The stage-dependent roles of Ldb1 and functional redundancy with Ldb2 in mammalian retinogenesis

Abstract

The Lim domain-binding proteins are key co-factor proteins that assemble with LIM domains of the LMO/LIM-HD family to form functional complexes that regulate cell proliferation and differentiation. Using conditional mutagenesis and comparative phenotypic analysis, we analyze the function of Ldb1 and Ldb2 in mouse retinal development, and demonstrate overlapping and specific functions of both proteins. Ldb1 interacts with Lhx2 in the embryonic retina and both Ldb1 and Ldb2 play a key role in maintaining the pool of retinal progenitor cells. This is accomplished by controlling the expression of the Vsx2 and Rax, and components of the Notch and Hedgehog signaling pathways. Furthermore, the Ldb1/Ldb2-mediated complex is essential for generation of early-born photoreceptors through the regulation of Rax and Crx. Finally, we demonstrate functional redundancy between Ldb1 and Ldb2. Ldb1 can fully compensate the loss of Ldb2 during all phases of retinal development, whereas Ldb2 alone is sufficient to sustain activity of Lhx2 in both early- and late-stage RPCs and in Müller glia. By contrast, loss of Ldb1 disrupts activity of the LIM domain factors in neuronal precursors. An intricate regulatory network exists that is mediated by Ldb1 and Ldb2, and promotes RPC proliferation and multipotency; it also controls specification of mammalian retina cells.

Keywords: Isl1; Ldb1; Lhx2; Retinogenesis.

Fig. 1.

Ldb1 and Ldb2 are required for maintaining proliferation and multipotency of RPCs. Gene expression and tissue morphology were monitored in control (A-E) and in Ldb1^loxP/loxP;Ldb2^−/−;α-Cre (F-J) eyes, determined by immunofluorescence analyses (A-D,F-I) and Hematoxylin and Eosin staining (E,J) for Ldb1 (red in A-C,F-H), Ap2a (green in B,C,G,H) and Ki67 (green in D,I) at E12.5 (A,F) and P0 (B-E,G-J). Counterstaining was with DAPI (blue, A,D,F,I). C,H are higher magnifications of staining for Ldb1 and Ap2a. White arrows in F,G,I mark the mutation area. CB, ciliary body; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; Ir, iris; NBL, neuroblastic layer; Re, retina. Scale bars: 100 μm in A,B,D-G,I,J; 50 μm in C,H.

Fig. 2.

The loss of Ldb1 results in premature cell cycle exit. (A-D′) BrdU was administered at E14.5 and was followed by analysis at E15.5 in control OCs (A-B′) and in Ldb1^loxP/loxP;Ldb2^−/−;α-Cre OCs (C-D′). Immunofluorescence analyses of Ldb1 (A,C, red), BrdU and Ki67 (red and green; B,B′,D,D′). The Ldb1/Ldb2 mutated area is marked with white arrows. (E) The percentage of BrdU⁺/Ki67⁻ from total BrdU⁺ cells was calculated in the control and Ldb1^loxP/loxP;Ldb2^−/−;α-Cre distal retina at E15.5, representing cell cycle exit. Data are mean±s.d., n=3, *P<0.001 calculated using a two-tailed t-test. NBL, neuroblastic layer. Scale bars: in A, 100 μm in A,B,C,D; in B′, 50 μm in B′,D′.

Fig. 3.

Ldb proteins are required first for preventing premature differentiation into ganglion and amacrine lineages and later for ganglion cell survival. (A-H) In control (A-D) and Ldb1^loxP/loxP;Ldb2^−/−;α-Cre (E-H) retinas, immunofluorescence analyses show the expression of Vc1.1 and Ki67 (red and green; A,E), Elavl3 (B,F), Ldb1 and Pou4f2 (red and green; C,G), and Ldb1 and Ap2a (red and green; D,H). Arrows indicate mutated area. (I) The percentage of Pou4f2⁺ cells at E15.5 in the control retina (34.7±1.55) and in Ldb1/Ldb2 mutant retina (50.4±0.21). Data are mean±s.d., n=3, *P<0.002 calculated using a two-tailed t-test. (J) The percentage of Ap2a⁺ cells at E16.5 in the control retina (15.8±0.56%) and in Ldb1/Ldb2 mutant retina (32.6±1.3%). Data are mean±s.d., n=3, *P<0.001 calculated using a two-tailed t-test. (K-R) In control (K-N) and Ldb1^loxP/loxP;Ldb2^−/−;α-Cre (O-R) retinas, immunofluorescence analyses show the expression of Isl1 and Pou4f2 at E16.5 (red and green, K,O), Pou4f2 and Ldb1 (green and red, L,P), cCasp3 (red, M,Q) and Ki67 (green, N,R) at E18.5. GCL, ganglion cell layer; NBL, neuroblastic layer. Scale bar: 100 μm.

Fig. 4.

Loss of Ldb1/2 alters the expression pattern of Notch and Hedgehog pathway genes, and impairs specification of PR cells. Control (A-F) and Ldb1^loxP/loxP;Ldb2^−/−;α-Cre (G-L) retinas from E14.5 (A-C,G-I) and E16.5 (D-F,J-L) eyes labeled with antibodies to Vsx2 (A,G), and Ldb1 and Crx (red and green; C,I). The expression of Rx (B,H), Hes1 (D,J), Hes5 (E,K) and Gli1 (F,L) were analyzed by in situ hybridization. Arrows indicate mutated area. GCL, ganglion cell layer; NBL, neuroblastic layer. Scale bar: 100 μm.

Fig. 5.

Ldb1 interacts with Lhx2 in the eye, is bound to the same regulatory regions as Lhx2 in Vsx2 and Rax genes, but is dispensable for Lhx2 stability. (A) Endogenous Ldb1 was immunoprecipitated (IP) from a lysate prepared from E15.5 eyes and P0 retinas (IP anti LDB1). As a negative control, parallel lysate was incubated with normal rabbit IgG control (IgG). The precipitated complex, whole cell lysates (Input) and supernatant (Sup; P0) were subjected to western analysis for detection of endogenous Lhx2 (IB-LHX2) and Ldb1 (IB-LDB1). (B-G) In control retinas (B-D) and in Ldb1^loxP/loxP;Ldb2^−/−;α-Cre retinas (E-G), antibody labeling was used to detect Lhx2 (B,C,E,F) and Ccnd1 (cyclin D1, D,G) during retinogenesis. The arrows mark the peripheral retina where α-Cre transgenes are active. Scale bar: 100 μm. (H) Ldb1 ChIP was performed on retinal tissue collected at P0. Scatter plot represents fold enrichment for the immunoprecipitated fractions relative to the isotype controls at regulatory sites of Vsx2 and Rax genes that were found to be bound by Lhx2 at positive sites (blue dots; de Melo et al., 2016) and at negative sites (orange diamonds). The horizontal lines indicate the mean fold enrichment of Vsx2 (n=3, *P=0.02) and Rax (n=4, *P=0.037) calculated using a one-tailed paired-t-test.

Fig. 6.

Ldb1 is not essential for maintaining the retinal progenitors but is required for activities of LIM domain proteins in retinal precursors. Control (A-E,K-O) and Ldb1^loxP/loxP;α-Cre (F-J,P-T) retinas were analyzed by antibody labeling, at the indicated developmental stages, for detection of Vsx2 (A,F), Lhx2 and Crx (red and green; B,G), Sox9 and Lhx2 (red and green; D,I), cyclin D3 and p27 (red and green; E,J), Isl1 and Pou4f2 (red and green; K,P), Ldb1 and Isl1 (red and green; L,Q,N,S, yellow arrowheads indicate a few cells that escape Cre activity based on maintained expression of Ldb1), Pou4f2 (M,R), and GABA (red; O,T). Hematoxylin and Eosin staining (C,H). CB, ciliary body; GCL, ganglion cell layer; INL, inner nuclear layer; NBL, neuroblastic layer; OC, optic cup; ONL, outer nuclear layer. White arrows indicate mutated areas. Scale bars: in A and K, 100 μm for A-M,P-R; in N, 50 μm in N,O,S,T.

Fig. 7.

The proposed dose-dependent roles of Ldb complexes in retinogenesis. (A) The expression of Ldb2 alone (Ldb^low) is sufficient to execute activities of the Ldb complex, which, probably through an interaction with Lhx2, is required for maintaining the RPC pool and Müller glia differentiation and function. High Ldb (Ldb1 alone; Ldb^high) is required for maturation of ganglion and amacrine (AC) precursors, and for generation of a subset of bipolar cells (BPL). Ldb1 probably contributes to these activities by maintaining the stability and function of LIM proteins that are expressed in these precursors: Isl1 and Lmo4. (B) The levels of Ldb available for interaction with the neuronal LIM proteins (Lhx2 vs Isl1) may be involved in regulating the balance between progenitors and precursors, and eventually in generating the correct numbers of retinal cell types.