Overlapping expression patterns and redundant roles for AP-2 transcription factors in the developing mammalian retina

Abstract

Background: We have previously shown that the transcription factor AP-2α (Tcfap2a) is expressed in postmitotic developing amacrine cells in the mouse retina. Although retina-specific deletion of Tcfap2a did not affect retinogenesis, two other family members, AP-2β and AP-2γ, showed expression patterns similar to AP-2α.

Results: Here we show that, in addition to their highly overlapping expression patterns in amacrine cells, AP-2α and AP-2β are also co-expressed in developing horizontal cells. AP-2γ expression is restricted to amacrine cells, in a subset that is partially distinct from the AP-2α/β-immunopositive population. To address possible redundant roles for AP-2α and AP-2β during retinogenesis, Tcfap2a/b-deficient retinas were examined. These double mutants showed a striking loss of horizontal cells and an altered staining pattern in amacrine cells that were not detected upon deletion of either family member alone.

Conclusions: These studies have uncovered critical roles for AP-2 activity in retinogenesis, delineating the overlapping expression patterns of Tcfap2a, Tcfap2b, and Tcfap2c in the neural retina, and revealing a redundant requirement for Tcfap2a and Tcfap2b in horizontal and amacrine cell development.

Fig. 1. AP-2β is expressed in postmitotic amacrine and horizontal cells in the developing mouse retina

A: Co-immunostain with anti-AP-2α (green) and anti-AP-2β (red) on horizontal section of E15.5 wild-type eye. B–G: Co-immunostain with anti-AP-2β (green) and either anti-PCNA (B), anti-STX1A (C–E), anti-GAT-1 (F), or anti-GLYT1 (G) antibodies (red) on horizontal sections of wild-type eyes, at the stages indicated. Arrowheads in C–E point to cells co-labeled by anti-AP-2β and anti-STX1A. Boxed areas in E–G are magnified in inset. i/onbl, inner/outer neuroblast layer; INL, inner nuclear layer; GCL, ganglion cell layer; IPL, inner plexiform layer; OPL, outer plexiform layer. Scale bars = 100 µm.

Fig. 2. Both AP-2α and AP-2β are expressed in developing mouse horizontal cells, although AP-2α expression is transient

A–F: Co-immunolabeling with anti-LIM1 (green) and anti-AP-2β (red; A–C); anti-AP-2α (green) and anti-AP-2β (red; D); anti-AP-2α (green) and anti-PROX1 (red; E); or anti-AP-2α (green) and anti-PGP9.5 (red; F) on horizontal sections of wild-type eyes, at the stages indicated. All LIM1-positive horizontal cells are AP-2β-positive (A–C, arrowheads). Arrowheads in D denote cells that are strongly AP-2β-immunoreactive and also more weakly AP-2α-immunoreactive (inset). Arrowheads in E and F point to PROX1-positive or PGP9.5-positive horizontal cells that co-express AP-2α. G: Quantification of the proportions of co-labeled versus singly labeled cells in the total AP-2 population examined (AP-2α and/or AP-2β-positive), at the stages indicated. Note that the relative proportions of co-labeled versus singly labeled cells were not significantly affected by the stage examined. H: Quantification of the proportion of PGP9.5-positive horizontal cells expressing AP-2α and proportion of LIM1-positive cells expressing AP-2β at P0. Bars represent the mean ±SEM of counts from 3 animals. onbl, outer neuroblast layer; INL, inner nuclear layer; GCL, ganglion cell layer; IPL, inner plexiform layer. ***P < 0.001; **P < 0.01; *P < 0.05. Scale bars = 100 µm.

Fig. 3. AP-2γ is not a horizontal cell marker

A–D: Immunofluorescence using anti-AP-2γ clone 6E4 (green) on horizontal sections of wild-type mouse heads or eyes counterstained with DAPI (4,6-diamino-2-phenylindole (DAPI; blue), at the stages indicated. Arrowheads in B–D indicate AP-2γ-immunoreactive cells in the GCL. E,F: Co-immunostain with anti-AP-2γ (green) and either anti-PROX1 (E) or anti-PGP9.5 (F) antibodies (red) on horizontal sections of wild-type eyes, at the stages indicated. i/onbl, inner/outer neuroblast layer; INL, inner nuclear layer; GCL, ganglion cell layer; IPL, inner plexiform layer; OPL, outer plexiform layer. Scale bars = 100 µm.

Fig. 4. AP-2γ is expressed in postmitotic amacrine cells

A–C: Co-immunostain with anti-AP-2γ (green) and either anti-PH3 (A), anti-GAT-1 (B), or anti-GLYT1 (C) antibodies (red) on horizontal sections of wild-type eyes, at the stages indicated. Boxed areas in B and C are magnified in inset. D–G: Co-immunostain with anti-AP-2γ (green) and anti-PAX6 (red) on horizontal sections of wild-type eyes at the stages indicated. D shows PAX6 alone while E shows double merge of the same image. Arrowheads in D and E denote strongly immunoreactive PAX6-positive cells in the neuroblast layer. Brackets in D and E indicate PAX6-positive amacrine cells in the INL. H–M: Co-immunostains of different AP-2 family members indicated (green) with BHLHB5 or SOX2 (red) at P0. Arrowheads in K–M point to SOX2-positive cholinergic amacrine cells, which co-express AP-2α and AP-2β (L,M) but not AP-2γ (K). i/onbl, inner/outer neuroblast layer; INL, inner nuclear layer; GCL, ganglion cell layer; IPL, inner plexiform layer; ONL, outer nuclear layer. Scale bars = 100 µm.

Fig. 5. The AP-2γ-immunopositive amacrine cell population only partially overlaps with the AP-2β-immunopositive amacrine cell population

A–D: Co-immunolabeling with anti-AP-2γ (green) and anti-AP-2β (red) on horizontal sections of wild-type eyes, at the stages indicated. Arrowheads denote double-labeled cells. E: Quantification of the proportions of co-labeled versus singly labeled cells in the total AP-2 population examined (AP-2γ and/or AP-2β-positive), at the stages indicated. Bars represent the mean ±SEM of counts from 3 animals. ***P < 0.001; **P < 0.01; *P < 0.05. onbl, outer neuroblast layer; INL, inner nuclear layer; GCL, ganglion cell layer; IPL, inner plexiform layer; ONL, outer nuclear layer. Scale bars = 100 µm.

Fig. 6

Subtle disorganization and diminished syntaxin immunoreactivity in the retina in Tcfap2a/b mutants. A–F: Horizontal H&E-stained sections of control (A–C) and Tcfap2a/b mutant (D–F) peripheral retinas, at the stages indicated. Black arrowheads (E,F) show regions of mutant retinas that appear less organized than controls. Red boxes in B and E are magnified in insets. G–L: Horizontal sections of control (G–I) and Tcfap2a/b mutant (J–L) peripheral retinas immunolabeled with anti-STX1A (green). Asterisks (H,I,K,L) indicate the band of STX1A-labeled amacrine cell processes in the IPL. The STX1A immunoreactivity detected in the outer retina of control mice (G–I, arrowheads) was diminished in Tcfap2a/b mutants (J–L, arrowheads). INL, inner nuclear layer; GCL, ganglion cell layer; IPL, inner plexiform layer. Scale bars = 100 µm.

Fig. 7

Deletion of Tcfap2a and Tcfap2b leads to loss of retinal horizontal cells. A–H: Horizontal sections of control (A,C,E,G) and Tcfap2a/b mutant (B,D,F,H) peripheral retinas immunolabeled (green) with anti-LIM1 (A–D), anti-neurofilament medium chain (NF-M; E,F) or anti-PGP9.5 (G,H) antibodies and counterstained with DAPI (4,6-diamino-2-phenylindole; blue) at E16.5 (A,B) or P0 (C–H). onbl, outer neuroblast layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars = 100 µm.

Fig. 8

Altered immunostaining patterns of amacrine cell markers in Tcfap2a/b mutant retinas. A–J: Horizontal sections of control (A,C,E,G,I) and Tcfap2a/b mutant (B,D,F,H,J) peripheral retinas immunolabeled (red) with anti-PAX6 (A,B), anti-BHLHB5 (C,D), anti-ISL1/2 (E,F), anti-SOX2 (G,H) or anti-AP-2γ (I,J) and counterstained with DAPI (4,6-diamino-2-phenylindole; blue), at the stages indicated. Closed arrows in C and I denote the band of BHLHB5-positive or AP-2γ-positive cells, respectively, clearly forming in the control INL, which was not observed in mutants (open arrows in D,J). Open arrowheads in E and G point to the developing cholinergic amacrine mosaic in control retinas, whereas asterisks in F and H indicate cholinergic cells that are failing to exhibit regular spacing in mutant retinas. onbl, outer neuroblast layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars = 100 µm.