought about by persistent expression of the anti-retinogenic ventral homeodomain gene Vax2 in the dorso-proximal and both Vax1 and Vax2 in the ventral RPE. We further show that dorso-proximal RPE respecification in Vax2/Mitf double mutants and dorso-proximal and ventral RPE respecification in Vax1/2/Mitf triple mutants result from increased FGF/MAP kinase signaling. In none of the mutants, however, does the distal RPE show signs of hyperproliferation or respecification, likely due to local JAGGED1/NOTCH signaling. Expression studies and optic vesicle culture experiments also suggest a role for NOTCH signaling within the mutant dorsal RPE domains, where ectopic JAGGED1 expression may partially counteract the effects of FGF/ERK1/2 signaling on RPE respecification. The results indicate the presence of complex interplays between distinct transcription factors and signaling molecules during eye development and show how RPE phenotypes associated with mutations in one gene are modulated by expression changes in other genes.

Figure 1. Expression patterns of MITF, VSX2, VAX1 and VAX2 in wild-type and Mitf mutant optic vesicles and cups.

Embryos of the indicated genotypes were harvested at the indicated times, cryosectioned, and labeled for the indicated proteins. Dorsal is up, and ventral down. The dotted lines mark presumptive RPE. (A–F) In both wild type (A–C) and mutants expressing non-functional MITF protein (D–F), optic vesicles initially show pan-vesicular MITF expression that in optic cups is extinguished in the presumptive retina and so becomes restricted to the presumptive RPE. Note that in mutants, MITF is downregulated in a portion of the dorsal RPE at E12.5 (F, arrow). Mitf downregulation in the retina is due to complimentary retinal expression of VSX2 (G–L). Note that the area of dorsal RPE thickening in mutants also expresses VSX2 (arrow in L). (M–R) VAX1 protein, present in wild type in presumptive ventral RPE at early stages (M,N) but absent later on (O, arrows) remains present in ventral RPE in mutant (R, arrow). Dorsally, VAX1 expression barely extends into the RPE (R, arrowhead). (S–X) VAX2 shows prominent ventral retina expression in wild type and mutant at E12.5 (U,X). In addition, it extends into both the dorsal as well as the ventral RPE in mutant (X, arrows). Single channel images of (R) and (X) are provided in Figure S1. Scale bar: 60 µm.

Figure 2. Vax mutations exacerbate the dorsal RPE phenotypes in Mitf ^−/− optic cups.

Coronal sections are from E12.5 embryos (A–F; G–J) and E14.5 embryos (K,L). (A,B,D,E) In Vax1 ^−/− mutants, VAX2 extends into the ventral RPE (D, arrow), and in Vax2^−/− mutants, VAX1 extends into the ventral RPE (B, arrow). Absence of labeling on control sections (Vax1^−/− labeled for VAX1, A, or Vax2^−/− labeled for VAX2, E) indicates antibody-specificity. (C,F) Vax2^−/−;Mitf ^−/− embryos show massive dorsal RPE hyperproliferation in areas negative for VAX1, but Vax1^−/−;Mitf ^−/− embryos show little dorsal RPE hyperproliferation. (G,H,I,J) Corresponding sections labeled for VSX2. Note that VSX2 expression is absent in the dorsal RPE of Vax1^−/− and barely visible in the dorsal RPE of Vax1^−/−;Mitf ^−/− mutants (G,I), but present in the hyperproliferating dorsal RPE region of Vax2^−/−;Mitf ^−/− mutants (J). Also note VSX2 expression at the ventro-proximal OS/RPE boundary in G,I (arrows) but absence of VSX2 expression in the ventral RPE. (K,L) Milder dorsal RPE thickening in E14.5 Vax1^−/−;Mitf ^−/− mutants (K) compared to Vax2^−/−;Mitf ^−/− mutants (L). Note that in both K,L, the ventral RPE remains largely unchanged at this stage (arrow). Scale bar: 60 µm (A–J); 130 µm (K,L).

Figure 3. Vax1 and Vax2 redundantly limit retinogenesis in the presumptive dorso-proximal and ventral RPE domains of the Mitf mutant optic cups.

(A–E) In E14.5 Mitf ^+/− heterozygotes, RPE defects are Vax1 and Vax2 gene dose-dependent. In the total absence of Mitf (F–I), VSX2 and PAX6 expression are seen in the ventral RPE regardless of whether only one copy of Vax2 (F,H) or one copy of Vax1 (G,I) is present. Also note that dorsal RPE thickening and VSX2 and PAX6 expression are more prominent when VAX2 is totally missing (G,I) as opposed to when VAX1 is totally missing (F,H). Scale bar: 200 µm (A–E); 130 µm (F–I). Coordinates in (A): D – dorsal; V – ventral; T – temporal; N – nasal.

Figure 4. FGF-MAP kinase signaling regulates RPE-to-retina transition in Mitf mutants.

(A–E) In situ hybridization for Fgf15. In wild type (A), Fgf15 is normally restricted to the neural retina but is absent in the distal retina (arrow). (B–E) Ectopic expression of Fgf15 in the dorsal RPE is seen in Mit ^−/− (B), Vax2^−/−;Mitf ^−/− (D), and Vax1^−/−;Vax2^−/−;Mitf ^+/− (E) though not in Vax1^−/−;Mitf ^−/− mutants (C). Note that as in wild type, the dorsal future ciliary margin shows little Fgf15 labeling (arrow in B–E). (F–H) Increased numbers of p-H3/p-ERK double-positive cells in the RPE of E10−10.5 Mitf ^−/− (G) and Vax2^−/−;Mitf ^−/− mutant (H) as compared to wild-type RPE (F). (I) Quantitation of the results obtained from sections as in F–G. Box plots show minimal, 25^th percentile, median, 75^th percentile and maximal percentages. Significance determined by Student’s t-test: *: p<0.05; ***: p<0.001 (2–3 sections per embryo, 6–10 embryos per genotype). Scale bar: 150 µm (A–E), 25 µm (F–H).

Figure 5. JAGGED1-NOTCH regulates RPE proliferation and specification.

(A–F) Optic cups of the indicated genotypes were harvested at the indicated times and stained for JAGGED1. Note that in wild type and all indicated mutants, JAGGED1 is expressed in the dorsal distal retina and in (F) also in the E12.0 ventral distal retina and RPE (arrow), in addition to its prominent expression in the lens vesicle. Also note that a dorsal RPE subdomain in Mitf single or Vax1/2/Mitf triple mutants expresses JAGGED1 at both E10.5 and E12.0. (G) Western blots for the indicated proteins from wild-type OV cultures eight hours after exposure to human FGF2 and/or MEK1/2 inhibitor (MEKi). Note decrease in the level of JAGGED1 in the presence of MEKi. (H–J) JAGGED1 expression in sections of E10.5 Mitf ^−/− optic cups 36 hours after exposure to control beads (H, marked by o), MEKi bead (I, marked by +) or after co-implantation of an FGF2 bead (J, marked by *) and a bead coated with both FGF2 and MEKi (J, marked by +). Note that these are horizontal sections and that FGF2 and FGF2+MEKi have differential effects within the same OV culture. (K–N) Effects of antibody neutralization of JAGGED1 on VSX2 expression in Vax2^−/−;Mitf ^−/− cultures. Compared to control antibodies (K), anti-JAGGED1 antibodies modestly increased VSX2 expression in the RPE (L) and further increased it when the beads were double-coated with FGF2 (N). Note that the anti-JAGGED1 effect in (L) is seen only in a subdomain of the RPE, consistent with the fact that JAGGED1 expression was confined to the thickening portion of the Mitf single and Vax/Mitf compound mutant RPE. (O) Quantitation of VSX2-positive cells in optic cup cultures. Box plots show minimal, 25^th percentile, median, 75^th percentile and maximal percentages of VSX2-positive cells in the RPE per section (one section per embryo, and 8–9 embryos for each treatment). Significance determined by Student’s t-test: p<0.01 for control versus FGF2, and FGF2 versus FGF2+anti-JAGGED1; p<0.001 for control versus anti-JAGGED1, anti-JAGGED1 versus FGF2+anti-JAGGED1. (P–Y) Wild-type whole embryo cultures after 36-hour exposure to DMSO or NOTCH antagonist γ-secretase inhibitor 1. Note that under control conditions, RPE markers MITF (P) and TYROSINASE (TYR)(Q) were normally expressed, the numbers of phosphorylated histone H3 (p-H3) positive (R) and Ki67 positive (S) cells in the RPE were low, and JAGGED1 expression (T) was normal in the distal future retina. After γ-secretase 1 incubation, however, MITF and TYR expression was greatly reduced, the number of proliferative cells was elevated, and the expression territory of JAGGED1 expanded into the distal RPE (U–Y). Scale bar: 80 µm (A–F), 60 µm (H–N), 25 µm (P–Y).

Figure 6. VAX, MITF and JAGGED1-NOTCH counteract FGF-ERK signaling in mediating proper compartmentalization of the optic neuroepithelium.

The genetic analyses presented in this paper suggest that at the optic cup stage, the gradients of VAX and MITF protein restrict the future neuroretinal domain to the central distal portion of the optic neuroepithelium. JAGGED1 expression, first dorsal and then ventral, counteract retinogenic signals in the future ciliary margin. Loss of MITF functions, such as due to loss-of-function mutations in its gene, lead to RPE abnormalities including a dorsally restricted RPE-to-retina transition mediated by strong local retinogenic FGF signals. These retinogenic signals are counteracted by the antiretinogenic VAX proteins that remain present in the RPE, helping to dorsally limit RPE respecification.